Resolving forebrain developmental organisation by analysis of differential growth patterns

Domain: biology
Published: 2025-12-21
Editorial outcome: major_revision

Abstract
The forebrain is the most complex region of the vertebrate central nervous system, and its developmental organisation is controversial. We fate-mapped the embryonic chick anterior neural tube and built a 4D model of brain growth. We reveal modular patterns of anisotropic growth, ascribed to progenitor regions through multiplex hybridisation chain reaction. Morphogenesis is dominated by directional growth towards the eye, more isometric expansion of the prethalamus and dorsal telencephalon, and anterior movement of ventral cells into the hypothalamus. Comparative gene expression analysis and cell mixing experiments suggest the existence of a contiguous transverse boundary region, encompassing the zona limitans intrathalamica and retro-mammillary hypothalamus, that divides the anterior and posterior forebrain, and becomes distorted at the base of the zona limitans intrathalamica. Fate conversion experiments indicate that the hypothalamus is topologically tripartite, lying ventral to the telencephalon, prethalamus and zona limitans intrathalamica. Our findings challenge the widely accepted prosomere model of forebrain organisation, do not support a segmented anterior forebrain, and instead suggest a ‘tripartite hypothalamus’ model.

Introduction
In recent years, remarkable progress has been made in the profiling and classification of central nervous system cell types, and in understanding their specification and differentiation from neural progenitors1–3. Nonetheless, the fundamental layout of the developing vertebrate brain is still disputed, reflected in a confused and contradictory contemporary literature, with the extent and position of the hypothalamus at the centre of the debate. In classic columnar models of forebrain organisation, the thalamus, prethalamus, hypothalamus and eye were held to belong to the diencephalon, with the telencephalon positioned anterior to these structures4. However, an alternative view, in the form of the ‘prosomere’ model, has since gained significant traction. In this scheme, the forebrain consists of seven transverse segments—‘prosomeres’—analogous to the neuromeres of the hindbrain and spinal cord. Each prosomere possesses both roof plate and floor plate, a conceptual necessity to meet the prosomere definition of a segment. The hypothalamus is grouped with the telencephalon, together occupying two anteriormost prosomeres (hp2, hp1). Posterior to these are three diencephalic prosomeres (p3-p1) housing the prethalamus (p3), thalamus (p2), pretectum (p1) and associated ventral structures5,6. Most recently the midbrain has been designated as forebrain, contributing two more prosomeres7.

Although influential, the prosomere model is controversial, relying heavily for its support on gene expression patterns in the mid-to-late stage embryonic mouse. Classic features of segment boundaries, such as lineage restriction, have not been identified at any purported prosomere boundaries anterior to the ZLI (the prosomere dorsal p2/p3 boundary), nor have the locations of prosomere boundaries in the early ventral forebrain been precisely defined. And while the model incorporates detailed fate-mapping data—which can contribute valuable insight into topological relationships within the developing forebrain—this evidence is largely restricted to the dorsal aspect of the chicken neural tube, leaving ventral and basal regions under-characterized. A further challenge has arisen from recent studies showing that hypothalamic patterning is closely coordinated with prethalamic, rather than telencephalic, patterning8–12.

Here we clarify forebrain developmental organisation. We completed the fate map for the Hamburger-Hamilton stage 10 (HH10) chicken anterior neural tube while systematically visualising growth patterns, enabling a 4D reconstruction of forebrain growth. DiI/DiO ‘growth lines’ reveal how region-specific patterns of anisotropic growth sculpt the amniote forebrain. We reveal progenitors that originate in close proximity, and therefore receives similar positional cues during critical patterning periods, but then move apart, their original topological relations obscured by differential and directional growth. Molecular studies of the early chicken and mouse forebrain, supported by fate conversion and cell mixing experiments, contradict key predictions of the prosomere model and suggest alternative models. Our findings indicate a central role of eye morphogenesis in separating the telencephalon from the prethalamus and hypothalamus, all within a common anterior forebrain subdivision. A contiguous transverse boundary region, encompassing the retromammillary hypothalamus and zona limitans intrathalamica (ZLI), divides the anterior and posterior forebrain; region-specific growth distorts this transverse morphology, creating a flexure in the D-V axis at the base of the ZLI. Our work resolves prior misinterpretation and provides a new model for forebrain developmental organisation that we term the ‘tripartite hypothalamus’ model.

Results
DiI/DiO ‘growth lines’ reveal forebrain anisotropic growth
We fate-mapped the chicken anterior neural tube at HH10, aided by prominent morphological landmarks including a series of transient epithelial folds in the ventral midline (Figs. 1A-C and S1A-D). Embryos were injected with DiI/DiO, targeting the twelve zones depicted in Fig. 1D, and analysed after 48 hours (HH17-20), with a subset analysed after 24 hours (HH14-15). We labelled relatively large numbers of cells to provide insight into tissue-level growth patterns, and describe the resulting DiI/DiO distributions on the basis of morphology and marker genes (Figs. 1E, and S1E)8,13,14. We use a standard definition of hypothalamus, namely the paraventricular (PV), tuberal, mammillary (MM) and retromammillary (RM, also known as supramammillary) domains, omitting the preoptic region which is now usually considered part of the telencephalic subpallium. We define the A-P axis as running parallel to the hypothalamic NKX2-2 expression domain and the D-V axis as orthogonal to it (Fig. 1F).

Labelled neuroectoderm reproducibly gave rise to distinctively shaped territories, referred to as ‘growth lines’ (Fig. 1G) that could be allocated to one or more of seventeen forebrain regions (Fig. 1H), together revealing highly anisotropic region-specific growth. Injection zones 7-9 give rise to growth lines that reproducibly stretch from the telencephalon to the dorsal optic stalk or optic midline (Figs. 1I-L and S2)—zone 9 also skirting the prethalamic eminence (Fig. S2). Growth lines extend into the nasal retina where injections approached or overlapped with zone 10 (Fig. 1K).

Growth lines originating from ventral zones are elongated in an A-P direction (Figs. 1L-O and S3 and S4). Those originating from lateral parts of zones 4 and 5 (not previously fate-mapped) stretch from the SHH+ve hypothalamus, across the alar-basal boundary (ABB), into the ventral optic stalk and temporal retina, mirroring the growth lines connecting the telencephalon with nasal retina (Figs. 1L, M and S3). The long growth lines originating in zones 7-10 and 4-5 therefore converge towards the eye, and tend to lie within either FOXG1+ve or FOXD1+ve domains, respectively (Figs. 1P and S3R, S). They outline a wedge-shaped area of more isometric growth encompassing the prethalamus, which originates in area 11 (Figs. 1Q and S4C, D).

Zones 1 and 2 label the ventral hypothalamus. As previously demonstrated13,14, cells closest to the midline remain near the ventral midline while maintaining their relative A-P positions (Figs. 1R, S and S2O, P). Wider midline injections form an anteroventrally-sloping V-shape, showing that midline cells are displaced anteriorly relative to their more lateral counterparts (Figs. 1T, yellow arrow; S3E, S4J-K; 1U). This pattern is also clearly seen in growth lines arising from zones 2/6, which slope anteroventrally towards the tuberal hypothalamic midline, curving around the end of the SHH+ve floor plate and becoming distorted as they cross the PITX2+ve RM hypothalamus (Figs. 1V and S3K, L). The anterior-most SHH+ve floor plate arises from zone 3 (Fig. 1W).

Zone 12 gives rise to growth lines in the anterior thalamus and pretectum, which align in a D-V direction (Fig. S2K, zone 12). Large/multiple injections that simultaneously target zones 2/6/12 show that the D-V growth lines from zone 12 are continuous with the A-P growth lines in the basal hypothalamus: the orientation of growth lines changes abruptly close to the ABB, just posterior to the ZLI (Figs. 1W, X and S4H, I). Zone 6 injections give rise to a small tricorn shape at this point (Fig. 1Z).

In summary, our data shows that the hypothalamus arises in zones 1, 2, 4 and 5 (Fig. 1A, full data in Fig. S5), and that morphogenesis of the forebrain occurs in a stereotyped pattern of anisotropic growth, which is manifest as a series of highly reproducible, overlapping growth lines (Figs. 1G, AB). Growth lines maintain their relative topology, with minimal to no mixing of dye observed from different injection sites (Figs. 1AA, AB, S2-S5, for example see Fig. S4K). Key growth patterns, including the V-shaped and A-P oriented lines in the hypothalamus, and D-V oriented lines in dorsal regions posterior to the ZLI, were confirmed by DiI-DiO labelling of HH7-9 neuroectoderm (Fig. S6), by genetic clonal analysis using the Cre-Cytbow transgenic line (Fig. S7A-C)15, and in RFP-electroporated embryos (Fig. S7D-F).

Forebrain morphogenesis through region-specific growth patterns
We built a four-dimensional model of forebrain growth, based on the above findings combined with existing fate maps for dorsal regions16–18 (Fig. S8). This provides the most complete fate map yet of the HH10 chicken forebrain (Figs. 2A and S9, Movie S1) and recapitulates the key growth patterns in ‘digital DiI’ simulations (Fig. 2B, Movie S2). In sum, the model describes the morphological transformation of the anterior neural tube to the characteristic form of the amniote forebrain.

Anterior to the ZLI, elongated growth lines converge towards the optic stalk, demonstrating that eye outgrowth is associated with strongly directional growth over a wide area of the forebrain (Fig. 2B, Movie S3 0:16-0:29). Up to HH20, anterior forebrain morphogenesis is dominated by the combination of this directional growth, and a more uniform expansion of the telencephalic pallium and the prethalamus (Fig. 2C, Movie S3 0:35-1.00). The telencephalon thus takes on a balloon-like shape, narrowing towards the optic stalk; the prethalamus and PV hypothalamus together become wedge-shaped, narrowing oblique toward the dorsal midline and optic stalk (Fig. 2C); the optic vesicle/cup/stalk opening holds to a progressively smaller corner of the anterior forebrain (Fig. S10A-D, Movie S3 1:03-1:20). Telencephalic and basal hypothalamic lineages are largely segregated from an early stage by the intervening prethalamus, PV hypothalamus and eye field (Movie S3 1:22-1:47; compare growth lines in Fig. 2B to HH20 regions in Fig. 2A). Growth lines connect regions sharing either FOXG1 (telencephalon and nasal retina) or SIX6/FOXD1 (hypothalamus and temporal retina) expression (Figs. 2D, E, 1K, S10E, Movie S3 1:48-2:30).

More posteriorly, the thalamus also expands, contributing to the dorsal curvature of the neural tube (Fig. 2F, pink asterisks; Movie S4 0:08-0:18). Cells ventral to the ABB are displaced anteriorly by extension of the ventral midline, bringing them toward the telencephalon and creating V-shaped growth lines (Fig. 2F, green/orange asterisks; Movie S4 0:35-1:35). This movement resembles an anterior rotation of ventral regions, with a ‘hinge’ point at the intersection of the ABB and ZLI (Fig. 2F; Movie S4 1:34-1:48). The epithelial folds in the HH10 ventral midline contribute to the ventral tuberal hypothalamus; the ventral inflection point of the HH10 neural tube does not correspond to the HH20 cephalic flexure (Fig. 2G, asterisks; Movie S4 1:51-end), as is sometimes assumed.

The dorsoventral axis becomes distorted around the ZLI base
Proponents of the prosomere model argue that higher proliferation in the dorsal neural tube17,19 distorts block-shaped proto-segments into wedge-shaped prosomeres, causing the neural tube to pivot around the cephalic flexure (Fig. 3A)20–23. This provides the explanation for the position of the floor plate and roof plate, and hence the rationale for the orientation of the A-P and D-V axes (Fig. 3B), and the resulting placement of the hypothalamus ventral to the telencephalon and anterior to the prethalamus (Fig. 3C). However, when purported prosomere boundaries, based on earlier fate mapping16, are drawn onto our model at HH10 and HH20 (Fig. 3D top panels), their positions are not as predicted when viewed at HH20 and HH10, respectively (Fig. 3D bottom panels). Instead, our fate-mapping shows that much of the ventral hypothalamus arises posterior to the telencephalon, and ventral to areas that will give rise to the PV hypothalamus, prethalamus and thalamus (Figs. 2A, F and 3D, E). The anterior tuberal hypothalamus is an exception, originating ventral to the telencephalon.

We hypothesised that the early proximity of prospective hypothalamic and diencephalic cells results in a similar positional identity, due to similar exposure to fate-determining cues. If this is the case, it should be reflected in the shapes of progenitor domains toward the end of patterning stages (by ~HH20). Intriguingly, several genes in the HH20 posterior hypothalamus run approximately parallel to the ZLI, a lineage-restricted dorsal diencephalic signalling centre that divides the vertebrate nervous system into anterior SIX/FEZ+ve and posterior IRX+ve regions24–26. These include PITX2 and LMX1B in the RM hypothalamus, EMX2, OLIG28, EPHA7, and FOXD1 in the MM hypothalamus, and LHX6 and ARX in the prethalamic-like tuberomammillary terminal (TT) (Figs. 3F-J; 1E, P; S1E; S11A-F and ref. 8). Notably, PITX2 overlaps with a distinct FOXA1/LMX1B/SHH+ve ventral midline domain that abuts the ARX+ve floor plate27 at a flexure point (Fig. 3H, arrowhead; S11B, G). The PITX2+ve RM is flanked by, and partly overlaps with, WNT8B, SIM1, EPHA7 and DBX1; these genes converge at the ZLI and ventral midline (Figs. 3I; S11C-E), WNT8B resolving from a wider uniform domain at earlier stages (Fig. S11H).

Strikingly, WNT8B and BARHL2 encompass the ZLI dorsally and extend continuously through the hypothalamus to the PITX2/SHH+ve ventral midline (Figs. 3I, J and S11I-J). A slender, straight LFNG-ve domain encompasses the ZLI28, PITX2+ve SM, and part of the hypothalamic WNT8B/BARHL2+ve domain (Figs. 3I, J and S11I-K). The PITX2+ve RM is flanked by, and partly overlaps with, IRX1/IRX3+ve and FEZF2-ve, as in mouse9,29,30 (Figs. 3L, and S11L-M), i.e. has a ‘posterior’ molecular profile. BARHL2 expression straddles the edge of IRX3 in the RM and ZLI (a slight kink is less pronounced at earlier stages, prior to the onset of PITX2 expression) (Figs. 3L, M and S11L). In keeping with the posterior origin of these cells (Fig. 2A), this ‘posterior’ molecular profile places the RM hypothalamus both posterior to the prethalamus and contiguous with the ZLI (selected expression patterns are summarised in Fig. 3N). The D-V axis within the hypothalamus is therefore rotated compared to the prosomere model, in which the prethalamus is posterior to the RM hypothalamus (Fig. 3C).

To investigate whether continued growth may disguise these topological relations, we examined selected gene expression patterns at later developmental stages. At E7-E10 many genes including PITX2, ARX, WNT8B, LFNG, EPHA7, FOXA1, OTP, SIM1 and FOXA2 bear similar relations to one another as at HH20 (Figs. 3P and S12A-E). However, recalling the anterior movement of ventral cells at earlier stages (Fig. 2), the PITX2+ve ventral midline has shifted anteriorly relative to the ZLI. This creates an obtuse angle between the SM domain and the ZLI, recalling the ‘hinge point’ in our growth model (Figs. 3P inserts; 2F). The D-V/transverse alignment of these regions becomes distorted and the MM/RM domains start to take on an A-P oriented/longitudinal appearance (Fig. 3Q), the anterior edge of WNT8B aligning with a characteristic morphological fold, the hypothalamic periventricular organ (Fig. S12F)31,32. The topology of the chick embryonic forebrain thus becomes obscured by differential growth.

A possible dorsoventral axis distortion in mouse
We asked whether our findings might translate to mouse forebrain development, analysing selected gene expression patterns in mouse embryos of different stages. Similar to the HH20 chicken, the anterior edge of Foxa1 forms an early straight line, encompassing the Shh+ve ZLI and continuing to the ventral midline in the E11 mouse (Fig. 4A). Pitx2 is co-expressed with Foxa1 ventral to the ZLI and anterior to the Arx+ve floor plate (Fig. 4A, B). Thus, our proposed D-V axis (Fig. 3O) translates straightforwardly to the E11 mouse forebrain (Fig. 4C). As the ZLI is intercalated between the prethalamus and thalamus—both diencephalic regions—this implicates the posterior hypothalamus as part of the ventral diencephalon.

In support of this, at E8-E9 a transverse band of Barhl2 connects ventral posterior hypothalamus with prospective thalamus (Fig. 4D). By E11 an angle develops in the Barhl2 expression domain, between the ZLI and hypothalamus. To explore whether this may point towards an axis distortion as seen in chicken, we tracked the positions of ventral midline domains with respect to the ZLI, from E10 to E13. Lines were drawn along the ZLI and from the base of the ZLI to the anterior and posterior limits of the Pitx2+ve ventral midline (Fig. 4E; dotted lines are shown for the anterior markers, *A, and for ZLI position). As in chicken (Fig. 3P), the angles formed between these lines became more acute between E11 and E13 (Fig. 4F). This was also seen when Shh and Foxa1 ventral midline positions were tracked between E11 and E15 (Fig. 4G). Judging by the size of the thalamus, which forms a large bulge at the ventricular surface at E13 and is even more prominent at E15 (Fig. 4H), thalamic growth may contribute to this change by displacing the dorsal ZLI anteriorly. Although it is not possible to resolve this question solely by analysing gene expression patterns, we propose that the D-V axis runs through the ZLI and RM hypothalamus and may become distorted in mice as in chicken.

