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# Jagged-mediated lateral induction patterns Notch3 signaling within adult neural stem cell populations

Sara Ortica, Miguel Martinez Herrera, Louis Degroux, Bastian Rochette, Nicolas Dray, Laure Bally-Cuif

► To cite this version:

Sara Ortica, Miguel Martinez Herrera, Louis Degroux, Bastian Rochette, Nicolas Dray, et al. Jagged-mediated lateral induction patterns Notch3 signaling within adult neural stem cell populations. 2025. pasteur-05262132

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https://pasteur.hal.science/pasteur-05262132v1

Preprint submitted on 15 Sep 2025

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Distributed under a Creative Commons Attribution 4.0 International License
1 Jagged-mediated lateral induction patterns Notch3
2 signaling within adult neural stem cell populations

3 Sara Ortica ¹, Miguel Martinez Herrera ¹, Louis Degroux ¹, Bastian Rochette ¹, Nicolas Dray ¹,* and

4 Laure Bally-Cuif ¹,*

5

6 ¹Institut Pasteur, Université Paris Cité, CNRS UMR3738, Zebrafish Neurogenetics Unit, F-75015 Paris, France

7 * Co-corresponding authors at: laure.bally-cuif@pasteur.fr; nicolas.dray@pasteur.de

8 Lead contact: laure.bally-cuif@pasteur.fr

9

10

11

12 Abstract

13 In the adult brain, Notch3 signaling promotes neural stem cell (NSC) quiescence and stemness. It
14 remains unknown how Notch3 signaling levels are controlled and relate to these NSC decisions. Here
15 we directly measure the nuclear translocation of the Notch3 intracellular fragment (N3ICD) and
16 quantify Notch3 signaling in NSCs of the zebrafish adult telencephalon in situ. We report that Notch3
17 signaling levels match NSC quiescence and stemness levels. In physical space, Notch3 signaling is
18 patterned and high signaling levels surround N3ICD<sup>low</sup> cells, which also express the <i>deltaA</i> (<i>dla</i>)
19 ligand. Another ligand, <i>jagged1b</i> (<i>jag1b</i>), expressed in all NSCs, positively interacts with Notch3 and
20 sustains expression of the stemness factor Sox2. Finally, lowering <i>jag1b</i> preserves the structured
21 distribution of Notch3 signaling levels in space but attenuates their variance. We propose that Notch3
22 signaling integrates Dla-mediated lateral inhibition and Jag1b-mediated lateral induction to control
23 quiescence and stemness and their spatiotemporal dynamics in adult NSCs.

24

25

26 Keywords

27 Adult neural stem cells, Notch3, Jagged, pallium, zebrafish, intrinsic niche

28
29 **Introduction**

30 Neural stem cells (NSCs) generate neurons in the adult vertebrate brain, contributing to brain
31 plasticity, repair, and growth. NSC potential ("stemness") follows individual NSC decisions such as
32 quiescence exit, self-renewal or neurogenesis commitment, and is also tightly regulated temporally
33 and spatially across the niche at population scale¹,². Numerous regulators of individual NSC activity
34 have been identified, but it remains largely unknown how the spatiotemporal nature of stemness is
35 encoded.