The prosomere mamillary domain cannot be identified in the chicken hypothalamus
The D-V axis position we propose in the posterior hypothalamus (Figs. 3Q and 4C) is nearly orthogonal to that in the prosomere model (Figs. 3C and 5A, B). To understand this discrepancy, we conducted a comparative study of prosomere boundaries in chicken and mouse.

In the prosomere model, the pretectum, thalamus and prethalamus occupy the dorsal portion of diencephalic prosomeres p1-p3, respectively. The entire hypothalamus is positioned ventral to the telencephalon within two anterior prosomeres (segments), hp2 and hp1 (Fig. 5A)—together comprising the ‘secondary prosencephalon’—and the hypothalamus is partitioned along intersecting transverse (interprosomeric; D-V) and longitudinal (A-P) boundaries (Fig. 5B-D). As these boundaries were deduced partly on the basis of mouse gene expression patterns between E11-E19, we first examined defining prosomere regional markers (Fig. 5D) in the E13 mouse brain, focussing on the Periretromammillary (PRM; in hp1), Retromammillary (RM, in hp1), Perimammillary (PM, in hp2) and Mammillary (M, in hp2) hypothalamic regions (see Figure. S13 for comparative hypothalamic terminology, axes, and selected regional marker genes)6–9,11,23,30.

Confirming previous findings, we readily identified a Nkx2-1/Sim1/Foxb1+ve (M) domain situated in between Pitx2/Foxa1+ve (RM) and Sim1/Otp+ve (PRM/PM) domains, (Fig. 5E-G), including a small Shh/Foxa1+ve midline region corresponding to the hp2 floor plate (arrowhead, Fig. 5F inset; note the prosomere floor plate is defined as extending beyond Arx, up to the anterior limit of Shh/Foxa1). Pitx2 was largely confined to the RM domain, but expression extended into the hp2 floor plate, this analysis revealing that the Pitx2+ve floor plate underlies both the M and RM domains (white line in Fig. 5G).

However, when we attempted to map prosomere model boundaries onto the HH20 embryonic chicken brain using the same markers, this proved challenging: putative transverse (using SIM1/NKX2-1/FOXA1, Fig. 5H, I) and longitudinal (using OTP/PITX2, Fig. 5J, K) boundaries appeared to take an identical course, instead of intersecting orthogonally6,7,23,30, so that FOXA1 and OTP directly abutted (Fig. 5K). Therefore, contrary to published accounts22,33, a SIM1/NKX2-1/FOXA1+ve, FOXA1/OTP-ve M domain (Fig. 5C, D) could not be identified in the HH20 chicken (Fig. 5L).

Placing prosomere boundaries onto the E7 chick brain was likewise problematic. By E7, FOXB1 is expressed and a slim SIM1/FOXB1+ve region could be identified (Fig. 5M), but it overlapped substantially with the RM marker FOXA1 (compare position of M label in Fig. 5M with RM label in Fig. 5N), and so could not be designated as the prosomere model M domain with certainty. Furthermore, similar to our findings at HH20, putative transverse and longitudinal boundaries (cyan and magenta dotted lines, respectively), intersected obliquely (Fig. 5O, middle panel), the A-P and D-V axes therefore crossing the posterior hypothalamus in similar directions (Fig. 5O, right panel). Notably, the edges of the hypothalamic Sim1/Otp/Nkx2-1/Pitx2 expression domains are highly curved in mouse, jutting out almost perpendicularly to the ZLI (e.g. Fig. 5E-G). The straighter edges of the equivalent expression domains in chicken more closely resemble those in Xenopus, zebrafish and lamprey22,34–37, and indeed the E11 mouse (Fig. 4A, B, D). Together this suggests that the morphology of the E13 mouse posterior hypothalamus is derived, the supposedly orthogonal boundaries and axes suggested by the prosomere model attributable to this. We conclude that the prosomere model misinterprets the hypothalamic axes due to an over-reliance on gene expression patterns from a single species at late developmental stages, combined with a flawed growth model arising from a lack of fate mapping information for ventral regions.

No evidence for a prosomere boundary within the hypothalamus
As our findings challenge both axes and boundaries in and around the M/RM hypothalamus, we expanded our interrogation of the prosomere model by revisiting the evidence in support of the hp1-hp2 transverse boundary, which purportedly cuts through the hypothalamus and telencephalon6,7,23,30. The position of this boundary was inferred partly from the course of the fornix tract, a structure appearing late in development, and justified by the small M floor plate, which is considered a conceptual requirement for the hp2 segment6, but the presence of which in chicken we could not confirm. A number of markers distinguishing hp1 from hp2 within hypothalamic longitudinal zones have been suggested from single colour in situ hybridisation on mouse brain sections (Fig. S14A, B)30. Taking advantage of newer techniques, we applied wholemount multiplex HCR to E13 mouse embryos.

Within the hypothalamus, the hp1-hp2 segmental boundary divides the Paraventricular (Pa) zone into anterior (terminal) and posterior (peduncular) subpopulations (Figs. 5C and S14 A, B). As predicted, the peduncular markers Lmo4 and Rgs4 showed a posteriorly restricted distribution (Fig. S14C, D). The terminal marker Zic1 was stronger anteriorly, as described, but showed graded expression rather than a distinct posterior limit (Fig. S14C, E). However, the peduncular marker Mfap4 colocalised with the canonical pan-Pa marker Otp, rather than being posteriorly restricted (Fig. 5P), and the terminal marker Fgf15 was expressed throughout the Pa area (Figs. 5P and S14E), as was Six3 albeit at a very low level (Fig. S14D).

In comparing our results with E13.5 sections from the Allen Brain Atlas38, we noted that the terminal markers Fgf15, Six3 and Zic1/5 were expressed most medially (i.e. the ventricular zone), followed by Otp, Sim1 and Mfap4, with Lmo4 and Rgs4 expressed most laterally (Fig. S14F). As proliferative progenitor cells reside at the hypothalamic ventricular surface, the mediolateral distribution of these genes likely reflects a maturity gradient. We sought to confirm this using our recent single cell RNA-sequencing (scRNA-Seq) dataset for pooled E11-E14 mouse hypothalamus39. Expression of Fgf15 was restricted to proliferative neural progenitor cells, while Otp was found mainly in differentiating neurons (Fig. 5Q).

To explore whether terminal and peduncular Pa markers may correlate with cell maturity, we extracted and reclustered Pa neural progenitor cells (NPCs) and PV/supraoptic nucleus (PVN/SON) neurons from the scRNA-Seq dataset. Eight clusters resulted (Fig. S15A), of which cluster 6 showed strong enrichment for progenitor/mitotic markers (Mki67, Top2a, Pcna; summarised as ‘Progenitor score’), the remaining six clusters scoring highly for neuronal markers (Neurod1, Dcx, Tubb3; summarised as ‘’Neuronal score’, Fig. S15B). The TPa markers Fgf15 and Six3 were most highly expressed in progenitor cluster 6, while Zic1 showed variable expression across the eight clusters (Fig. S15C, D). The PPa markers Mfap4, Rgs4, and Lmo4 were also variably expressed, all three genes on balance being lower in cluster 6. Terminal and peduncular ‘scores’ were calculated based on the TPa and PPa genes displayed in Fig. S15C; progenitor cluster 6 and neuronal cluster 3 scored highest for the terminal profile (Fig. S15B). This suggests that TPa and PPa markers may in part reflect differentiation status rather than segmental identity.

Finally, turning to the Subparaventricular (SPa) zone, we were not able to confirm differential expression of Vax1 in the TSpa versus PSPa (Figs. S14A and S15E), and Nkx6-2 was not excluded from the Six6+ve acroterminal SPa as previously thought (Fig. S15F, see Fig. S15G for the acroterminal concept). While Meis2 expression did taper anteriorly within the SPa (Fig. S15E), it is unclear why this—or indeed any of the above genes showing graded differential expression—should indicate a segmental boundary, rather than being the result of any other patterning process. Overall, the weak evidence for the hp2-hp1 boundary leads us to question its existence. Along with our axis revisions, these conclusions nullify the key prosomere concept of the secondary prosencephalon and begin to return the hypothalamus to the diencephalon, as per the classical (columnar) view.

A new model of forebrain developmental organisation
Our results re-orient the position of the D-V axis suggested by the prosomere model, and consequently call into question the A-P axis. The prosomere model takes the anterior limit of ventral midline Shh/Foxa1 as the anterior limit of the neuraxis. However, while Shh/Foxa1 expression does indeed terminate ventral to the ZLI in mid-embryogenesis, it extends far more anteriorly at neural tube patterning stages, into and including cells that give rise to the tuberal hypothalamus13,40. This suggests that the A-P axis continues into the tuberal hypothalamus. Using our revised axis positions as a starting point, we explored further ways of testing forebrain positional relationships. One approach is to use fate conversion, as cells with similar positional identities often share competence to express a restricted set of factors. We therefore dorsalised the chicken hypothalamus by applying SHH inhibitors (cyclopamine or Sonidegib), with or without follistatin8, at the neural tube stage and looked for ectopic expression of regional markers.

Dorsalisation treatment at this stage resulted in a small SHH+ve basal hypothalamus and decreased expression of NKX2-1 (Fig. S16A, B). The telencephalic marker FOXG1 was ectopically expressed, but only in part of the anterior-most tuberal hypothalamus (Figs. 6A and S16A-C). Although FOXG1 is also expressed in the anterior (nasal) retina, no change was detected in the optic stalk marker PAX2, suggesting that the converted cells are similar to telencephalon rather than anterior retina (Fig. S16C). Altered cell movements were not responsible for the presence of FOXG1+ve cells within the hypothalamus, as DiI outcomes from zone 1/7 injections (in combination with dorsalising treatment) were not altered in position or shape with respect to the optic region (Fig. 6C). This result therefore appears to be a genuine fate conversion of anterior hypothalamus towards a telencephalic identity.

Notably, however, more posteriorly within the tuberal hypothalamus, the prethalamic/PV hypothalamic marker PAX6 was ectopically expressed (Fig. S16A, C), and the prethalamic marker ARX was significantly expanded (Figs. 6A, B, and S16A). Similar results were obtained when HH10 zones 1, 2, 4 and 5 were isolated and cultured in the presence or absence of Sonidegib (Fig. S17). OLIG2 remained confined to the prethalamus, posterior tuberal and MM hypothalamus (Fig. S16A). Considered alongside our fate map (Fig. 2A, Movie S1), these results suggest that proximity to prospective telencephalon at HH10 predicts competence to express FOXG1, and proximity to prospective prethalamus/PV hypothalamus predicts competence to express PAX6/ARX. Accordingly, we suggest that the anterior-most tuberal hypothalamus is topologically ventral to the telencephalon, in partial agreement with the prosomere model, but that the remainder of the tuberal region is ventral to the PV hypothalamus and prethalamus, and is part of the diencephalon.

Classically, the PV hypothalamus is included as part of the diencephalon, but the development of the PV hypothalamus remains particularly poorly understood. Our fate mapping revealed a close association between PV progenitors and prethalamus, although growth lines also connect smaller parts of the PV to the telencephalon and basal hypothalamus. Elsewhere in the nervous system, cells with a common identity intermingle, whereas those from different regions segregate. We isolated PV and telencephalic cells (Fig. S18), then disaggregated and reaggregated them in in vitro cultures. PV hypothalamic cells intermingled with prethalamic cells, but segregated from telencephalic cells (Fig. 6D, E combinations A-B, A-C; controls shown in Fig. S19). As prethalamic cells showed poor viability when mixed with telencephalic cells, we could not assess interspersion for this combination (Fig. 6D, E combinations B-C). Similar to the classical view, we therefore tentatively group the PV hypothalamus with the prethalamus as part of the diencephalon, posterior to the telencephalon.

Finally we applied the cell mixing approach to the posterior hypothalamus (Fig. S18), where our D-V axis runs through the ZLI and RM hypothalamus. Dorsally, cells taken from anterior and posterior to the ZLI segregated, albeit only partially (Fig. 6D, E combination B-D. Importantly, basal plate cells taken from anterior and posterior to the RM hypothalamus segregated in culture (Fig. 6D, E combinations E-F and E-G), while those from posterior and anterior or ventral positions within the basal hypothalamus intermingled (Fig. 6D, E combinations G-H, F-G). We therefore suggest that lineage restriction may operate ventral to the ZLI, as shown for an En1-Dbx1 floor plate boundary in mouse41.

In conclusion, we forward a ‘tripartite hypothalamus’ model of forebrain organisation in which the basal hypothalamus sits ventral to three regions—the telencephalon, prethalamus/PV hypothalamus, and the ZLI (Fig. 6F). The LFNG-ve ZLI and posterior hypothalamus form a topological boundary between the anterior SIX/FEZ+ve and posterior IRX+ve forebrain, with the RM hypothalamus part of a ventral boundary region associated with lineage restriction. Informed by high resolution fate mapping, the topological relations of forebrain regions in our model reflect their early spatial origins—and therefore the environment seen by their progenitor cells. Inevitably, emerging and future studies will put this concept to the test.

Methods
Embryosourcing and incubation
Fertilised Bovan Brown eggs (Henry Stewart & Co., Norfolk, UK), and transgenic Chameleon (cytbow)15, Cytoplasmic GFP66, and Flamingo (tdTomato)15 eggs (The Roslin Institute, Edinburgh, UK) were incubated in a humidified incubator at 37°C until the desired stage, according to67,68. Time-mated pregnant wildtype C57B1/6 mice were sourced from Envigo RMS (UK) Limited, Shaws Farm, Bicester.

Immunolabelling
Embryos were fixed in 4% PFA for 2 hours at 4°C and washed in PBS. Samples were incubated in block solution (1% TritonX, 10% heat-inactivated goat serum (HINGS) in PBS) overnight, then primary antibody solution (Rabbit anti-laminin α (LAMA), Sigma L9393 at 1:1000 in block solution) overnight. Embryos were washed twice in PBS at room temperature, and transferred to 10% HINGS in PBS and left overnight. Alexa 488-conjugated anti-rabbit IgG secondary antibody (1:500, Jackson ImmunoResearch) and phalloidin-TRITC (1:100, 1% TritonX, 1% HINGS in PBS) were applied at 4°C overnight, washed the following day in PBS at room temperature, then washed overnight in PBS with 0.1% TritonX. All steps were performed at 4°C except where stated.

DiI/DiO injections
Eggs were windowed and Coomassie Blue (0.5μl/ml in L-15 - Fisher Scientific, Cat No 11580396) was injected under the embryo to aid visualisation. The forebrain inner neuroectoderm was accessed via a dorsal midsagittal incision. CellTrackerTM CM-DiI Dye (Invitrogen, Cat No. C7000) in ethanol (50μg per 30μl), or Vybrant™ DiO cell-labeling solution (Invitrogen, Cat No. V22886) was loaded into a fine glass needle and injected into embryos by hand using a Parker Picospritzer II (10-20 msec pulses, 15 psi). Dye location was noted and imaged using a Leica MZ16F stereomicroscope. Embryos were incubated for 48h as stated, fixed in 4% buffered paraformaldehyde, hemisected and reimaged prior to processing for HCR. Some DiI/DiO signal is lost during the HCR procedure, therefore dye and gene expression patterns are shown separately for some examples.

HCR analysis
Embryos were fixed overnight in 4% paraformaldehyde at 4°C and stored in methanol for a minimum of one night before rehydrating in PBS. HCR v3.069 was performed on intact embryos according to the manufacturer’s RNA-FISH protocol for whole-mount chicken and mouse embryos (as shown on Molecular Instruments website), using reagents obtained from Molecular Instruments, Inc. (Los Angeles, CA, USA). HCR probes were designed by the manufacturer based on the following accession numbers or the manufacturer’s database (‘infinite catalogue’): Chick: ARX (XM_025146483.1), BARHL2 (XM_015290665.4), EMX2 (XM_025152058.1 and XM_025152057.), EPHA7 (NM_205083.1), FEZF2 (XM_414411.5), FOXA1 (XM_004941922.3), FOXA2 (NM_204770.1), FOXD1 (NM_205192.3), FOXG1 (NM_205193.1), IRX3 (XM_015292372.4), LFNG (NM_204948.1), LHX6 (XM_015279838.2), LMX1B (NM_205358.1), NKX2-1 (NM_204616.1), NKX2-2 (XM_015283379.2). OLIG2 (NM_001031526.1 with extra sequence from NC_006088.5|:106522977-106525323#256 chromosome 1, GRCg6a), PAX6 (NM_205066.1), PITX2 (NM_205010.1 with additional 5’ exons from XM_025149516.1/ XM_025149515.1), SHH (NM_204821.1), SIM1 (XM_004940357.3), SIX6 (NM_001389365.1), WNT8B (XM_025151998.1).

Mouse: Arx (NM_001305940.1), Barhl2 (NM_001005477.1), Fgf15 (Infinite catalogue), Foxa1 (NM_008259.4), Foxb1 (NM_022378.3), Lmo4 (Infinite catalogue), Meis2 (Infinite catalogue), Mfap4 (Infinite catalogue), Nkx2.1 (NM_009385.4), Nkx6.2 (NM_183248.4), Nr5a1 (Infinite catalogue), Otp (NM_011021.5), Pitx2 (NM_001287048.1), Rgs4 (Infinite catalogue), Six3 (Infinite catalogue), Shh (NM_009170), Sim1 (NM_011376.3), Vax1 (Infinite catalogue), Zic1 (Infinite catalogue). Proteinase K treatment (10µg/ml) was performed for 2-30min and embryos were re-fixed for 20 minutes, washed in PBST and transferred to 5x SSCT on ice. Embryos were preincubated in hybridisation buffer for 30 mins, then hybridised overnight in 10 nM (1:100) probe solution in hybridisation buffer, both steps at 37°C. Samples were washed 4×15 minutes in wash buffer at 37°C, then 2×5 minutes in 5x SSCT at room temperature. Embryos were equilibrated in amplification buffer for 5 minutes before adding the hairpins solution. Even and odd hairpins were melted (90 secs, 96°C) and cooled (30 minutes, room temperature) separately before mixing in amplification buffer (1:50). Samples were incubated in the dark overnight and washed in 5x SSCT (2×5 mins, 3×30 mins, 1×5 mins). Embryos were hemisected or the neuroectoderm isolated (see below) as necessary. Tissue was counterstained with DAPI prior to imaging (Cell Signaling Technology, Cat no. 4083, 1:1000).

Quantification of DiI/DiO results
Seventeen DiI/DiO outcome areas (Fig. 1H), and the growth line shapes associated with each (Fig. 1G), were developed based on extensive analysis of >300 DiI/DiO patterns. Areas were named according to the major region in which they were located (T1-T2-telencephalon, D1-D7—diencephalon excluding hypothalamus, H1-H5—hypothalamus and Hm1-Hm3—hypothalamic midline), but as these were based on DiI/DiO patterns and not gene expression, in some cases the areas overlap more than one major region.

Quantification was performed only on samples injected at HH10. Positional outcomes were scored by assigning each result to one or more of these seventeen outcome areas. For each of the twelve injection zones, and the meeting points of adjacent zones, the percentage of samples showing labelling in each area was calculated. Results were displayed as thumbnail images where all outcome areas with at least one instance were coloured according to frequency, i.e. white means no examples were seen. Concurrently with assigning positional outcomes, it was judged whether the shape of each resulting DiI/DiO pattern (growth line) conformed to the description for each area. In some cases the location of labelling was clear but the shape of the growth line was not; these cases were pooled with those examples where the described shape was not seen.

Cre recombination and neuroectoderm isolation
Chameleon (Cytbow) eggs were incubated until HH9, windowed, staged, and screened for the Cytbow transgene, using a handheld 365nm black-light torch. 0.5-1.5μl TAT-Cre recombinase (1500U) (Merck Life Science UK, SCR508) diluted 1:20 to 1:200 in L-15 was injected into the anterior neural tube using a pulled capillary needle. Eggs were sealed and incubated for a further 24-48 hours before fixing and staining for NKX2-2/WNT8B by HCR. Following HCR, forebrains were hemisected and the neuroectoderm isolated by microdissection aided by treatment with Dispase (1mg/ml) for approximately 10 minutes to help separate tissue layers. Highly curved regions were nicked with a scalpel to enable flat mounting on a standard microscope slide.

Electroporations
A Microelectrode holder (World precision Instruments, MEH6RFW) with a 0.25mm silver wire was used as the cathode. A capillary needle containing 1-2 ug/ul pCAGGS-RFP with 0.1% Fast Green dye was threaded over the silver wire, and a mouth pipette attached to the side inlet valve to control the internal pressure. The anode, made from a tungsten needle, was positioned under the target cells and 2×3 pulses of 40V were applied using a TSS20 Ovodyne electroporator (Intracel), whilst simultaneously increasing the pressure in the needle to release the DNA mixture. Eggs were then re-sealed and incubated for a further 24-48 hours.

Imaging and image processing
Images were taken on a Zeiss Apotome 2 (10x objective) with ZEN 2 Pro, blue edition software (Zeiss), Leica MZ16F using LAS X 1.1.0.12420 software, Zeiss Lightsheet (5x, 10x objectives) with ZEN software (Zeiss), and Nikon W1 Spinning Disk confocal (4x, 10x, 20x objectives) with Nikon NIS-Elements AR version 5.42.03 (build 1812) software. For Apotome, Lightsheet and Spinning Disc imaging, embryos were mounted in 1% agarose, and z-stacks are presented as maximum intensity projections. Image processing was performed using Fiji (ImageJ, version 2.7.0/1.53t) and Adobe Photoshop 2022. Hemisected embryos are presented as the right side of the embryo, for ease of comparison. Gamma levels have been altered for clarity.

4-dimensional modelling of forebrain morphogenesis
Neuroectoderms were isolated (see above) from HH10, HH11, HH14 and HH20 chicken embryos, stained with DAPI, and imaged on a Zeiss Lightsheet confocal (Fig. S6, step 1). Image stacks were converted to binary, denoised, and exported as a wavefront (.obj file) using FIJI software and the 3D viewer plugin (Fig. S6, step 2). The .obj was imported into the open source animation software Blender (blender.org) and the smooth tool (in sculpting mode) was used to remove the artefactual contour lines. The objects were remeshed to reduce file size, and these meshes were used as morphological templates to build the model around (Fig. S6, step 3).

A simple mesh was built around the HH10 template by first adding a single plane and enabling snapping to the surface of the template (using Face Project, with ‘Project individual elements’ enabled, as well as the Shrinkwrap modifier; Backface culling was enabled, as was ‘in front’ under Object Properties/Viewport Display). Geometry was added using the modelling tools, until the entire right hand side of the neuroectoderm was represented by a simple quad mesh. The mirror modifier was used to view the entire neuroectoderm to assist progress, but never actually applied to the mesh. Different faces of the mesh were given different colours, to assist subsequent stages by making them more easily identifiable. The simple quad mesh was then duplicated (ensuring the new object was not linked to the original) and snapped to the surface of the HH20 template mesh. The vertices were moved using the tweak tool, so that their positions at HH10 and HH20 roughly corresponded, according to the DiI/DiO results from the present study and the fate maps of Garcia-Lopez et al.16 and Pombero et al.17. In a similar manner, the meshes were copied and snapped to the HH11 and HH14 template meshes, and the vertices positioned to intermediate points (between the HH10 and HH20 meshes) (Fig. S6, step 4). The meshes were periodically subdivided by applying the subdivision modifier (simple setting, Fig. S6, step 5) and adjusting further, until the level of detail and precision, was deemed sufficient. All such changes to the mesh geometry were applied identically to all four meshes in order to enable joining later. Progress was monitored along the way by joining the meshes (usually only the HH10 and HH20 meshes) as shape keys (‘Join as shapes’ option); selected faces were then coloured according to HH10 position or HH20 region, and the outcome at the opposite time point was viewed to assess the fate map or ‘digital dye’ growth lines.

Once the vertex positioning was complete, the four meshes were duplicated to the insides of the template meshes and adjusted for optimal fit. Inner and outer meshes were joined and subdivided further, so that they closely followed the surfaces of the template meshes (Fig. S6, step 6). The four meshes were then joined as shape keys to enable warping between the four stages (Fig. S6, step 7). Fate map and ‘digital dye’ colours were applied to the surface by creating a UV map and using the texture painting options (Fig. S6, step 8). A procedural material was assigned to generate a surface texture. Forebrain animations were created and exported from Blender, and annotated movies were compiled using Adobe Premier Pro 2024 software. Note that the modelling approach does not allow for any ‘cell mixing’ between adjacent parts of the mesh, hence ‘digital growth lines’ are slightly smaller than in vivo.

Quantification: Maximum projections of spinning disk images taken at the same settings were opened in Fiji and the selection tool used to manually select the hypothalamus, the resulting shapes saved in the ROI manager. FOXG1 HCR images were de-speckled using the Fiji Remove outliers tool before applying an equal threshold to all images. The hypothalamus ROI was reapplied and the area of positive expression within measured. FOXG1 expression has been de-speckled using the photoshop ‘dust and scratches filter’ in Fig. 6A panel 1, but the same images shown at higher magnification in panel 2 has been left unaltered. Two-sided Welch’s t tests were run as the variances differed between groups (p<0.00001 FOXG1, p=0.0333 ARX), using Graphpad Prism 10.6.1.

Morphometric analysis
To measure chicken forebrain dimensions (Fig. S10), HH10-HH19 forebrains were stained by HCR for FOXG1, OLIG2 and WNT8B and hemisected and imaged on a Nikon W1 Spinning Disc confocal. Morphometric measurements were performed on maximum projections of z-stacks using FIJI (ImageJ, version 2.7.0/1.53t). Distance measurements were obtained using the line tool and ‘measure’ function. First, line A (Fig. S10B) was drawn between two trackable points X and Y: respectively, the midline at the ventral limit of FOXG1+ve, and a corner of the OLIG2 expression domain (Fig. S10B-C); our DiI results demonstrate that these points correspond at HH10 and HH20 (X-zone 1/7 boundary, Y-zone 6, Fig. 1D). A perpendicular line was then positioned to intersect both line A and the posterior margin of the optic vesicle/stalk, which was clearly visible in hemisections as an edge (brightness/contrast levels were adjusted as needed). Line B was drawn between the posterior limit of A and the point of intersection of A and the perpendicular line. Results were exported to Excel using the Read_And_Write_Excel Plugin, the B:A ratio was calculated, and a logarithmic trend line was added.

To track mouse dorsoventral axis distortion (Fig. 4E-G), lines were drawn on HCR-stained mouse E11/E13 forebrains along the ZLI, and from the base of the ZLI to the ventral midline: point *A marked the anterior limit of Pitx2 ventral midline expression, and point *B marked the Pitx2 posterior limit and the Arx anterior limit. Angles between the two lines were measured using the angle tool in Adobe Photoshop. Two-sided t tests were run using Graphpad Prism 10.6.1.

scRNA-Seq analysis of mouse E11-14 PVN lineage
We analysed single-cell RNA-Seq (scRNA-Seq) resources generated previously in the lab, a whole-hypothalamus E11-E14 10x Genomics dataset (GEO accession GSE284492), to subset paraventricular/supraoptic neurons and their local progenitors (“PVN+NPC”). All analyses were performed as previously described39.

Cells expressing Otp and/or Sim1 were retained, but NPCs lacking these markers were also included to represent earlier stages of the lineage. Contaminating ventral/posterior populations were removed by excluding any cell with >0 UMIs for Foxa1, Foxa2, Pitx2, or Shh. Additional marker-based filtering removed cells expressing Nr5a1, Foxb1, Tbr1, Agrp, or Pomc. The remaining cells were reclustered de novo and visualised using UMAP. Gene-set module scores were computed using Seurat’s AddModuleScore() function70. The anterior/terminal (hp2) module included Fgf15, Six3, Zic1, and Zic5, while the posterior/peduncular (hp1) module included Mfap4, Lmo4, and Rgs4.

To capture proliferative and differentiating states, a cycling progenitor score was calculated using Mki67, Top2a, and Pcna, and an early neuronal differentiation score was computed using Neurod1, Dcx, and Tubb3. For each cell, genes of interest were overlaid on UMAPs or summarised in dot plots.

Dorsalisation of chicken embryos
Embryos were windowed at HH10-11 and the vitelline membrane torn above the head. Dorsalisation was induced by treatment with the SHH antagonist Sonidegib (Cat No. S2151, Selleckchem). 2 mM in DMSO was diluted 1:4 in L15 to 0.5 mM, or DMSO only diluted 1:4 in L15, and 2ul pipetted onto embryo and the egg re-sealed. Alternatively, Cyclopamine (1μg/μl; Sigma - C4116, dissolved in PBS with carrier 45% 2-hydroxypropyl-β-cyclodextrin, Sigma - H5784), with or without the TGF-β inhibitor Follistatin, recently shown to repress ventral identity8 (15μg/μl Recombinant Mouse Follistatin 288; 769-FS, Bio-Techne). 5μl of cyclopamine only or 5μl of Cyclopamine/FST (1:1) was pipetted on top of the embryo and the egg was re-sealed. Embryos were incubated at 37°C and were dissected and fixed 24-48 hours later and processed for analysis by HCR.

Quantification: Maximum projections of spinning disk images taken at the same settings were opened in Fiji and the selection tool used to manually select the hypothalamus, the resulting shapes saved in the ROI manager. FOXG1 HCR images were de-speckled using the Fiji Remove outliers tool before applying an equal threshold to all images. The hypothalamus ROI was reapplied and the area of positive expression within measured. FOXG1 expression has been de-speckled using the photoshop ‘dust and scratches filter’ in Fig. 6A panel 1, but the same images shown at higher magnification in panel 2 has been left unaltered. Two-sided Welch’s t tests were run as the variances differed between groups (p<0.00001 FOXG1, p=0.0333 ARX), using Graphpad Prism 10.6.1.

Cell mixing analysis
Transgenic Cytoplasmic GFP and Flamingo (tdTomato) chicks were dissected at E5 or E6 into L-15 on ice. Neuroectoderms were isolated following Dispase (Sigma-Aldrich, Cat No. 4942086001) treatment and forebrain regions microdissected. A set of explants from individual forebrain regions were immediately fixed, and processed for HCR in situ to confirm accuracy of dissection. A second set of explants from each region (unfixed) were incubated in Papain (2mg/ml) for 10 mins at 37°C and dissociated into single cells by trituration using fire polished glass pipette with different capillary openings (large and small). Following dissociation, the enzyme was inactivated with 5mls of L-15 and cells gently centrifuged at 600 RPM for 5 mins at 4°C. Cells were resuspended in 1ml of explant medium71 and counted using a haemocytometer. Hanging drops were formed from 15ul cell suspension containing 20,000 cells/drop, and incubated at 37°C for 24-48hrs to form pellets. Cell pellets were fixed, cryosectioned and imaged without further staining.

Explant culture
HH10 neuroectoderms were isolated following dispase treatment (Cat no 4942086001, Roche, 1mg/ml in L15 medium at room temperature, 5-15 minutes)72. Explant encompassing zones 1-2-4-5 was carefully sub-dissected and mounted on matrigel (11523550, Fisher Scientific UK) beds and cultured for 24hrs at 37°C71. Explants were treated with DMSO control or Sonidegib (200μM). Explants were then fixed and processed for analysis by HCR.