36 In the major adult NSC niches (sub-ependymal zone of the lateral ventricle (SEZ) and sub-granular
37 zone of the dentate gyrus (SGZ) in mouse, ventricular zone of the pallium in zebrafish3-5), NSCs are
38 quiescent radial glia-like cells (RGs). They activate to divide and self-renew or generate further
39 committed neural progenitors (NPs) fated to delamination and neuronal differentiation6-13. The Notch
40 signaling pathway is a major regulator of NSC decisions along this lineage, in a complex receptor-
41 dependent and context-dependent manner13-15. Several Notch receptors are expressed in NSCs
42 (Notch1, 2 and 3 in mouse16-22, and Notch1a, 1b and 3 in zebrafish23-25). Notch1 expression and
43 signaling are predominant after NSC activation to limit neuronal differentiation16,24. In contrast,
44 Notch2 and 3 are strongly expressed in quiescent NSCs and promote quiescence: upon lowering
45 Notch3 activity in zebrafish, quiescent pallial NSCs enter proliferation²⁴; likewise, abrogation of
46 Notch2 and Notch3 also lead to NSC quiescence exit and proliferation in the mouse SEZ and SGZ20-22.
47 In the zebrafish pallium, Notch3 signaling also maintains the NSC progenitor state, likely via a
48 distinct downstream pathway involving the transcription factor Hey126. Finally, in physical space,
49 Notch signaling also influences the spatial distribution of NSC activation decisions: in the zebrafish
50 pallium, NPs prevent NSC activation in their vicinity, and this is abolished when Notch signaling is
51 abrogated27. Together, these results identify Notch signaling, in particular Notch3, as a major regulator
52 of the spatiotemporal aspects of stemness, contributing to controlling quiescence, progenitor
53 properties, and the proper distribution of NSC decisions in physical space.

54 The challenge now is to understand Notch3 signaling *in situ* in quantitative, dynamic and mechanistic
55 terms across adult NSC niches. Upon binding a ligand (Delta or Jagged) expressed by a contacting
56 cell, a major output of Notch signaling in the receiving cell is transcriptional¹⁴,²⁸,²⁹: Notch cleavage by
57 the gamma-secretase complex releases its intracellular fragment (NICD), which translocates to the
58 nucleus where it binds the cofactor RBPj to activate target genes. This is the case in adult NSCs, where
59 NICD overexpression rescues Notch signaling blockade to promote quiescence²²,²³, and Notch
60 invalidations generate phenotypes¹⁶,²¹,²²,³⁰ that resemble RBPj invalidations³¹⁻³³. Canonical Notch
61 signaling is a direct pathway that does not involve second messengers³⁴,³⁵, and quantitative measures
62 in Drosophila and C. elegans indicate that nuclear NICD levels correlate with the duration of
63 transcriptional bursts in target genes, thus are quantitative indicators of Notch signaling potential³⁶⁻³⁸ -
64 although, depending on context, additional regulators may be needed³⁹⁻. We exploited these findings
65 to design a direct tracer of Notch3 signaling activity in zebrafish adult pallial NSCs *in situ*. Inspired by
66 pioneering work in Drosophila⁴⁰⁻⁴², we knocked-in fluorophores into the C-terminal end of Notch3-
67 ICD (N3ICD) and monitored N3ICD-fluo translocation to the NSC nucleus. We show how the
68 generated lines allow a reliable quantification of nuclear N3ICD in NSCs in real time and in physical
69 space across the NSC population, and use this tool to identify the ligands controlling Notch3 signaling
70 *in situ*. Our results indicate that the Notch3 signaling pattern at the population level responds to both
71 lateral inhibition and lateral induction, the latter being mediated by Jagged1b, expressed by most
72 NSCs, and controlling stemness. Our results further support a role for Jag1b in potentiating lateral
73 inhibition. These results reveal how the different ligand-receptor interaction modes permitted by
74 Notch signaling are integrated to encode the spatiotemporal dynamics of Notch3 signaling and ensure
75 quiescence and stemness within the adult pallial NSC niche.

76

77 **Results**

78
79 Generation of direct *in vivo* reporters of Notch3 expression

80 We designed two strategies to generate Notch3 signaling readout lines, which together provide
81 versatile technical applications: to quantify nuclear N3ICD in transgenic backgrounds with various
82 fluorophores, to track Notch3 in different subcellular compartments, and to test the effect of *notch3*
83 gene dosage.