Ethics
All work was performed at our licensed establishment (UK Home Office under the Animals (Scientific Procedures) Act 1986) and was approved by the University of Sheffield Animal Welfare and Ethical Review Body (AWERB). Named Animal Care and Welfare Officers (NACWOs) had oversight of all incubated eggs. Pregnant female mice were killed by a Schedule 1 method.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a very useful resource for the developmental neuroscience research community. The authors present a set of nice lineage tracing experiments in the chick forebrain. Based on those, 3D computational models (fantastic movies describing likely 3D temporal progression of domains) and restricted set of HCR mRNA detection, the authors refute the current prosomeric model of forebrain organisation, proposing a modified model. Although the work is very carefully done and of general interest for the field, we have difficulties with the lack of focus of the narrative and some lack of evidence for the model proposed (while agreeing on finding the prosomeric model problematic for the anterior and ventral forebrain). Overall, the work as it is presented currently does not support strongly enough the conclusions made by the authors.\nSpecific points to be addressed:\n- The most precious part of the data is the collection of lineage tracing results. Although the stated n for each of the injected forebrain areas described on Figure 1 is high, there is no quantification of the clone size, shape and position at HH20 across embryos injected for each area at HH10. This quantification is essential to assess variability across injections. Figure S2 suggests a fair amount of variability and same embryos are used for two distinct data points, using different left and right positions of injection (eg. Fig. S2 E & F, H & I, J & K).\n- The last frame of Movie S2 should be used in an improved Figure 2 to visually match the findings with the model of clonal shapes (if the above quantification of clone size, shape and final position confirms the current conclusion per position on the HH10 map).\n- The dynamic model for the yellow area in Movie S4 (not equivalent to the yellow area of Figure 1) is key to the author’s model but does not have enough lineage tracing evidence for its proposed origin. Specific lineage tracing of this area (with enough n) is needed.\n- The attempt to refute the prosomeric model is not well articulated, offering partial evidence for the proposed alternative. The most important data would be extensive tracing in area 1, 2, 5 and 7. Fig S4D is key and yet is not showing the position of the injected cells at HH10.\n- A substantial portion of the argument for the proposed ‘compartmental’ origins of the hypothalamic areas is based on HCR analyses. However, dynamics in gene expression can’t be used as rigorous support for clonal dynamics. This is a big limitation of the manuscript. Higher resolution lineage tracing is needed to back the HCR conclusion up.\n- The cell mixing experiment (Fig 4J), where cells separate based on their positional identity (anterior hypothalamic vs posterior thalamic), is key to the proposed model and is missing key controls and ambition. It would need i) mixing the posterior hypothalamic cells with their own (A with A in 4J, matching the P with P) and with cells from the proposed anterior hypothalamic compartment, to see if mixing results match their proposed model.\n- The quality of the DiI/DiO images is very low. And the Cre/tdTomato images are also very hard to interpret because of overabundance of positive cells. A better approach could be to inject fluorescently labelled viruses in regions of interest and see lines of cells that way.\n- The point about dorsalization of ventral diencephalon region with cyclopamine is weak, as the markers they use to indicate telencephalon (Foxg1) and prethalamic/PV hypothalamic (Pax6) are also expressed in the eye. Given that this treatment is known to lead to cyclopic phenotypes, the markers may be present in ‘trapped’ retinal cells, instead of proving a fate conversion to TC and pre-thalamus. Much more needs to be done to conclude anything from this experiment.\n- The comparison between chick and mouse is potentially powerful but only basing it on HCR of a few markers introduces too many possible caveats and shouldn’t be considered here unless adding clonal evidence.\nSmaller issues:\n- They refer to figure 5K in the discussion. That panel doesn’t exist in figure 5.\n- Figure S5 is not useful as the Cre clones can’t really be informative if not quantified carefully (size and shape) across many embryos.\n- Some of the HCRs are hard to see especially when they’ve used grey colour on top of the faint blue background.\n- The most important focus of the manuscript is the organisation of the hypothalamus. Yet, the results are written in a very complicated fashion, distracting the reader from the most important conclusions. We would advise to reshape the text to facilitate understanding and clarity of thoughts.", "reviewer_2": "This manuscript describes a comprehensive and detailed fate-mapping of the chicken forebrain at HH10 using fluorescent dye labeling along with spatial expression pattern analysis of multiple marker genes.\nThe following new knowledge has been generated from this study:\n1) Generation of detailed fate map of the chick forebrain at HH10.\n2) Generation of a 4D model of forebrain growth.\n4) Establishing that eye morphogenesis plays a key role in separation of the telencephalon from the prethalamus and the hypothalamus.\n3) Proposal of a \"tripartite\" model for the hypothalamus.\n4) Identification of a major forebrain boundary that cuts through the MM hypothalamus and the ZLI.\nThis study does not contain any functional characterization that may shed light on the genes and mechanisms involved in the morphogenesis of the forebrain, particularly the hypothalamus. However, it has brought significant clarity to this phenomenon with respect to the growth movements and the changes in the orientation and positioning of the AP and DV axes. In fact this study has challenged some aspects of the existing prosomere model and proposed a new \"tripartite\" model for the formation of the hypothalamus. Thus significant new knowledge has been add to the field.\nThe experiments that have been carried out largely support the conclusions made and the data has been analyzed in depth and interpreted correctly.\nThe methodology used meets the expected standards in the field and sufficient details have been provided in the supplementary materials for reproduction.\nHence this manuscript is suitable for publication without the requirement of any major changes.", "reviewer_3": "The authors of “Resolving forebrain development organization by analysis of differential growth patterns” proposed a common anterior compartment of the forebrain with telencephalon, hypothalamus and prethalamus. In addition, the authors claim a new model of forebrain development with a tripartite hypothalamus. This is a detailed study that provides extensive data that aims to support their hypothesis. However, after a detailed analysis of the manuscript, some key points need to be resolved by the authors, including the need to better clarify whether they are proposing a new model or suggesting some changes to the prosomeric model, but also other conceptual and interpretive points (see below).\nThe main novelty of this work is the improvement of fate map data at stages that have not been sufficiently explored or resolved in the chicken. Fate map information is an essential tool to resolve questions about the specific origin of cell populations and addresses questions such as those raised here. These fate map data compared with molecular data provide the authors with the possibility of reaching some conclusions that are interesting, but not definitive and that in some points require a deep understanding and exploration due to the complexity of the adult brain that seems not to be sufficient in the proposed analysis at these early stages (see below for specific details). On of the controversial aspect supported by the authors is their claimed ZLI distortion. Although the work does not definitively demonstrate its proposal, it is an interesting alternative that should be taken into account to definitively resolve this problem in the future.\nGeneral comments:\nI need to highlight three main aspects that the authors need to improve in the manuscript, one related to the model, another related to the conceptual assumption of the hypothalamus and third one the validity of the selected strategy to resolve the fate map and combining it with the molecular map.\nThe first point is related to the model. There are two main and somewhat antagonistic models to understand how, starting from the stages of the neural plate and going through the different stages of the neural tube, the brain is parceled out and the main derivatives of each region have emerged. These models are the columnar model and the prosomeric model. Both models are different because, for example, they assume different axial dimensions that determine different dorsoventral relationships along the anteroposterior axis, but in addition, the prosomeric model establishes an anteroposterior segmentation in terms of neuromeres. These differences concern, for example, the relationship of the roof and floor plates to the components of the neural tube (for details of both models, see, for example, in Puelles 1995; A segmental morphological paradigm for understanding vertebrate forebrains). If the authors propose a new model that is different from the columnar or segmental prosomeric model, they should indicate this in the text but also explain in detail all the features of their new model in relation to why it is not a variation of either the prosomeric or the columnar model. In my opinion, considering how it is presented in the text and the details added so far, the authors propose some changes at specific points in the prosomeric model, but not a new model. In summary, in my opinion, the paper sounds like a claim for a new model, but they provide some sort of variations for the basal rostral and floor plate components of the prosomeric model. The authors should either describe in depth the fundamental basis of the model they are proposing or assume that they are proposing changes to that model. However, they need to include details on why some works showed a complex partition with two rostral neuromeres related to the hypothalamus and one to the prethalamus. How will the authors address this complexity in the adult with this new simplified version that mixes prethalamic and hypothalamic territories? (see below for more details and take into account these articles to think if this simplified version is consistent with the complexity of the adult as can be seen as an example in Ferran et al., 2015-Frontiers for hypothalamus-, Bilbao et al.2022 for the hypothalamus, and Puelles et al., 2021-Jcomp neurology for the prethalamus).\nRegarding the concept of hypothalamus, the authors do not provide a definition of what the hypothalamus is and why it is. If they have a model, they need to argue conceptually based on which characteristics the derivatives will be hypothalamic. In addition, there is a problem because the authors mix the prethalamic territory with the telencephalon and the hypothalamus in one unit (according to their explanation in the text). They do not explain if it is an entity like a neuromere and in that case they do not explain how this large segment will be divided in future stages. Furthermore, the authors do not reconcile their proposal with the abundant literature that follows the prosomeric description that provides a complex framework of the simplest ones proposed by the authors. This point needs to be discussed and explained carefully throughout the text (see the articles on hypothalamus related to the prosomeric model included in their bibliography -refs 4 and 6- and the previous one I mentioned).\nRegarding the third point, classical fate maps in chickens used quail-chicken grafts (Cobos et al., 2001; Sanchez-Arrones et al., 2009; García-López et al., 2004; Pombero et al, 2009; Alonso et al., 2021) using a precise frame to determine the graft location on the neural plate or neural tube to more accurately correlate territory with fate at later stages. However, the authors used as fate map strategy Dil/DiO injections to determine the way as the forebrain is growing. Usually, this kind of early fate map requires the use of a grid to measure the distance that gives more specificity to the graft in relation to the proposed position of the graft (in that case the DiI/DiO injections) (See Fig. 1 in Pombero and Martinez to know better my question and Fig. 1 in García-López et al., 2004). I couldn’t find any information about the use of a coordinate system that warranties the position of the injection that you are assuming in your mapping. Another point is related to assumptions about growth patterns when using a technique that includes relatively large areas of injections (see line 76 in their work). In that case, my impression is that there is no reference to ensure that the injections were carried out in the claimed regions or if the authors interpreted the injection point considering where they observed the mark in later states (Fig S2 is useful to know in same way the position but not with enough detail that will give grid coupled to this figures). These experiments need a grid at the beginning that includes images of the initial injection (at least for validation) so that they can be compared in later stages (see previous comments and papers cited). This precise information is essential to consider the specificity of the proposed fate map. In short, the authors must find a way to demonstrate that they are placing the injection in the location they claim.\nSpecific comments:\nTitle: This title does not exactly reflect the results obtained. The study focuses solely on the organization of the rostral forebrain and, above all, on the arrangement of the components of the basal plate and the floor of the hypothalamic and prethalamic regions. If the authors are “solving” anything, it is not about the organization of the forebrain, but rather they are suggesting an alternative option for these parts of the forebrain at some specific stages of development. Furthermore, it gives the impression that the study is a suggestion that will modify some aspects of the prosomeric model, in that sense this work would be improving the prosomeric model or proposing a variant of it. The authors propose an alternative view for the arrangement of mainly rostral neuromeres of the basal plate, as they do not explain in detail whether this is a new model, suggesting that they are proposing changes to the prosomeric model.\nAbstract:\nLine 26. Authors are following the definition of forebrain including diencephalon proper (p1p3 prosomeres) and secondary prosencephalon (peduncular and terminal neuromeres). However, the fate map is mainly devoted to the secondary prosencephalon, p3, and alar plate of p2, not covering the prosomere 1. Authors fate mapped large region of different forebrain domains\nLine 36. According to the data provided by authors they are proposing changes in the prosomeric model of the rostral forebrain mainly in the basal and floor plates. The proposed tripartite model if is matched with the prosomeric ideas that need to be better explained and developed. Some concerns are that these basal plate subdivisions are not continuous dorsally and all of them necessarily need to be connected with floor plate.\nMain text:\n-Line 50. These sentences need to be revised because they do not exactly apply to the ideas defined by the prosomeric model (check the literature cited in your bibliography and the previous one I mentioned here that deals with the general principles of the prosomeric model - references 4 and 6, but also Puelles 1995 and other articles mentioned above -). Some confusing ideas may have developed in these sentences, probably due to their excessive simplification (check and rephrase the ideas). The prosomeric model suggests that there are two segments (each segment is defined as a transverse unit with roof, wing, basal, and floor plates) related to the origin of the telencephalon and hypothalamus. Furthermore, the model proposes that the evaginated telencephalon derives from the most dorsal alar plate near the roof plate of the peduncular neuromere (the neuromere in front of p3). This peduncular neuromere according to the model includes an alar portion that is close to the alar-basal boundary (nk2.2 positive) and is related to the origin of hypothalamic derivatives such as the paraventricular nucleus. The basal plate of the peduncular neuromere is related to the origin of hypothalamic derivatives such as the dorsomedian or subthalamic nucleus among others. From the dorsal to the ventral position there are differences between the territories that are related to the distance at which they are located, for example, from the organizers of the roof plate or the floor plate. The most rostral neuromere called the terminal neuromere, includes a portion of the dorsal alar plate that will be the preoptic area (close to the roof plate) and a portion of the alar plate close to the alar-basal limit that will be the origin of the retinal territory (not the eye that involves ectodermal and mesodermal components), supraoptic and suprachiasmatic nucleus among others. In addition, the basal plate of the terminal neuromere will be the origin of the ventromedian, arcuate and mammillary nuclei among others. In summary, the prosomeric model proposes that neuromeres have a clear transverse boundary that should be molecularly identified at some point in early development. Furthermore, the prosomeric model proposes that components, for example, such as the preoptic area (unpaired telencephalon) and the ocular territory, but also the anterior paraventricular area, share the alar plate of the same neuromere, but that does not imply molecular similarity with the components of the basal plate. The great complexity of the hypothalamic region added to the complexity of prosomere 3 must be considered from early stages and a simplified version may give some confusing ideas. Components of the basal plate or alar plate between adjacent neuromeres are expected to share some molecular features because they share ventral and dorsal inductions from the floor or roof plates, respectively. Also, the last sentence of this paragraph is a bit odd because the telencephalon is the most dorsal wing plate near the roof plate and does not necessarily have to be the result of a coordinated pattern with the alar or basal plate of the hypothalamus to be included in the same prosomere. The authors' interesting observation in this paper is related to the behavior of different neuronal clones during neural tube growth. However, the authors do not explain in the text why clonal coordination is required to define neuromeres. Also, the clonal relation doesn’t mean that they finally will derive in the same components.\n-Line 67. A new model of forebrain development cannot be defined in terms of a tripartite hypothalamus. It does not sound correct that the model of forebrain development is a tripartite hypothalamus. The authors need to review this proposition better. The authors propose a new model (if it really is a new model or a modified version of the prosomeric model) of hypothalamic development or hypothalamic-prethalamic development, at least taking into account the explanations given in the text.\nResults:\n-Line 78. The authors: “For descriptive purposes, we define the A-P axis as running parallel to the hypothalamic NKX2-2 expression domain and the D-V axis as orthogonal to it (Fig. 1F)”. The authors propose a confusing model that has some unresolved problems as a model. For example, they considered a dorsoventral position in scheme 1 F that extends to the rostral tuberal region. The blue line going to the tuberal region runs from dorsal to anterior and not ventrally (unless the authors are considering that there is a rostral floor plate at that point that was not postulated in the text). Furthermore, following this assumption implies that this part of the brain that will be influenced by the prechordal plate will give rise to the floor plate. Does that mean that the alar-basal boundary is in contact with the floor plate? For example, to improve the ideas of this part of the scheme (even if they want to propose a new model), see Ferran et al 2021 (Is there a prechordal region…) for the importance of the prechordal plate and the notochord in establishing the main partitions of the brain bauplan. If the authors want to propose another option, they must consider this conceptual framework which must also be resolved in their model.\n-Line 98. The growth lines should be interpreted as representing the patterns of the neurepithelial clones growing throughout longitudinal domains involving several neuromeres (cases 4-5 for example) and inner portions of the neuromeres as the case of 8-9 (inner pallial and subpallial subdivision that can be visible at clonal levels). In summary, the same information can be used to reinforce prosomeric interpretations. I don’t understand or is not conceptually clear defined why the authors consider that the way of patterning growing will be used to redefine the relationship between the territories. The authors need to provide the mechanistic support or evidence to validate this assumption.