84 First, a BAC encompassing the entire *notch3* locus, plus 150kb of upstream sequence, was recombined
85 in *E. coli* to insert a *GFP-P2A-nlsRFP* cassette in position +6823 of the coding *notch3* region (Fig.1A).
86 This fuses GFP to Notch3 within N3ICD after amino acid 2274 (MLLHQ), upstream of the PEST
87 sequence (Fig.S1A-C). nlsRFP will translocate to the nucleus and provide a nuclear marker for *notch3- *
88 expressing cells, useful for nuclear segmentation and quantification. This line is referred to as
89 TgBAC(*notch3:notch3-GFP-P2A-nlsRFP*)<sup>ip13T<sub>8</sub></sup>, abbreviated in TgBAC<sup>n3-GFP/+</sup> (for heterozygote animals),
90 and is intact at its endogenous *notch3* locus.

91 Second, we used Crispr/Cas9 and Homology Directed Repair (HDR) recombination to knock-in an
92 *Azamigreen-P2A-nlsRFP* cassette in position +29155 of the endogenous *notch3* locus (Figs.1B, S1A,B).
93 Azamigreen (AG) was chosen for: (i) its green fluorescence, combinable with nlsRFP in direct imaging,
94 (ii) its detection by a specific anti-AG antibody, for combination with GFP lines by
95 immunohistochemistry (IHC), and (iii) its different sensitivity to pH compared to GFP. The
96 recombination site was selected upon comparing the efficiency of several gRNAs and led to a
97 manageable efficiency of germline transmission (Fig.S1D-F). It generates a fusion of AG to Notch3
98 within N3ICD after amino acid 2382, C-terminal to the first PEST sequence (GSTAG) (Fig.S1B). This
99 line is referred to as TgKI(*notch3:notch3-mAG-P2A-nlsRFP*)<sup>ip14T<sub>8</sub></sup>, abbreviated in TgKI<sup>n3-AG/+</sup> (for
100 heterozygotes) or TgKI<sup>n3-AG/n3-AG</sup> (for homozygotes).

101 We first verified that GFP and AG displayed the expected expression pattern in the embryonic and
102 adult brain. In situ hybridization for *gfp* in TgBAC<sup>n3-GFP/+</sup> embryos, and for *azamigreen* in TgKI<sup>n3-AG/+</sup>
103 embryos, revealed a pattern identical to *notch3* at 24 hours-post-fertilization (hpf) (Fig.S2A). Direct
104 imaging of GFP and nlsRFP expression in the forebrain of 24hpf TgBAC<sup>n3-GFP/+</sup> embryos also showed
105 nlsRFP confined to cell nuclei in neural progenitors, while GFP intensely labeled cell membranes and
106 more weakly nuclei (Fig.S2B). Finally, direct whole-mount imaging of adult brains at 3 months-post-
107 fertilization (mpf), with focus on pallial ventricular neural stem and progenitor cells (NSPCs),
108 revealed a similar pattern for nlsRFP/GFP and nlsRFP/AG in TgBAC<sup>n3-GFP/+</sup> and TgKI<sup>n3-AG/+</sup> animals,
109 with visible membrane and nuclear staining (Fig.1C,D).

110 In addition, we observed fluorescence in numerous cytoplasmic dots in both lines, very motile in
111 embryonic neural progenitors imaged dynamically (Fig.S2C). Based on previous observations in
112 Drosophila<sup>42</sup>, we tested whether they correspond to the endocytic trafficking of Notch3-fluo. We
113 compared the localization of Notch3-GFP and Notch3-AG with Rab proteins in early/sorting (Rab5<sup>pos</sup>),
114 recycling (Rab11<sup>pos</sup>) or late (Rab7<sup>pos</sup>) endosomes, or the Lysosome-associated membrane protein 1 for
115 lysosomes (Lamp1<sup>pos</sup>), in NSPCs. Expression of Zona occludens 1 (Zo1) was used to delineate the
116 NSPC apical surface. We could detect colocalization of some Notch3-GFP with recycling endosomes
117 and lysosomes (Fig.S2D), while Notch3-AG, in addition, colocalized with early/sorting endosomes
118 (Fig.S2E). Thus, Notch3 is also engaged in dynamic endocytosis, recycling and degradation in adult
119 pallial NSPCs.