\n-Line182. This interpretation is not clear. What does it mean bulk of hypothalamus? According to your interpretation some basal plate components but not from the prosomeric model are in a caudal neuromere. Some portions of the tuberal region are ventrally positioned to the telencephalon according to your schema.\n-Line 202. Authors “The claimed segmental identity between the telencephalon and hypothalamus implies that fate conversion should be possible only between these identities.” According to the prosomeric model, FoxG1 is expressed in the unpaired and evaginated telencephalon (terminal and peduncular neuromeres, respectively). However, the alar hypothalamus (absence of Shh expression) is found in most of the paraventricular territory and subparaventricular territory characterized by the absence of FoxG1. In the animals of this study treated with Cyclopamine it seems as if both FoxG1 and PAX6 were ventralized (See in Ferran et al., 2007 Fig. 1 and 5E, PAX6 expression is also observed at stage HH24 in the paraventricular territory. At early stages an increase in signal can be seen in this territory). In my opinion, these results can be perfectly interpreted considering the detailed description of the forebrain regionalization supported by the prosomeric model.\n-Line 220. This paragraph shows an interesting observation that the authors use to propose a different boundary compared to the prosomeric boundaries. This is an interesting observation but not a definitive demonstration that this distortion represents the actual interprosomeric boundary. The peculiar clonal expansion of the basal plate below the ZLI (assuming that the ZLI is only on the wing plate or if we extend it to the basal plate, it does not necessarily have to follow these lines) needs to be cautiously interpreted and defined precisely if the identity is changes or not on those stages and with the impact in adult brain. There are several patterns like Lim1 (see Ferran et al., 2007 Fig. 6 A that could be interpreted as the interprosomeric boundary between p2/p3). The authors make an interesting observation but do not imply that it represents the boundary between territories. The fate map through later stages with definitive derivatives will provide better support for understanding common subsequent identities to recognize possible derivatives of each neuromere. However, if the authors wish to propose such a boundary, they need to develop a model that indicates the basal plate position for the redefined regions. In addition, more details need to be provided on the components derived of each territory.\n-Line 276: The authors propose a change in the interpretation of the pattern observed in HH20 that will help them reinterpret the interprosomeric boundaries. This is an interesting point of view, but not a definitive demonstration of the actual prosomeric interpretations. Of course, it remains an interesting possibility to be considered but requires more detail from the authors on what the model they propose looks like. The authors assume that all bands are specifically related to the proposed derivatives. Molecular maps at this early stage do not necessarily reflect the complete or definitive fate of these territories. Further work examining detailed fate maps will be necessary to demonstrate the true value of early mapping with accurate comparisons of fate maps reaching late stages when definitive derivatives are already found. As mentioned above, some patterns suggest a p2/p3 interprosomeric boundary below ZLI (Lim1), which adds confusion to the resolution of this issue. This distortion observed by the authors could be a behavior of the basal plate near the midline that is then definitively specified and be a longitudinal band.\nFigure 1:\nFor example, Fig. 1 L indicates the injection supposedly in area 11, marking the prethalamus. However, prethalamic region touch the roof plate, and it is worth asking whether the extension to the roof is not representing an expected identification of the fate map reaching the dorsal midline that was not considered in your schemas.\nFig. S3 F. Is green Shh expression?\nDiscussion:\nThe discussion should be enriched by aspects such as what are the differences between the proposed model and the prosomeric one (or is it a variation of the prosomeric model?). In addition, a discussion that considers the complexity of the adult brain is necessary because the author proposes a simple model that does not consider all the derivatives proposed in the prosomeric model. Some discussion will also be necessary, for example, on the dorsoventral position of the peduncular fibers in the peduncular prosomere, because with this model some things have become difficult to explain.\nSome general considerations need to be made in this regard. It is true that clonal growing is the key to define the boundaries? To what extent do they help or not?\nFinal comment to consider in your discussion:\nDo related clones reflect the identity of the territory at each stage? We see the behavior of related clones, but that does not necessarily mean that they belong to the same neuromere at all stages. If the territory is expanded and exposed to different organizers, it may be differentially affected in different dorsoventral domains. In summary, clonal relatedness suggests possible identities conserved through time, but may not necessarily be the key to defining anteroposterior neuromeric boundaries. In my opinion, future fate maps should be performed on individual cellular components to better understand clonal relationships. Then, at different stages, those patterns should be compared with molecular maps, but later stages with defined derivatives are essential because at early stages molecular maps do not always represent precise boundaries (see figure 14 in Sánchez Arrones et al 2009, for a better idea about the problem).", "reviewer_1_attached": "Thank you for the opportunity to respond to the reviewers’ concerns. Our detailed responses are below:\nREVIEWER COMMENTS\nReviewer #1\nThis is a very useful resource for the developmental neuroscience research community. The authors\npresent a set of nice lineage tracing experiments in the chick forebrain. Based on those, 3D\ncomputational models (fantastic movies describing likely 3D temporal progression of domains) and\nrestricted set of HCR mRNA detection, the authors refute the current prosomeric model of forebrain\norganisation, proposing a modified model. Although the work is very carefully done and of general\ninterest for the field, we have difficulties with the lack of focus of the narrative and some lack of evidence\nfor the model proposed (while agreeing on finding the prosomeric model problematic for the anterior\nand ventral forebrain). Overall, the work as it is presented currently does not support strongly enough\nthe conclusions made by the authors.\nReviewer 1 accepts that the prosomere model is problematic, but requests additional evidence to support our\nclaims for a new model. We thank the reviewer for their very constructive and helpful feedback, which has led\nus to perform a number of additional experiments and analyses to strengthen our conclusions"}, "author_response": "Thank you for the opportunity to respond to the reviewers’ concerns. Our detailed responses are below:\nREVIEWER COMMENTS\nReviewer #1\nThis is a very useful resource for the developmental neuroscience research community. The authors\npresent a set of nice lineage tracing experiments in the chick forebrain. Based on those, 3D\ncomputational models (fantastic movies describing likely 3D temporal progression of domains) and\nrestricted set of HCR mRNA detection, the authors refute the current prosomeric model of forebrain\norganisation, proposing a modified model. Although the work is very carefully done and of general\ninterest for the field, we have difficulties with the lack of focus of the narrative and some lack of evidence\nfor the model proposed (while agreeing on finding the prosomeric model problematic for the anterior\nand ventral forebrain). Overall, the work as it is presented currently does not support strongly enough\nthe conclusions made by the authors.\nReviewer 1 accepts that the prosomere model is problematic, but requests additional evidence to support our\nclaims for a new model. We thank the reviewer for their very constructive and helpful feedback, which has led\nus to perform a number of additional experiments and analyses to strengthen our conclusions, including:\n● Quantitative analysis of DiI/DiO lineage tracing data. The new analyses fully support our original\nconclusions, which were based on qualitative analysis of the same data.\n● New mouse HCR data, essential for translating our detailed findings from chicken, revealing close\nsimilarities between the two species.\n● Additional chick HCR analysis of prosomere model boundaries, and brand new scRNA-Seq analysis,\nhighlighting the lack of evidence for a segmental boundary within the hypothalamus.\n● New evidence in support of our model, from additional fate conversion experiments and increased tests\nin cell mixing experiments.\n● Limited re-ordering of results, and extensive text changes, for a simpler narrative. Tighter focus on our\nargument concerning the position and distortion of the forebrain dorsoventral axis.\nSpecific points to be addressed:\nR1 Q1 The most precious part of the data is the collection of lineage tracing results. Although the stated n for\neach of the injected forebrain areas described on Figure 1 is high, there is no quantification of the clone size,\nshape and position at HH20 across embryos injected for each area at HH10. This quantification is essential to\nassess variability across injections. Figure S2 suggests a fair amount of variability and same embryos are used\nfor two distinct data points, using different left and right positions of injection (eg. Fig. S2 E & F, H & I, J &\nK).\nWe now provide a detailed quantitative analysis of our fate mapping results, describing outcomes in terms of\nposition, shape and relative size. The new data is shown in Fig 1G-H, AA-AB and Supplementary fig. S5.\nIn addition to providing new quantification from the twelve HH10 injection zones, we have included data from\ninjections placed at the intersections of these zones, providing higher spatial resolution, totalling 290 examples.\n(note that as a consequence of showing more data, original Supplementary fig. S2 is now expanded into new\nSupplementary figs S2-S4). These numbers exclude results from injections covering more than two injection\nzones, and a further approx. 80 patterns resulting from earlier (HH7-9) injections, which presented very similar\npatterns (some HH9 examples are newly shown in Supplementary fig. S6). Critically, and as shown in new\nSupplementary fig. S5, variability was minimal, and the results confirm our previous conclusions. As pointed\nout, in some instances, we used the same embryo for multiple injection sites: this provided internal consistency,\nfurther supporting the robustness of our interpretation.\nR1 Q2 The last frame of Movie S2 should be used in an improved Figure 2 to visually match the findings with\nthe model of clonal shapes (if the above quantification of clone size, shape and final position confirms the\ncurrent conclusion per position on the HH10 map).\nThank you for this suggestion. We have improved Figure 2 accordingly.\nR1 Q3 The dynamic model for the yellow area in Movie S4 … is key to the author’s model but does not have\nenough lineage tracing evidence for its proposed origin. Specific lineage tracing of this area (with enough n) is\nneeded.\nWe generated a lot of data for this region, as we focussed our fate mapping efforts on the barely explored ventral\nforebrain, and apologise that this was not obvious in the previous version. The yellow area (the SHH-negative\ntuberal hypothalamus) closely corresponds to our outcome area H5. As shown in new Fig 1AA, the majority of\nthis zone arises from anterior zone 2 (level with zone 5; please refer to zone 2, 1-2 and 2-5 thumbnails in new\nSupplementary fig. S5). This is accurately represented in our fate map (Fig. 2A).\nR1 Q4 The attempt to refute the prosomeric model is not well articulated, offering partial evidence for the\nproposed alternative. The most important data would be extensive tracing in area 1, 2, 5 and 7. Fig S4D is key\nand yet is not showing the position of the injected cells at HH10.\nThank you for encouraging us to provide additional evidence to better refute the prosomere model and build the\ncase for our alternative model. We have performed a number of additional experiments (see detailed responses\nto questions below), and have substantially re-ordered the manuscript in order to better articulate our findings.\nIn terms of the lineage tracing queried here, we considered 15 injections in area 1, 29 in area 2, 17 in area 5 and\n20 in area 7, plus 21 further injections at the intersections of these zones. Lineage tracing of these regions is\nshown in Fig. 1I,R-T, Supplementary figures S2-S3, and summarised in new Figure 1AA-AB and new\nSupplementary fig. S5. We have updated Supplementary figure S4D (now S6D) to include the injection area,\nhowever this specimen was injected at HH8. Equivalent outcomes arise from injections in HH10 zone 7-8 (DiO\nspot, result is similar to meeting point of T1 and T2 outcome areas, Fig. 1H) and zone 4-5 (DiI spot, result is\nsimilar to H4 outcome area, new Supplementary fig. S5).\nR1 Q5 A substantial portion of the argument for the proposed ‘compartmental’ origins of the hypothalamic\nareas is based on HCR analyses. However, dynamics in gene expression can’t be used as rigorous support for\nclonal dynamics. This is a big limitation of the manuscript. Higher resolution lineage tracing is needed to back\nthe HCR conclusion up.\nWe agree that HCRs do not provide clonal information and wish to clarify what we are suggesting. Primarily,\nour model revises the forebrain A-P and D-V axis positions. We propose a new boundary region that extends\nventral to the ZLI, and propose that the D-V axis in this area then becomes distorted. Importantly, we provide\nnew evidence (new Fig. 4A-C,E-G) that indicates that a similar D-V axis distortion at the base of the ZLI\noccurs in mouse. But we have never suggested a compartment boundary within the tuberal hypothalamus. The\ndefining feature of our model is that it places the anteriormost portion of the hypothalamus ventral to the\ntelencephalon and the remainder of the hypothalamus ventral to two different diencephalic compartments\n(prethalamus and ZLI), hence ‘tripartite’.\nWe have been unable to perform high resolution lineage tracing at the boundary ventral to the ZLI, for\nbiological and technical reasons. Lineage restriction is not established in our boundary region at HH10 (growth\nlines cross the supramammillary hypothalamus), requiring that injections are performed at later stages. This\nproved impossible: the third ventricle narrows to a cleft where left and right sides are closely apposed, so\nmanoeuvring within this space spreads dye around and causes the needle to snap. Instead, and as detailed in the\nresponse to Q6, we have performed a series of novel mixing experiments.\nR1 Q6 The cell mixing experiment (Fig 4J), where cells separate based on their positional identity (anterior\nhypothalamic vs posterior thalamic), is key to the proposed model and is missing key controls and ambition. It\nwould need i) mixing the posterior hypothalamic cells with their own (A with A in 4J, matching the P with P)\nand with cells from the proposed anterior hypothalamic compartment, to see if mixing results match their\nproposed model.\nThank you for this suggestion; we apologise for having omitted the controls from the first version of the\nmanuscript (despite having done them). As well as including these, we have now performed a much more\nextensive analysis, testing the ability of cells from seven different forebrain regions to intermingle or separate.\nThe mixing results support our key claim of a ventral boundary region contiguous with the ZLI. Second, the\nresults reveal that PV hypothalamic cells mix with prethalamic, but not telencephalic cells (data shown in new\nFig. 6D-E and new Supplementary figs. S18-S19).\nTogether, the new data that we have added in response to Qs1-6 provides substantial new evidence that refutes\nthe prosomere model. As additional evidence, we provide brand new analyses, including new HCRs and\nscRNA-Seq analysis, that again highlights the lack of evidence for a segmental boundary within the\nhypothalamus (new Fig. 5P-Q, new Supplementary figs. S14-15).\nR1 Q7 The quality of the DiI/DiO images is very low. And the Cre/tdTomato images are also very hard to\ninterpret because of overabundance of positive cells. A better approach could be to inject fluorescently labelled\nviruses in regions of interest and see lines of cells that way.\nWe have improved the quality of the DiI/DiO images. We discuss the Cre/tdTomato images in the response to\nQ10.\nR1 Q8 The point about dorsalization of ventral diencephalon region with cyclopamine is weak, as the markers\nthey use to indicate telencephalon (Foxg1) and prethalamic/PV hypothalamic (Pax6) are also expressed in the\neye. Given that this treatment is known to lead to cyclopic phenotypes, the markers may be present in ‘trapped’\nretinal cells, instead of proving a fate conversion to TC and pre-thalamus. Much more needs to be done to\nconclude anything from this experiment.\nThank you for this insightful comment: we agree that we need to exclude the possibility that these are trapped\nretinal cells. Since our embryos were analysed at too early a stage to detect mature retinal markers (VSX2 was\nattempted), we analysed PAX2, finding no evidence for expansion (new Supplementary fig.16B). In addition\nwe performed three further experiments. First, we analysed lineage at the same time as inhibiting Shh signaling.\nThese studies (new Fig. 6C) showed that growth patterns are not altered. Second, we performed additional\ndorsalisation experiments, analysed with a broader set of markers. These reveal an upregulation of the ARX in\nthe basal hypothalamus of dorsalised embryos. Over the time period of analysis ARX is largely confined to the\nprethalamus (just overlapping into the basal hypothalamus) - and is not detected in either eye or telencephalon.\nIts upregulation after dorsalisation fits with previous studies linking hypothalamic to prethalamic development\n(new Fig. 6A-B, new Supplementary fig. S16B). Third, we have performed in vitro experiments that likewise\nreveal an upregulation of ARX in explants of prospective prethalamus/hypothalamus (new Supplementary fig.\nS17). Together these experiments support a fate conversion rather than a cell trapping.\nR1 Q9 The comparison between chick and mouse is potentially powerful but only basing it on HCR of a few\nmarkers introduces too many possible caveats and shouldn’t be considered here unless adding clonal evidence.\nWe agree that clonal lineage tracing would provide the strongest evidence, but this is beyond the scope of the\ncurrent study - although we now note that DiI patterns in mouse, similar to some of those we describe in chick,\nhave been published (https://doi.org/10.1006/dbio.2000.9616). However, we were extremely keen to better\nunderstand the extent to which our studies may translate to the mouse, and so have instead included additional\nHCR evidence to support our point about possible evolutionary conservation, focussing our narrative\nspecifically on the position and distortion of the dorsoventral axis. New Figure 4A-C,E-G strongly supports the\ncontention that growth of the thalamus distorts the ZLI. The ideas raised in this section are important, because\nthey help to explain the arrangement of progenitor regions in mice, and suggest how they may have been\nmisinterpreted in the prosomere model.\nSmaller issues:\nR1 Q10 They refer to figure 5K in the discussion. That panel doesn’t exist in figure 5.\nThank you for spotting this; all figure references in the new version of the manuscript have been carefully\nchecked.\nR1 Q11 Figure S5 is not useful as the Cre clones can’t really be informative if not quantified carefully (size and\nshape) across many embryos.\nWe spent months attempting to perform such studies, prior to our submitting the original manuscript.\nUnfortunately, despite putting in extensive time and effort, it was not possible to provide a detailed quantitative\nanalysis of these samples. This was due to embryo quality (many non-viable), only 50% being positive for the\ntransgene, and because many of the recombined embryos had either too few or too many clones to be useful,\neven after optimisation of the TAT-Cre recombinase dose and delivery. We performed, but decided to omit, an\nanalysis of clone size 24 hours after treatment, because there were not enough well-separated clones to provide\ninsights beyond the observation that dorsal clones tended to be larger than ventral clones. We felt nonetheless\nthat Figure S5 was worth including as we could readily discern shapes in the 48 hour samples that were\nconsistent with the DiI/DiO patterns.\nR1 Q12 Some of the HCRs are hard to see especially when they’ve used grey colour on top of the faint blue\nbackground.\nThank you for this feedback. We have improved the quality of the HCR images as requested.\nR1 Q13 The most important focus of the manuscript is the organisation of the hypothalamus. Yet, the results are\nwritten in a very complicated fashion, distracting the reader from the most important conclusions. We would\nadvise to reshape the text to facilitate understanding and clarity of thoughts.\nThank you for this important prompt. We have made a number of changes and rearrangements to the manuscript\nto improve our narrative. These include:\n● Providing extra context in the introduction\n● Consolidating all of our work relating to the chicken D-V axis position and distortion within figure 3\n● Using figure 4 to explore the same (D-V axis position/distortion) in mice. Our key novel findings are\nthereby all delivered early on.\n● Figure 5 now exclusively deals with our rebuttal of the prosomere model, expanded in scope for\nrobustness\n● Relocating the cyclopamine (/FST/sonidigib) and cell mixing experiments to Figure 6, which is now\ndedicated to developing our new model\n● Expanding our discussion, including clarifying our claims about our new model, as per points made\nabove\nWe very much hope that these changes have improved the narrative, and help the reader to follow our key\nconclusions. Again, we are grateful for your careful reading of the original manuscript, and very helpful\ncomments.\nReviewer #2\nThis manuscript describes a comprehensive and detailed fate-mapping of the chicken forebrain at HH10 using\nfluorescent dye labeling along with spatial expression pattern analysis of multiple marker genes.\nThe following new knowledge has been generated from this study:\n1) Generation of detailed fate map of the chick forebrain at HH10.\n2) Generation of a 4D model of forebrain growth.\n4) Establishing that eye morphogenesis plays a key role in separation of the telencephalon from the\nprethalamus and the hypothalamus.\n3) Proposal of a \"tripartite\" model for the hypothalamus.\n4) Identification of a major forebrain boundary that cuts through the MM hypothalamus and the ZLI.\nThis study does not contain any functional characterization that may shed light on the genes and\nmechanisms involved in the morphogenesis of the forebrain, particularly the hypothalamus. However, it\nhas brought significant clarity to this phenomenon with respect to the growth movements and the\nchanges in the orientation and positioning of the AP and DV axes. In fact this study has challenged some\naspects of the existing prosomere model and proposed a new \"tripartite\" model for the formation of the\nhypothalamus. Thus significant new knowledge has been add to the field.\nThe experiments that have been carried out largely support the conclusions made and the data has been\nanalyzed in depth and interpreted correctly.\nThe methodology used meets the expected standards in the field and sufficient details have been provided\nin the supplementary materials for reproduction.\nHence this manuscript is suitable for publication without the requirement of any major changes.\nThis reviewer had no suggestions, and we thank them for their positive remarks.\nReviewer #3\nThe authors of “Resolving forebrain development organization by analysis of differential growth patterns”\nproposed a common anterior compartment of the forebrain with telencephalon, hypothalamus and prethalamus.\nIn addition, the authors claim a new model of forebrain development with a tripartite hypothalamus. This is a\ndetailed study that provides extensive data that aims to support their hypothesis. However, after a detailed\nanalysis of the manuscript, some key points need to be resolved by the authors, including the need to better\nclarify whether they are proposing a new model or suggesting some changes to the prosomeric model, but also\nother conceptual and interpretive points (see below).\nThe main novelty of this work is the improvement of fate map data at stages that have not been\nsufficiently explored or resolved in the chicken. Fate map information is an essential tool to resolve\nquestions about the specific origin of cell populations and addresses questions such as those raised here.\nThese fate map data compared with molecular data provide the authors with the possibility of reaching\nsome conclusions that are interesting, but not definitive and that in some points require a deep\nunderstanding and exploration due to the complexity of the adult brain that seems not to be sufficient in\nthe proposed analysis at these early stages (see below for specific details). On of the controversial\naspect supported by the authors is their claimed ZLI distortion. Although the work does not definitively\ndemonstrate its proposal, it is an interesting alternative that should be taken into account to definitively\nresolve this problem in the future.\nWe thank the reviewer for taking the time to read our manuscript so thoughtfully, and provide positive\ncomments. We are pleased to provide a detailed point-by-point response to the issues raised.\nGeneral comments:\nI need to highlight three main aspects that the authors need to improve in the manuscript, one related to the\nmodel, another related to the conceptual assumption of the hypothalamus and third one the validity of the\nselected strategy to resolve the fate map and combining it with the molecular map.\nThe Reviewer raises a number of points under his/her ‘General comments’, which we have broken down into\nsections Q1-Q8, responding to each in turn.\nR3 Q1 The first point is related to the model. There are two main and somewhat antagonistic models to\nunderstand how, starting from the stages of the neural plate and going through the different stages of the neural\ntube, the brain is parceled out and the main derivatives of each region have emerged. These models are the\ncolumnar model and the prosomeric model. Both models are different because, for example, they assume\ndifferent axial dimensions that determine different dorsoventral relationships along the anteroposterior axis, but\nin addition, the prosomeric model establishes an anteroposterior segmentation in terms of neuromeres. These\ndifferences concern, for example, the relationship of the roof and floor plates to the components of the neural\ntube (for details of both models, see, for example, in Puelles 1995; A segmental morphological paradigm for\nunderstanding vertebrate forebrains). If the authors propose a new model that is different from the columnar or\nsegmental prosomeric model, they should indicate this in the text but also explain in detail all the features of\ntheir new model in relation to why it is not a variation of either the prosomeric or the columnar model. In my\nopinion, considering how it is presented in the text and the details added so far, the authors propose some\nchanges at specific points in the prosomeric model, but not a new model.\nThank you for this important suggestion. We have made several text additions to better explain our model and\nhow it differs from the prosomere model. In brief, our model differs from the prosomere model in the following\nmajor respects:\n1. Our proposed topological boundary within the supramammillary hypothalamus, ventral to the ZLI,\nexcludes floor plate from the anterior forebrain. The anterior forebrain therefore does not meet the\nprosomere definition of a segmented tissue. Further, while our model describes two forebrain\ntransverse subdivisions, we find no evidence that hp1, hp2 or p3 are segments, in the commonly\nunderstood sense in developmental biology, such as lineage restriction or differential expression of\nsignalling pathways associated with cell segregation.\n2. As well as disagreeing over the topological relations of basal plate structures, our model emphasises\nthe closer relationship of the paraventricular hypothalamus to the prethalamus, as opposed to the\ntelencephalon, placing this region too in the diencephalon. We also relate the temporal side of the\nretina to these diencephalic structures, whereas the prosomere model considers the eye an acroterminal\n(anteriormost) specialisation.\n3. The repositioning of most of the hypothalamus within the diencephalon fundamentally undermines the\nmajor concept of the secondary prosencephalon.\nPlease refer to the following added text from the discussion, which follows our conclusions in the results\nsection.\n● “We do not propose a ‘hard’ (lineage restricted) boundary between anterior tuberal/telencephalon and\nmore posterior regions, and view these relationships as topological rather than segmental.”\n● “Fundamentally, however, we do not consider the forebrain to be segmented as suggested by the\nprosomere model. And whereas the prosomere model claims a segment boundary between the\nprethalamus and PV hypothalamus, we group these regions, reflecting the intimate connection of their\nprogenitors and relative separation from telencephalon by the eye field. Indeed, we further speculate\nthat the eye itself has a 'split' identity, the anterior (nasal) side relating to the telencephalon, and the\nposterior (temporal) side relating to the prethalamus and hypothalamus - consistent with the expansion\nof both telencephalon and prethalamus into the eye field of eyeless Rax mutants.”\n● “In conclusion, we forward a ‘tripartite hypothalamus’ model in which the hypothalamus sits ventral to\nthree regions – the telencephalon, prethalamus/PV hypothalamus, and the ZLI (Fig. 6F). The LFNG-ve\nZLI and posterior hypothalamus form a topological boundary between the anterior SIX/FEZ+ve and\nposterior IRX+ve forebrain, with the SM hypothalamus part of a ventral boundary region associated with\nlineage restriction.”\nR3 Q2 In summary, in my opinion, the paper sounds like a claim for a new model, but they provide some sort of\nvariations for the basal rostral and floor plate components of the prosomeric model. The authors should either\ndescribe in depth the fundamental basis of the model they are proposing or assume that they are proposing\nchanges to that model. However, they need to include details on why some works showed a complex partition\nwith two rostral neuromeres related to the hypothalamus and one to the prethalamus. How will the authors\naddress this complexity in the adult with this new simplified version that mixes prethalamic and hypothalamic\nterritories? (see below for more details and take into account these articles to think if this simplified version is\nconsistent with the complexity of the adult as can be seen as an example in Ferran et al., 2015-Frontiers for\nhypothalamus-, Bilbao et al.2022 for the hypothalamus, and Puelles et al., 2021-Jcomp neurology for the\nprethalamus).\nThank you for your comment. As we have now clarified (above), ours is fundamentally a new model, and we\nelaborate on this in further comments below. Although it will be important to publish a wide-ranging discussion\nand critique comparing our model with the prosomere and columnar models, we believe a greater level of detail\nis better suited to a review. We have attempted to balance the competing needs of detail and accessibility, and\nhave taken into account feedback from colleagues, who have advised us that to include extra detail would make\nunderstanding difficult for non-expert readers.\nHowever, to respond to your comment, we do not agree that these papers - although valuable -\ndemonstrated the existence of two rostral neuromeres. We have conducted our own analysis of a selection of the\ngenes reported in Ferran et al. 2017 to distinguish hp1 from hp2, failing to discern evidence for the existence of\nthese two compartments. New analysis of large-scale scRNA-Seq from the developing mouse hypothalamus\nlikewise does not support the segregation of terminal and peduncular paraventricular markers, and instead\nsuggests that some reported regional differences in gene expression patterns may be simply attributed to\ndifferences in cell maturity (new Fig. 5P-Q, new Supplementary figures S14-15). Although purported\nprosomere boundaries have twice been redrawn (2003, 2012), no definitive evidence for their existence has been\nprovided. Furthermore, in the absence of features generally associated with boundary functions, such as lineage\nrestriction or differential expression of signalling pathways associated with cell segregation, it is debatable\nwhether the language of segments should be employed, rather than that of regional subdivisions (which we\nfavour).\nWe have added the following text to clarify our views:\n● Introduction: “Although influential, the prosomere model is controversial, relying heavily for its\nsupport on gene expression patterns in the mid-to-late stage embryonic mouse. Classic features of\nsegment boundaries, such as lineage restriction, have not been identified at any purported prosomere\nboundaries anterior to the ZLI (the prosomere dorsal p2/p3 boundary), while the locations of prosomere\nboundaries in the early ventral forebrain are not precisely defined. And while the model incorporates\ndetailed fate-mapping data - which can contribute valuable insight into topological relationships within\nthe developing forebrain - this evidence is largely restricted to the dorsal aspect of the chicken neural\ntube, leaving ventral and basal regions under-characterized. A further challenge has arisen from recent\nstudies showing that hypothalamic patterning is closely coordinated with prethalamic, rather than\ntelencephalic, patterning (7–11).”\nR3 Q3 How will the authors address this complexity in the adult with this new simplified version that mixes\nprethalamic and hypothalamic territories? (see below for more details and take into account these articles to\nthink if this simplified version is consistent with the complexity of the adult as can be seen as an example in\nFerran et al., 2015-Frontiers for hypothalamus-, Bilbao et al.2022 for the hypothalamus, and Puelles et al.,\n2021-Jcomp neurology for the prethalamus).\nOur model is mainly concerned with the topological relations of forebrain regions at patterning stages, which is\nto say their anterior/posterior and dorsal/ventral positions in relation to each other, and this is what our\nconcluding schematic illustrates. By using a simplified diagram we do not mean to imply that the forebrain is\nnot regionalised into complex territories. The existence of neuromeres and their boundaries is not in principle\nnecessary to explain this complex regionalisation, which we suppose arises through established (plus potentially\nnovel) mechanisms such as long-, medium- and short-range signalling from the neural tube itself and the\nmultitude of surrounding tissues. Our scheme therefore raises no special difficulty in explaining later\ncomplexity.\nAlthough the study does not deal directly with patterning mechanisms, we do suggest that the\nintersection of neural and non-neural tissues in our ventral ‘boundary region’ creates an environment rich in cell\nsignalling interactions that may help to explain the complexity of this part of the forebrain. We also refer in our\ndiscussion to the chimeric brain hypothesis, which holds that the anterior neural tube (broadly, Six3/Rax/Fezf-\nexpressing areas, i.e. corresponding to our anterior forebrain) represents the fusion of two ancestral nervous\nsystems, creating new combinations of co-expressed genes (and therefore functions) and potentially contributing\nto the extraordinary variety of cell types within the hypothalamus. See also Albuixech-Crespo et al. 2017, whose\nanterior ‘HyPTh’ segment in amphioxus appears equivalent to our anterior forebrain. Our scheme therefore\naligns well with contemporary evo-devo research, whereas it is difficult to see how the prosomere model fits\nwith these recent insights. We think the anterior forebrain is unique in its complexity and its evolutionary and\ndevelopmental origins, and that if anything, the prosomere contention that the entire nervous system is\nepichordal, possessing roof and floor plate as per the rest of the nervous system, is an oversimplification.\nR3 Q4 Regarding the concept of hypothalamus, the authors do not provide a definition of what the\nhypothalamus is and why it is. If they have a model, they need to argue conceptually based on which\ncharacteristics the derivatives will be hypothalamic.\nThank you for this suggestion. We have added the following text to the results section:\n“We use a standard definition of the hypothalamus, namely the paraventricular (PV), tuberal, mammillary (MM)\nand supramammillary (SM) domains, omitting the preoptic region which is now usually considered part of the\ntelencephalic subpallium.”\nIn the discussion, we now state:\n\"We therefore accept the subthalamic nucleus as part of the hypothalamus, a view that has gained considerable\ntraction recently.\"\nR3 Q5 In addition, there is a problem because the authors mix the prethalamic territory with the telencephalon\nand the hypothalamus in one unit (according to their explanation in the text). They do not explain if it is an\nentity like a neuromere and in that case they do not explain how this large segment will be divided in future\nstages.\nOur study is not concerned with the mechanisms of forebrain patterning, which fall outside the scope of the\nwork. Please refer to our above answers on the subjects of forebrain patterning (Q3) and the clarification of our\nproposed model (Q1).\nR3 Q6 Furthermore, the authors do not reconcile their proposal with the abundant literature that follows the\nprosomeric description that provides a complex framework of the simplest ones proposed by the authors. This\npoint needs to be discussed and explained carefully throughout the text (see the articles on hypothalamus\nrelated to the prosomeric model included in their bibliography -refs 4 and 6- and the previous one I mentioned).\nThe prosomere literature references (and incorporates) many excellent and impressive studies, but we disagree\nwith some of the reasoning and conclusions presented therein. Similar to our comment above, we believe that a\nreview is required to do justice to this subject, and that to provide this level of discussion within a primary\nresearch article unfortunately would distract from the most important messages and compromise readability.\nR3 Q7 Regarding the third point, classical fate maps in chickens used quail-chicken grafts (Cobos et al., 2001;\nSanchez-Arrones et al., 2009; García-López et al., 2004; Pombero et al, 2009; Alonso et al., 2021) using a\nprecise frame to determine the graft location on the neural plate or neural tube to more accurately correlate\nterritory with fate at later stages. However, the authors used as fate map strategy Dil/DiO injections to\ndetermine the way as the forebrain is growing. Usually, this kind of early fate map requires the use of a grid to\nmeasure the distance that gives more specificity to the graft in relation to the proposed position of the graft (in\nthat case the DiI/DiO injections) (See Fig. 1 in Pombero and Martinez to know better my question and Fig. 1 in\nGarcía-López et al., 2004). I couldn’t find any information about the use of a coordinate system that warranties\nthe position of the injection that you are assuming in your mapping. Another point is related to assumptions about growth patterns when using a technique that includes relatively large areas of injections (see line 76 in their work). In that case, my impression is that there is no reference to ensure that the injections were carried out in the claimed regions or if the authors interpreted the injection point considering where they observed the mark in later states (Fig S2 is useful to know in same way the position but not with enough detail that will give grid coupled to this figures). These experiments need a grid at the beginning that includes images of the initial injection (at least for validation) so that they can be compared in later stages (see previous comments and papers cited). This precise information is essential to consider the specificity of the proposed fate map. In short, the authors must find a way to demonstrate that they are placing the injection in the location they claim.\nThank you for this comment. We greatly admire these studies, and their conclusions have accurately predicted\nour DiI fate mapping results in dorsal regions. Reflecting this, we performed far fewer injections in dorsal\nregions than were required for the ventral neural tube (please refer to new Supplementary figure S5B).\nHowever, in our own, earlier work (Fu et al., 2017) we found that using a distance-based coordinate system led\nto inconsistent results for the ventral neural tube, and we therefore developed a morphology/landmark-based\nsystem. In the present study we extended this approach, using a grid based on morphological criteria (see Figure\n1D). Regardless of the respective merits of the two different approaches, you will see from our new quantitative\nanalysis (new Supplementary figure S5C) that the strategy we employed led to consistent results - and we\nconsider this to validate our method. Please also see supplementary fig. S1C, which provides examples of how\nwe confirmed the accuracy of our targeting at HH10.\nR3 Q8 Another point is related to assumptions about growth patterns when using a technique that includes\nrelatively large areas of injections (see line 76 in their work). In that case, my impression is that there is no\nreference to ensure that the injections were carried out in the claimed regions or if the authors interpreted the\ninjection point considering where they observed the mark in later states (Fig S2 is useful to know in same way\nthe position but not with enough detail that will give grid coupled to this figures). These experiments need a grid\nat the beginning that includes images of the initial injection (at least for validation) so that they can be\ncompared in later stages (see previous comments and papers cited). This precise information is essential to\nconsider the specificity of the proposed fate map. In short, the authors must find a way to demonstrate that they\nare placing the injection in the location they claim.