120

121 *notch3* expression in the adult pallium encompasses the entire NSC population

122 NSCs and NPs are intermingled at the pallial ventricular surface. NSCs express the radial glia marker
123 Glial fibrillary acidic protein (Gfap), while NPs do not. Both cell types express the progenitor marker
124 Sox2. To position Notch3 expression among NSPCs, we compared nlsRFP, Gfap and Sox2 using IHC
125 (Fig.1E-H). Among apical Sox2<sup>pos</sup> cells, over 96% of Gfap<sup>pos</sup> cells were nlsRFP<sup>pos</sup>, and over 94% of
126 nlsRFP<sup>pos</sup> cells appeared Gfap<sup>pos</sup> (Fig.1G). This confirms *notch3* expression in NSCs. The proportion of
127 strongly nlsRFP<sup>pos</sup> cells was 90%, with an additional 5% harboring low nlsRFP levels. This also
128 indicates the presence of nlsRFP in NPs (Fig.1H), either due to the stability of nlsRFP or to low *notch3*
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expression extending to NPs. The similar pattern of nlsRFP in the two lines also confirms that the regulatory elements contained in TgBAC<sup>n3-GFP/+</sup> drive proper Notch3-GFP expression in the adult brain.

## Notch3 fusion proteins are functional

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To assess the functionality of Notch3 fusion proteins, we used *notch3*<sup>fl332</sup> null mutants<sup>24</sup>. *notch3*<sup>fl332/fl332</sup> larvae display a “curved” phenotype at 7dpf<sup>43</sup> and generally die between 10 and 15dpf, while *notch3*<sup>fl332/+</sup> heterozygotes are morphologically normal and adult viable. Pallial RG in *notch3*<sup>fl332/fl332</sup> larvae also display decreased quiescence entry starting at 5dpf, in link with Notch3 promoting RG quiescence<sup>26</sup>.

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TgKI<sup>n3-AG/n3-AG</sup> homozygotes displayed no overt phenotype at larval stage and survived normally until adulthood. Focusing on neurogenesis, we assessed the proliferation and neurogenic activity of embryonic and larval RG, and adult NSPCs, in TgKI<sup>n3-AG/n3-AG</sup> fish. In embryos, RG proliferation rate was measured using a 1-hour BrdU pulse then immediate IHC (Fig.S3A,B), and neurogenesis with the neuronal protein HuC/D (Fig.S3C-E). TgKI<sup>n3-AG/n3-AG</sup> embryos behaved identically to wild-type (WT) for these parameters (Fig.S3B,D,E). In larvae, like for *notch3*<sup>fl332/fl332</sup> mutants<sup>24</sup>, we assessed telencephalic RG quiescence in TgKI<sup>n3-AG/n3-AG</sup> homozygotes by quantifying the proportion of RG (Brain lipid binding protein-positive, Blbp<sup>pos</sup>) expressing the proliferation marker Proliferating cell nuclear antigen (Pcna<sup>pos</sup>) at 8dpf. We found no difference with WT larvae (Fig.S3F,G). Finally, we quantified NSPC proliferation in the adult pallium. The proportion of Pcna<sup>pos</sup>, Sox2<sup>pos</sup> cells in TgKI<sup>n3-AG/n3-AG</sup> homozygotes was ~10-15%, identical to heterozygotes and to previous reports in WT, and in agreement with the long quiescence phases of adult NSCs (Fig.1I,J). Thus, Notch3-AG appears functional in its control of forebrain neurogenesis.