\nWe are happy to clarify our workflow for this process. The injection position for every sample was recorded\nindividually in a lab notebook and a photograph was also taken for every example (a number of examples are\npresented in new Supplementary figures S2-S4 - expanded from original figure S2). Both were referred to\nwhen interpreting the results later on, because the photographs carry only partial (2D) information that is\ninsufficient to distinguish. The experimenter who performed the injections designated the position with respect\nto the twelve zones in Figure 1D, and these were independently scored by two others. Concordance was\nextremely high and the few disagreements (approximately 5% of injections) were settled by discussion\nincluding reference back to lab notes.\nSpecific comments\nThe Reviewer raises a number of points under his/her ‘Specific comments’, which we have broken down into\nqueries Q9-Q25, responding to each in turn.\nR3 Q9 Title: This title does not exactly reflect the results obtained. The study focuses solely on the organization\nof the rostral forebrain and, above all, on the arrangement of the components of the basal plate and the floor of\nthe hypothalamic and prethalamic regions. If the authors are “solving” anything, it is not about the\norganization of the forebrain, but rather they are suggesting an alternative option for these parts of the\nforebrain at some specific stages of development. Furthermore, it gives the impression that the study is a\nsuggestion that will modify some aspects of the prosomeric model, in that sense this work would be improving\nthe prosomeric model or proposing a variant of it. The authors propose an alternative view for the arrangement\nof mainly rostral neuromeres of the basal plate, as they do not explain in detail whether this is a new model,\nsuggesting that they are proposing changes to the prosomeric model.\nAlthough we indeed focus most heavily on the position of the rostral forebrain, we do present DiI data as far\nback as the pretectum, and we interpret our results in a whole-forebrain context. We therefore consider it\nreasonable to use 'forebrain' in the title. As we have clarified above, our model is novel and not a variant of the\nprosomere model.\nAbstract:\nR3 Q10 Line 26. Authors are following the definition of forebrain including diencephalon proper (p1p3\nprosomeres) and secondary prosencephalon (peduncular and terminal neuromeres). However, the fate map is\nmainly devoted to the secondary prosencephalon, p3, and alar plate of p2, not covering the prosomere 1.\nAuthors fate mapped large region of different forebrain domains\nAs above, we do have some injections that labelled prospective pretectum (e.g. see zone 12 results, new\nSupplementary fig. S5C) so we think it a fair approximation to describe the work as fate mapping the\nforebrain.\nR3 Q11 Line 36. According to the data provided by authors they are proposing changes in the prosomeric\nmodel of the rostral forebrain mainly in the basal and floor plates. The proposed tripartite model if is matched\nwith the prosomeric ideas that need to be better explained and developed. Some concerns are that these basal\nplate subdivisions are not continuous dorsally and all of them necessarily need to be connected with floor plate.\nAs we have explained above, our model is not a version of the prosomere model and therefore we are not bound\nto restrictions based on the prosomere definition of segments.\nMain text:\nR3 Q12 -Line 50. These sentences need to be revised because they do not exactly apply to the ideas defined by\nthe prosomeric model (check the literature cited in your bibliography and the previous one I mentioned here\nthat deals with the general principles of the prosomeric model - references 4 and 6, but also Puelles 1995 and\nother articles mentioned above -). Some confusing ideas may have developed in these sentences, probably due to\ntheir excessive simplification (check and rephrase the ideas). The prosomeric model suggests that there are two\nsegments (each segment is defined as a transverse unit with roof, wing, basal, and floor plates) related to the\norigin of the telencephalon and hypothalamus. Furthermore, the model proposes that the evaginated\ntelencephalon derives from the most dorsal alar plate near the roof plate of the peduncular neuromere (the\nneuromere in front of p3). This peduncular neuromere according to the model includes an alar portion that is\nclose to the alar-basal boundary (nk2.2 positive) and is related to the origin of hypothalamic derivatives such as\nthe paraventricular nucleus. The basal plate of the peduncular neuromere is related to the origin of\nhypothalamic derivatives such as the dorsomedian or subthalamic nucleus among others. From the dorsal to the\nventral position there are differences between the territories that are related to the distance at which they are\nlocated, for example, from the organizers of the roof plate or the floor plate. The most rostral neuromere called\nthe terminal neuromere, includes a portion of the dorsal alar plate that will be the preoptic area (close to the\nroof plate) and a portion of the alar plate close to the alar-basal limit that will be the origin of the retinal\nterritory (not the eye that involves ectodermal and mesodermal components), supraoptic and suprachiasmatic\nnucleus among others. In addition, the basal plate of the terminal neuromere will be the origin of the\nventromedian, arcuate and mammillary nuclei among others. In summary, the prosomeric model proposes that\nneuromeres have a clear transverse boundary that should be molecularly identified at some point in early\ndevelopment. Furthermore, the prosomeric model proposes that components, for example, such as the preoptic\narea (unpaired telencephalon) and the ocular territory, but also the anterior paraventricular area, share the\nalar plate of the same neuromere, but that does not imply molecular similarity with the components of the basal\nplate. The great complexity of the hypothalamic region added to the complexity of prosomere 3 must be\nconsidered from early stages and a simplified version may give some confusing ideas. Components of the basal\nplate or alar plate between adjacent neuromeres are expected to share some molecular features because they\nshare ventral and dorsal inductions from the floor or roof plates, respectively. Also, the last sentence of this\nparagraph is a bit odd because the telencephalon is the most dorsal wing plate near the roof plate and does not\nnecessarily have to be the result of a coordinated pattern with the alar or basal plate of the hypothalamus to be\nincluded in the same prosomere.\nWe have updated our text to include more detail on the prosomere model, making clear that we are referring to\ntwo segments, for example:\nIntroduction:\n“Each prosomere possesses both roof plate and floor plate, a conceptual necessity to meet the prosomere\ndefinition of a segment. The hypothalamus is grouped with the telencephalon, together occupying two\nanteriormost prosomeres (hp2, hp1). Posterior to these are three diencephalic prosomeres (p3-p1) housing the\nprethalamus (p3), thalamus (p2), pretectum (p1) and associated ventral structures (4, 6).”\nAs our results are complex, we have been compelled to present only those core aspects of the prosomere model\nthat are necessary for the understanding of our manuscript.\nR3 Q13 The authors' interesting observation in this paper is related to the behavior of different neuronal clones\nduring neural tube growth. However, the authors do not explain in the text why clonal coordination is required\nto define neuromeres. Also, the clonal relation doesn’t mean that they finally will derive in the same\ncomponents.\nThank you for this comment. We do not claim that clonal patterns define regions in and of themselves, but this\ninformation should guide our interpretation of the relationships between regions because cells that arise close\ntogether experience similar patterning signals, which determine regional identity during early developmental\nstages. This point is strongly backed up by the results of our dorsalisation experiments (new Fig. 6A-C;\nSupplementary fig. S16), which indicate that early spatial origin predicts competence to express regional\nmarkers.\nWe have added the following comments to the discussion:\n“Growth patterns help us to understand the epigenetic history and environmental exposures of cells during a\ncrucial period of specification, relevant for both stem cell differentiation efforts and topological models. Our\nresults reveal early proximity of much of the developing basal hypothalamus to the diencephalic prethalamus\nand thalamus; confirm a close association of prospective prethalamus and PV hypothalamus; and suggest that\ntelencephalic and basal hypothalamic lineages are largely segregated. We conducted our fate mapping through\npatterning stages, when cell fate is initially plastic, but becomes progressively restricted according to the\npositional information encountered. Thus, although it must be stressed that growth lines do not directly delineate\nforebrain regions, the many similarities in the shapes of DiI growth lines and gene expression domains\ndemonstrate the close coupling of growth and fate.”\nWe have also improved our explanation of our results by adding the following statement to the corresponding\nresults section:\n“Considered alongside our fate map (Fig. 2A, Movie S1), these results suggest that proximity to prospective\ntelencephalon at HH10 predicts competence to express FOXG1, and proximity to prospective prethalamus/PV\nhypothalamus predicts competence to express PAX6/ARX.”\nOur suggestions are further backed up by cell mixing experiments, including new data presented in new Figure\n6D-E. Hence, our conclusions are based on several different lines of evidence.\nR3 Q14 -Line 67. A new model of forebrain development cannot be defined in terms of a tripartite\nhypothalamus. It does not sound correct that the model of forebrain development is a tripartite hypothalamus.\nThe authors need to review this proposition better. The authors propose a new model (if it really is a new model\nor a modified version of the prosomeric model) of hypothalamic development or hypothalamic-prethalamic\ndevelopment, at least taking into account the explanations given in the text.\nThank you for this suggestion. As we highlight in the Introduction “the fundamental layout of the developing\nvertebrate brain is still disputed, reflected in a confused and contradictory contemporary literature, with the\nextent and position of the hypothalamus at the centre of the debate”, therefore our placement of the\nhypothalamus clearly differentiates our model from both columnar and prosomere models – something we want\nto convey in the name. Importantly, the name we have chosen refers not to the hypothalamus in isolation, but to\nits position with respect to other structures. These topological relations are inseparable from the orientations of\nthe A-P and D-V axes, so considering that we have placed the hypothalamus ventral to three major regions,\nthe tripartite hypothalamus and its dorsal relations together constitute the majority of the forebrain, their described\nrelations revealing – simply but clearly – the axis directions across the same area.\nWe have added the following clarification to the discussion:\n“In conclusion, we forward a ‘tripartite hypothalamus’ model in which the basal hypothalamus sits ventral to\nthree regions – the telencephalon, prethalamus/PV hypothalamus, and the ZLI (Fig. 6F). The LFNG-ve\nZLI and posterior hypothalamus form a topological boundary between the anterior SIX/FEZ+ve and\nposterior IRX+ve forebrain, with the SM hypothalamus part of a ventral boundary region associated with\nlineage restriction.”\nResults:\nR3 Q15-Line 78. The authors: “For descriptive purposes, we define the A-P axis as running parallel to the\nhypothalamic NKX2-2 expression domain and the D-V axis as orthogonal to it (Fig. 1F)”. The authors propose\na confusing model that has some unresolved problems as a model. For example, they considered a dorsoventral\nposition in scheme 1 F that extends to the rostral tuberal region. The blue line going to the tuberal region runs\nfrom dorsal to anterior and not ventrally (unless the authors are considering that there is a rostral floor plate at\nthat point that was not postulated in the text). Furthermore, following this assumption implies that this part of\nthe brain that will be influenced by the prechordal plate will give rise to the floor plate. Does that mean that the\nalar-basal boundary is in contact with the floor plate? For example, to improve the ideas of this part of the\nscheme (even if they want to propose a new model), see Ferran et al 2021 (Is there a prechordal region…) for\nthe importance of the prechordal plate and the notochord in establishing the main partitions of the brain\nbauplan. If the authors want to propose another option, they must consider this conceptual framework which\nmust also be resolved in their model.\nWe included Figure 1F only to show the reader how to interpret the directional terms in our results section,\nalthough it does fit with our final scheme (please refer to Figure 6F). These comments appear to follow from the\nassumption that we have provided a prosomere update rather than a new model, which we have now clarified.\nAccordingly, by definition the blue line does not run from dorsal to anterior because this aligns with the\nproposed D-V axis in our model. Likewise, we are not suggesting that a floor plate exists in the anterior tuberal\nregion, because the anterior forebrain in our model does not possess a floor plate in the usual sense of the word.\nOn the question of how closely the ventral midline (rather than the Arx+ve floor plate) approaches the alar-basal\nboundary, this is an interesting unresolved question, and indeed, in principle the two could meet. We have added\nthe following comment to the discussion:\n“We do not define a specific point in the anterior tuberal hypothalamus where the ventral midline ends\nanteriorly, but note that in principle it could extend far enough to abut the ABB.”\nRegarding the prechordal plate and notochord, our discussion includes the comments:\n“We speculate that the SM hypothalamus and flanking WNT8B-positive regions represent an extensive ventral\n‘boundary region’ (Fig. 5K), where the juxtaposition of anterior (FEZF+ve) and posterior (IRX+ve) forebrain -\nplus underlying tissues such as notochord and prechordal mesoderm - provided an evolutionary substrate for the\ngeneration of high signalling and cell type complexity.”\n“The rest of the hypothalamic midline lacks floor plate markers at mid-embryogenesis but arises from Foxa1+ive\nShh+ive floor plate-like (HypFP) progenitors (12, 50), its uniqueness accounted for by the distinct evolutionarily\norigin of the anterior forebrain (51–54), and mechanistically by the distinct patterning influence of the\nprechordal mesoderm (55–57).”\nR3 Q16-Line 98. The growth lines should be interpreted as representing the patterns of the neurepithelial\nclones growing throughout longitudinal domains involving several neuromeres (cases 4-5 for example) and\ninner portions of the neuromeres as the case of 8-9 (inner pallial and subpallial subdivision that can be visible\nat clonal levels). In summary, the same information can be used to reinforce prosomeric interpretations. I don’t\nunderstand or is not conceptually clear defined why the authors consider that the way of patterning growing\nwill be used to redefine the relationship between the territories. The authors need to provide the mechanistic\nsupport or evidence to validate this assumption.\nPlease see our response to Q13 where we have outlined our thoughts on the relevance of the growth patterns to\ninterpreting the relationships between regions, including comments on new evidence presented (new Fig. 6D-\nE). Regarding the prosomere model, as we describe in our manuscript (Figure 3A-C, associated text and\nreferences therein), our work disproves the growth model invoked in support of the prosomere model.\nAlternative interpretations are welcome, but as we have suggested in various comments above (e.g. Q2), we do\nnot believe the prosomere model is well supported and believe a fresh proposition is warranted.\nR3 Q17-Line182. This interpretation is not clear. What does it mean bulk of hypothalamus? According to your\ninterpretation some basal plate components but not from the prosomeric model are in a caudal neuromere. Some portions of the tuberal region are ventrally positioned to the telencephalon according to your schema.\nThank you for requesting greater precision. We now refer specifically to the ventral hypothalamus and state that\nthe anterior tuberal hypothalamus originates ventral to the telencephalon:\n“Instead, our fate-mapping shows that much of the ventral hypothalamus arises posterior to the telencephalon,\nand ventral to areas that will give rise to the PV hypothalamus, prethalamus and thalamus (Fig. 2A,F; Fig. 3D-\nE). The anterior tuberal hypothalamus is an exception, originating ventral to the telencephalon.”\nR3 Q18-Line 202. Authors “The claimed segmental identity between the telencephalon and hypothalamus\nimplies that fate conversion should be possible only between these identities.” According to the prosomeric\nmodel, FoxG1 is expressed in the unpaired and evaginated telencephalon (terminal and peduncular\nneuromeres, respectively). However, the alar hypothalamus (absence of Shh expression) is found in most of the\nparaventricular territory and subparaventricular territory characterized by the absence of FoxG1. In the\nanimals of this study treated with Cyclopamine it seems as if both FoxG1 and PAX6 were ventralized (See in\nFerran et al., 2007 Fig. 1 and 5E, PAX6 expression is also observed at stage HH24 in the paraventricular\nterritory. At early stages an increase in signal can be seen in this territory). In my opinion, these results can be\nperfectly interpreted considering the detailed description of the forebrain regionalization supported by the\nprosomeric model.\nThank you for these thoughtful comments. In fact we have now rearranged our manuscript (for the sake of\nclarity), and the cyclopamine analysis is presented without reference to the prosomere model, as we have\nalready presented our reasons for proposing an alternative.\nR3 Q19 -Line 220. This paragraph shows an interesting observation that the authors use to propose a different\nboundary compared to the prosomeric boundaries. This is an interesting observation but not a definitive\ndemonstration that this distortion represents the actual interprosomeric boundary. The peculiar clonal\nexpansion of the basal plate below the ZLI (assuming that the ZLI is only on the wing plate or if we extend it to\nthe basal plate, it does not necessarily have to follow these lines) needs to be cautiously interpreted and defined\nprecisely if the identity is changes or not on those stages and with the impact in adult brain. There are several\npatterns like Lim1 (see Ferran et al., 2007 Fig. 6 A that could be interpreted as the interprosomeric boundary\nbetween p2/p3). The authors make an interesting observation but do not imply that it represents the boundary\nbetween territories. The fate map through later stages with definitive derivatives will provide better support for\nunderstanding common subsequent identities to recognize possible derivatives of each neuromere. However, if\nthe authors wish to propose such a boundary, they need to develop a model that indicates the basal plate\nposition for the redefined regions. In addition, more details need to be provided on the components derived of\neach territory.\nOur model is a first iteration, but one we believe is well-supported by our data. We agree that further work will\nbe necessary to elucidate the precise nature of our proposed boundary, but this would be beyond the scope of the\ncurrent study. We would like to highlight the numerous genes that we have analysed in the posterior\nhypothalamus, all of whose profiles support our model (thank you for mentioning the interesting Lim1\nexpression pattern, which appears to be excluded from our boundary region at its posterior edge). In contrast,\nmore than thirty years since its first iteration (Puelles & Rubenstein 1993), no such description exists of\nprosomere boundaries in the basal plate at stages equivalent to chicken HH20 (approximately mouse E11). We\nshow that the prosomere boundaries as described in mouse cannot be resolved correctly in the HH20 chicken\nembryo (Fig. 5H-L), or adequately in the E7 chicken (Fig. 5M-O).\nAlthough we agree that it would be interesting to analyse fate mapping results at later stages, we chose to focus\non a relatively early endpoint (HH20) for two reasons. First, this time-point marks the end of patterning stages,\ncertainly for the hypothalamus. Species conservation is high at this time point, and regional marker genes can\nthen be used to identify the spatial location of cells in single cell datasets for example (including mouse,\nhuman), whose lineages may then be reconstructed. We have added the following text to the discussion:\n“All major progenitor zones are established in the chicken hypothalamus by the experimental endpoints used,\nenabling the inference of adult nuclei origins by comparison to longer term fate mapping studies and\ndevelopmental single cell sequencing datasets, including to some extent cross-species data. Continued rapid\nprogress in understanding the ventral forebrain in particular should confirm these inferences, directly or\nindirectly.”\nRegarding the SM hypothalamus specifically, we draw the readers’ attention to the derivatives of this progenitor\nzone, as it occupies a key position in our model. From the discussion:\n“Cells originating in the SM region contribute widely to the posterior hypothalamus and beyond, with\nPITX2/LMX1B+ve progenitors in mouse contributing to the SM, subthalamic and ventral premammillary nuclei”\nSecondly, we chose to end our studies at an early stage to avoid the complication of the large tangential\nmigrations that occur within the forebrain. These movements do not necessarily help to elucidate developmental\norganisation, because lineage restriction (for example between rhombomeres) generally occurs in the ventricular\nzone but often not the mantle. We believe there is a limit to the usefulness of extrapolating from later to earlier\nstages, and consider the detailed focus on early stages a major strength of this study.\nR3 Q20-Line 276: The authors propose a change in the interpretation of the pattern observed in HH20 that will\nhelp them reinterpret the interprosomeric boundaries. This is an interesting point of view, but not a definitive\ndemonstration of the actual prosomeric interpretations. Of course, it remains an interesting possibility to be\nconsidered but requires more detail from the authors on what the model they propose looks like. The authors\nassume that all bands are specifically related to the proposed derivatives. Molecular maps at this early stage do\nnot necessarily reflect the complete or definitive fate of these territories. Further work examining detailed fate\nmaps will be necessary to demonstrate the true value of early mapping with accurate comparisons of fate maps\nreaching late stages when definitive derivatives are already found. As mentioned above, some patterns suggest a\np2/p3 interprosomeric boundary below ZLI (Lim1), which adds confusion to the resolution of this issue. This\ndistortion observed by the authors could be a behavior of the basal plate near the midline that is then\ndefinitively specified and be a longitudinal band.\nPlease see our response to questions 1 (extra detail on our model) and 19 (discussion on boundaries and stages\nanalysed).\nR3 Q21 Figure 1:\nFor example, Fig. 1 L indicates the injection supposedly in area 11, marking the prethalamus. However,\nprethalamic region touch the roof plate, and it is worth asking whether the extension to the roof is not\nrepresenting an expected identification of the fate map reaching the dorsal midline that was not considered in\nyour schemas.\nThank you for pointing this out - our original text was confusing as we did not distinguish between the\nprethalamus itself (which arises in zone 11) and the prethalamic eminence. We have now labelled these areas\nseparately in Fig. 1E, and provide text and images (new fig. S2F-G) describing the origin of the prethalamic\neminence in posterior zone 9:\n“Injection zones 7-9 give rise to growth lines that reproducibly stretch from the telencephalon to the dorsal optic\nstalk or optic midline (Fig. 1I-L; fig. S2) - zone 9 also skirting the prethalamic eminence (fig. S2).”\nR3 Q22 Fig. S3 F. Is green Shh expression?\nThank you, this has been fixed (now fig. S2K).\nR3 Q23 Discussion:\nThe discussion should be enriched by aspects such as what are the differences between the proposed model and\nthe prosomeric one (or is it a variation of the prosomeric model?). In addition, a discussion that considers the\ncomplexity of the adult brain is necessary because the author proposes a simple model that does not consider all\nthe derivatives proposed in the prosomeric model. Some discussion will also be necessary, for example, on the\ndorsoventral position of the peduncular fibers in the peduncular prosomere, because with this model some things\nhave become difficult to explain.\nThank you for this suggestion. We have improved our discussion on the difference between the two models\n(please see e.g. question 1). From the discussion:\n“The defining feature of our model is that it places the hypothalamus ventral to the telencephalon and to two\nparts of the diencephalon (prethalamus and ZLI), hence ‘tripartite’. Therefore, the majority of the hypothalamus\nis considered ventral diencephalon, similar to classical columnar views. However, we have adopted the curved\nABB from the prosomere model, and link the anterior tuberal hypothalamus and telencephalon. We also note\nthat earlier versions of the prosomere model placed axes more similarly to ours. Therefore, our model\nincorporates elements from both columnar and prosomere models.”\nOur model aims to describe early developmental organisation in terms of axes and major forebrain divisions,\nrather than to outline in detail the derivatives of the early hypothalamic (and other) progenitor zones. Similar to\nour comment for question 13, having worked out the HH10 fate map to ~HH18, existing work can be used to\nbroadly infer later outcomes, and rapid progress is being made in this area.\nRegarding the peduncular fibres, similar to our response to Q3, we do not accept that segment boundaries are\nrequired to explain the existence of complex patterned territories. In the absence of specific evidence for\nsegment boundaries at early developmental stages, we suppose that ordinary patterning mechanisms are\nresponsible for these phenomena. We include the following note in the results section:\n“While Meis2 expression did taper anteriorly within the SPa (fig. S15E), it is unclear why this - or indeed any of\nthe above genes showing graded differential expression - should indicate a segmental boundary, rather than\nbeing the result of any other patterning process.”\nR3 Q24 Some general considerations need to be made in this regard. It is true that clonal growing is the key to\ndefine the boundaries? To what extent do they help or not?\nPlease see our response to question 13.\nR3 Q25 Final comment to consider in your discussion:\nDo related clones reflect the identity of the territory at each stage? We see the behavior of related clones, but\nthat does not necessarily mean that they belong to the same neuromere at all stages. If the territory is expanded\nand exposed to different organizers, it may be differentially affected in different dorsoventral domains. In\nsummary, clonal relatedness suggests possible identities conserved through time, but may not necessarily be the\nkey to defining anteroposterior neuromeric boundaries. In my opinion, future fate maps should be performed on\nindividual cellular components to better understand clonal relationships. Then, at different stages, those patterns\nshould be compared with molecular maps, but later stages with defined derivatives are essential because at early\nstages molecular maps do not always represent precise boundaries (see figure 14 in Sánchez Arrones et al 2009,\nfor a better idea about the problem).\nWe agree that a range of evidence must be considered when approaching questions of organisation. Please see\nour earlier comments (question 13 on the relevance of growth patterns, and question 19 on boundaries and\nstages considered). We appreciate the point about the imprecision of using early stages, but the Sánchez Arrones\npaper performed forebrain fate mapping at HH4 and analysed at HH8. This is a very different proposition to the\nHH17-20 forebrain (= our main endpoint), in which patterning is largely complete and neurogenesis well\nunderway, having begun at approximately HH11. Forebrain cell clones labelled at HH18 tend to stay close\ntogether rather than dispersing widely (e.g. Larsen 2001).\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI congratulate the authors for vastly improving the manuscript. It is now a very clear and convincing\nwork of great interest for the dev bio community.\nWe thank the reviewer for this comment and their many helpful suggestions.\nThe only point I still am not comfortable with is the sole use of HCR results to claim growth pattern\ndistort DV axis in mouse (Figure 4) and would like the authors to make a more careful conclusion from\nthese data, stating that the staining suggests distortion rather than saying it shows that it happens.\nWe agree that this point should have been made cautiously. We have made the following changes.\nLine 302: subheading changed to “A possible dorsoventral axis distortion in mouse”\nLine 314: figure heading changed to “Growth patterns may distort the dorsoventral axis in mouse”\nLines 325-326: “Expansion of the thalamus may displace the Shh+ve ZLI, causing the angle between\nthe ZLI and posterior hypothalamic Shh expression domain to become more acute …”\nLines 332: “To explore whether this may point towards an axis distortion as seen in chicken …”\nLines 340-343: “… thalamic growth may contribute to this change by displacing the dorsal ZLI\nanteriorly. Although it is not possible to resolve this question solely by analysing gene expression\npatterns, we propose that the D-V axis runs through the ZLI and SM hypothalamus and may become\ndistorted in mice as in chicken.”\nReviewer #3 (Remarks to the Author):\nThe authors have made considerable improvements to the manuscript, and many of my initial\nquestions have been addressed. The authors have clarified several key points, which have\nsignificantly improved the clarity of the manuscript. However, several key points still require further\nclarification to ensure the work is fully understandable.\nWe thank the reviewer for their comments, and their helpful suggestions for improving the manuscript\nAs a general comment:\nThis is an interesting and provocative work that introduces an alternative hypothesis for how the\nhypothalamic region develops. The core contribution of this work is to present a new way to interpret\ndifferential growth in the context of neural tube specification. The authors propose one possible\ninterpretation, but for the moment the evidence didn’t discard other options. Differential growth is not a\nspecification map, and even when interpreted as a fate map, the growth patterns need to be analyzed\nmore carefully and in greater detail to support stronger conclusions. Nevertheless, this enormous\namount of data are a valuable contribution to the scientific community as they can be reinterpreted to\nsupport different conclusions, not only than those proposed by the authors.\nWe have been clear on this point: lines 587-590: “Thus, although it must be stressed that growth lines\ndo not directly delineate forebrain regions, the many similarities in the shapes of DiI growth lines and\ngene expression domains demonstrate the close coupling of growth and fate.”\nIn summary the proposal is interesting and provocative, and independently of the lack of maturity at\nsome points (a simple view about the complexity observed in adult brains), give some fresh ideas to\nhave in mind and to be deeply explored in further works.\nSome points to be revised.\nTitle: According to the data provided this work focuses mainly on hypothalamic organization but not\non the forebrain in detail. It is not true that hypothalamus is the main problem implying all the\nforebrain. The forebrain includes telencephalon (impar and evaginated) prethalamus, thalamus and\npretectum, but also the midbrain territory according to the finding by Albuixech-Crespo et al. 2017.\nMost current definitions include the midbrain as part of the forebrain.\nI recommend focusing the title on the hypothalamus, as this is the central challenge and main subject\nof the work. The results of this study have implications for how the forebrain develops. However, the\nauthors did not address the complex issue of how the pallium and subpallium are specified. My\nrecommendation at least is to focus the title on hypothalamic organization. Focusing this title on the\nhypothalamus will provide greater clarity on the authors' findings, key results, and the challenging\ndata.\nRespectfully, we disagree that our data focuses mainly on hypothalamic organisation, and that the\ntitle should be altered to reflect this. Both our fate-mapping and our cell mixing studies cover the\ntelencephalon, eyes, prethalamus, thalamus, pretectum, as well as the hypothalamus. The question\nof how the pallium and subpallium are specified is far beyond the scope of this manuscript. Altering\nthe title would minimize the importance of our study, and would not reflect its wider contextual\nimportance.\nAbstract: I think that the problem is the same, the focus of this study is on the hypothalamus not in the\nentire forebrain. Some little changes need to be made to clarify it.\nWe accept that we did not study the midbrain, which proponents of the prosomere model argue to be\nthe posterior forebrain. However, this is not yet common usage. To emphasise that our study is\nrelevant to the entire forebrain (our italics, below), we have made slight rearrangements to the\nabstract:\nLines 32-39: “Comparative gene expression analysis and cell mixing experiments suggest the\nexistence of a contiguous transverse boundary region, encompassing the zona limitans intrathalamica\n(ZLI) and retromammillary hypothalamus, that divides the anterior and posterior forebrain, and\nbecomes distorted at the ZLI base. Fate conversion experiments indicate that the hypothalamus is\ntopologically tripartite, lying ventral to the telencephalon, prethalamus and ZLI. Our findings challenge\nthe widely accepted prosomere model of forebrain organisation, do not support a segmented anterior\nforebrain, and instead suggest a ‘tripartite hypothalamus’ model.”\nLine 36: The authors “Our findings challenge the widely accepted prosomere model of forebrain\norganisation, and we propose an alternative ‘tripartite hypothalamus’ model.”\nThe findings of the authors doesn’t challenge the prosomeric model, the data of this manuscript\nchallenge the prosomeric interpretation of the hypothalamic organization. If the authors provide in\nfuture works additional details for other brain regions like telencephalon, midbrain and even the entire\nhindbrain they will claim that the challenge the prosomeric model.\nThe clearest part is the proposed alternative tripartite hypothalamus model. The title and the abstract\nwill benefit from focusing this work on the hypothalamus.\nLine 37-38: Again, we respectfully disagree. Our model is fundamentally incompatible with the\nprosomere model as it directly challenges the existence of the secondary prosencephalon and the\nexistence of segments anterior to the ZLI, i.e. p3, hp1 and hp2 prosomeres. Likewise it directly\nchallenges the orientation of the axes. Instead, our model substantially expands the diencephalon by\nincluding most of the hypothalamus and the temporal retina.\nMain text: The authors: “In this scheme, the forebrain consists of five transverse segments –\n‘prosomeres’ – analogous to the neuromeres of the hindbrain and spinal cord.”\nRecent studies based on the Albuixcetch-Crespo et al. 2017 describe that the midbrain is part of the\nforebrain (see a summary of it for clarification and reference about this change in Ferran et al., 2025,\nsee also its Fig. 1 to understand the idea).\nLines 51-58: we have updated the text to reflect this: “In this scheme, the forebrain consists of seven\ntransverse segments – ‘prosomeres’ … Most recently the midbrain has recently been designated as\nforebrain, therefore contributing two more prosomeres.”\nLine 69: The authors: “Here we clarify forebrain developmental organisation.” The authors propose\nsome point of view based on interesting data about hypothalamic organization. The study is not\ncompleted enough to claim that this study is about forebrain organization due that complex regions\nlike subpallium and pallium were not deeply explored. The most controversial aspect in this study\ngoes around the hypothalamic region.\nPlease see our response to requests with regard to the title and line 36.\nLine 83 The authors “Our work resolves prior misinterpretation and provides a new model for\nforebrain developmental organisation that we term the ‘tripartite hypothalamus’ model.” The authors\nare not providing a new model of forebrain organization; they are providing a new model of\nhypothalamus or a new interpretation about how the hypothalamic and prethalamic territories can be\ninterpreted. Sound so strange that the new model of the forebrain is the tripartite hypothalamus\nmodel. Rewrite this because it is confused and not clear. Forebrain is more than the portions\ndiscussed by the authors and more than hypothalamus.\nPlease see our response to requests with regard to the title and line 36.\nResults:\nLine 100: The authors: “For descriptive purposes, we define the A-P axis as running parallel to the\nhypothalamic NKX2-2 expression domain and the D-V axis as orthogonal to it (Fig. 1F)”. The\nexpression “descriptive purpose” is confused and not necessary in text. If for the authors, this is the\ntopological A-P axis is enough mentioning directly that this this the A-P axis based on the comparison\nwith Nkx2.2. Recognizing this axis is essential to deep understanding of this study and as\nconsequences in how to understand the data through the text.\nLine 103: we have removed the expression “For descriptive purposes”\nLine 150 “The anteriormost diencephalic floor plate arises from zone 3 (Fig. 1W).“ The authors need\nto define diencephalon, because their considering in their model that this is to rostral diencephalon\nand is different to other views. To better understand the position is a good point to define that this is\nthe anteriormost diencephalic floor plate defined as….\nThank you for pointing this out. We have altered the wording to read ‘The anteriormost SHH+ve floor\nplate arises from zone 3’. See also our response to your comment about line 304 regarding the\ndiencephalon.\nLine 162 change topography for topology, because the conserved are the relations with neighboring\nterritories (topological relations).\nThank you, this has been corrected\nLine 229: Authors need to explain that they elongate the roof plate close to the alar basal border (See\nfig. 3E). This detail is important to highlight because it implies that there is a rostral floor plate derived\nfrom the prechordal effects. Another curious thing from these schemes is that they appear to follow a\nsegmental partition of the neural tube like a modified prosomeric model.\nWe have added a legend to panel E in Figure 3 to clarify the meaning of the orange and green zones,\nwhich indeed represent the roof plate and floor plate in A, but in E they represent the dorsal and\nventral midlines. We have added new text to the figure legend to clarify the meaning of the dotted\nlines, which are intended to illustrate the growth pattern, not to indicate segments:\nLines 240-247: “A) … Transverse lines represent incipient interprosomeric boundaries. … E) …\nDotted lines represent cells at particular transverse positions at the earlier stage, and their\ndescendants’ positions after neural tube bending.”\nLine 255: “We hypothesised that the early proximity of prospective hypothalamic and diencephalic\ncells results in a shared topological identity, due to similar exposure to fate-determining cues”. This is\ndifficult to understand, “shared topological identity”… . The topology is conserved between all\nterritories along neural tube; some models can share the topology but it is not clear what the author is\nthinking about with share identity. If they are referring to sharing identity of neighbors’ territories it is\nok, but they don’t need to include the word topology. Or explain better the meaning of their idea.\nLines 261: We have improved the phrasing, to read: “a similar positional identity”\nLine 304. “implicating the posterior hypothalamus as part of the ventral diencephalon.” Again authors\nneeds to clarify what is the definition for them about diencephalon.\nWe have clarified this in lines 308-310: “As the ZLI is intercalated between the prethalamus and\nthalamus – both diencephalic regions – this implicates the posterior hypothalamus as part of the\nventral diencephalon.”\nSee also lines 547-549: “Similar to the classical view, we therefore tentatively group the PV\nhypothalamus with the prethalamus as part of the diencephalon, posterior to the telencephalon.”\nLines 638-640 clarify this further: “the ZLI and ventral boundary region cut through the diencephalon,\ndividing the forebrain into anterior and posterior parts.”\nLine 400 “so could not be designated as prosomere M with certainty” Check this description along the\ntext because there no any prosomere M. The mammillary domain is the most ventral domain of the\npeduncular prosoemer (or hp1). Check your text and explain better the idea.\nLine 400-402: We now refer back to the relevant information: “(21,32), a SIM1/NKX2-1+ve,\nFOXA1/OTP-ve M domain (Fig. 5C,D) could not be identified in the HH20 chicken (Fig. 5L).\nLine 407-408: This now reads: “and so could not be designated as the prosomere model M domain\nwith certainty”.\nLine 521 “sondegig”?? Shh?\nWe have clarified this in Lines 488-490: “We therefore dorsalised the chicken hypothalamus by\napplying SHH inhibitors (cyclopamine or Sonidegib)”\nSupplementary figure 1. E: Telencephalon include pallium and subpallium. Please, modify\naccordingly. G: Is green Shh gene expression pattern?\nThank you for spotting these omissions, which have been corrected.\nSupplementary figure 4B. According to the schema (dorsal view) injections were made in 10 or limit\n10/8 (red). However, the green one appears to be in 11 or limit 9/11 not 5.\nThank you for spotting this - it has been corrected"}]

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