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To assess Notch3-GFP, we crossed *notch3*<sup>fl332</sup> mutants and TgBAC<sup>n3-GFP/+</sup> fish to construct genetic contexts where *n3-GFP* is the only productive *notch3* copy (TgBAC<sup>n3-GFP/+;notch3*fl332/fl332</sup>) or is combined with one WT *notch3* copy (TgBAC<sup>n3-GFP/+;notch3*fl332/+</sup>). *notch3-GFP* alone was sufficient to rescue the curved larval phenotype at 7dpf (Fig.S3I), and viability until adulthood (Fig.S3J), thus behaved similarly to a single productive copy of endogenous *notch3* in *notch3*<sup>fl332/+</sup> heterozygotes. We also used TgBAC<sup>n3-GFP/+</sup>, carrying three copies of *notch3*, to assess the effect of Notch3-GFP overexpression on telencephalic neurogenesis. RG proliferation and neuronal generation were normal in TgBAC<sup>n3-GFP/+</sup> embryos (Fig.S3B,D,E), but there was a strong trend for RG activation decrease in 8dpf larvae (Fig.S3F,H). Thus, increased *notch3* gene dosage at this stage likely increases signaling and Notch3-GFP promotes quiescence entry, like WT Notch3. Finally, in the adult pallium, we quantified Pcna<sup>pos</sup> cells among all NSPCs (Sox2<sup>pos</sup>;Zo1<sup>pos</sup>) in these different genotypes. There were no biases compared to WT (Fig.1K,L). We conclude that Notch3-GFP is functional. The lack of a visible effect of *notch3* overexpression in the adult pallium of TgBAC<sup>n3-GFP/+;notch3*fl332/+</sup> fish will be discussed below.

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## Notch3 fusion lines are dynamic and quantitative readouts of Notch3 signaling in adult pallial NSCs

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We next assessed whether the TgKI<sup>n3-AG</sup> and TgGFP<sup>n3-GFP</sup> lines could reliably quantify canonical Notch3 signaling in adult pallial NSPCs. Depending on signaling strength, we expect to observe NSPCs with various levels of nuclear N3ICD-fluo (Fig.2A,B). At population scale in TgKI<sup>n3-AG</sup> and TgBAC<sup>n3-GFP</sup> fish, the AG or GFP fluorescence signals at the cell membrane appeared much higher than the nuclear signal, as expected due to the high instability of N3ICD (Fig.1C,D). We thus used nlsRFP, encoded in *notch3*-expressing cells, to develop a pipeline of nuclear segmentation to selectively quantify nuclear N3ICD-AG or N3ICD-GFP fluorescence (Fig.2C). In both lines, nuclear fluorescence levels, measured as pixel intensity levels in individual cells across the NSPC population, displayed a broad range of values with an approximately normal distribution. It was similar in direct fluorescence and when using whole-mount IHC with anti-AG or anti-GFP antibodies (Fig.2D,E), validating IHC for quantitative measurements.
178 We next tested whether N3ICD-fluo levels were quantitative readouts of Notch3 signaling. First, 179 nuclear N3ICD-AG levels appeared on average two-fold higher in TgKI<sup>n3-AG/n3-AG</sup> homozygotes than in 180 TgKI<sup>n3-AG/+</sup> heterozygotes (Fig.2F). Second, nuclear N3ICD-GFP levels were significantly lower in 181 proliferating (Pcna<sup>pos</sup>) than in quiescent (Pcna<sup>neg</sup>) cells, in agreement with Notch3 signaling promoting 182 quiescence (Fig.2G, left panel). In this experiment, measuring GFP levels in non-apical Sox2<sup>pos</sup>, 183 nlsRFP<sup>neg</sup> or nlsRFP<sup>low</sup> cells was also used to estimate the "zero" value of nuclear N3ICD-GFP, 184 corresponding to IHC and imaging noise (Fig.2G, right panel). By comparison, this further indicates 185 that Pcna<sup>pos</sup> cells have N3ICD-GFP values still above null values, thus do experience N3ICD signaling, 186 albeit at low level (Fig.2G). Finally, decreasing canonical Notch signaling for 24 hours using the 187 gamma-secretase inhibitor LY411575 *in vivo* led to an accumulation of N3ICD-GFP at the membrane 188 and a significant reduction of nuclear N3ICD-GFP levels (Fig.2H,I). The range of decrease (1.7-fold 189 from mean to mean at population level), compared to the zero value (Fig.2G, right panel), further 190 suggests that Notch3 signaling is lowered but not fully blocked by such a treatment.

191 Together, these results demonstrate that TgBAC<sup>n3-GFP</sup> and TgKI<sup>n3-AG</sup> permit a quantitative and direct 192 readout of transcriptional Notch3 signaling in adult NSPCs in situ.

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194 Notch3 signaling levels in adult pallial NSPCs are controlled by ligand availability in addition to 195 gene dosage

196 We took advantage of the quantitative power of our measures to assess the link between *notch3* gene 197 dosage and signaling levels in adult pallial NSPCs. For this, we quantified nuclear N3ICD-GFP levels 198 in TgBAC<sup>n3-GFP</sup> animals carrying one, two or three productive gene copies of *notch3* (as in Fig.1K). In 199 the case of non-limiting amounts of ligands and signaling levels determined by gene dosage, we 200 expect the same absolute amounts of nuclear N3ICD-GFP for these three genotypes (Fig.2J, top); in 201 contrast, if endogenous ligand availability is a limiting factor for signaling, the absolute levels of 202 N3ICD-GFP should progressively decrease as *notch3* gene copy number increases (Fig.2J, bottom). In 203 support of the latter hypothesis, we observed progressively decreasing N3ICD-GFP levels between 204 TgBAC<sup>n3-GFP/+</sup>;*notch3<sup>fl332/flh332</sup> , TgBAC<sup>n3-GFP/+</sup>;*notch3<sup>flh33/+</sup> , and TgBAC<sup>n3-GFP/+</sup>;*notch3<sup>+/+</sup> compound adults 205 (Fig.2K). Thus, ligand availability is a major determinant of N3ICD signaling levels in adult NSPCs, 206 limiting the effect of the extra gene dose in the TgBAC<sup>n3-GFP/+</sup>;*notch3<sup>+/+</sup> genotype.

207

208 Notch3 signaling levels are spatially patterned among adult NSPCs in situ

209 To gain insight into Notch3 signaling regulation, we used spatial statistics to describe the profile of 210 N3ICD levels in space in adult TgKI<sup>n3-AG/+</sup> and TgBAC<sup>n3-GFP/+</sup> pallia. With a marked L-function, we 211 scored Notch3 signaling levels in the respective neighborhood of cells with highest vs lowest N3ICD 212 values (highest or lowest 20% quantile of the whole distribution) and compared them with levels 213 expected assuming uncorrelation, i.e., upon random cell permutations (Fig.3A-C, S4C). Because such 214 analyses are based on closest neighborhood, we also considered apical cell size, measured using Zo1. 215 At population scale, cells of lowest Notch3 signaling levels tended to have smaller apical areas (and 216 conversely, cells of high Notch3 signaling levels tended to be large) (Fig.S4A,B). Nevertheless, there 217 was no size bias within a 30 µm distance from N3ICD<sup>low</sup> cells (Fig.3D, bottom panel) and, within the 218 first 7-8 µm surrounding each N3ICD<sup>low</sup> cell, N3ICD signaling levels (normalized to nuclear volume) 219 were overall higher than expected by chance (Fig.3D and S4D, top panels). The trend was similar 220 when considering absolute N3ICD values (within the hypothesis rejection threshold but significant at 221 5%, p=0.03) (Fig.3D and S4D, middle panels). A similar analysis conducted for the neighborhood of 222 N3ICD<sup>high</sup> cells was confounded by the effect of cell size and inconclusive (not shown). Thus, Notch3 223 signaling levels in individual cells across the germinal pallial population are spatially regulated: cells 224 receiving lowest Notch3 signaling tend to be surrounded by cells with highest signaling.

225

226 Jagged and Delta are differentially expressed in relation with NSC state
227 To exploit quantitative and spatial information and mechanistically dissect the regulation of Notch3
228 signaling, we used the most appropriate of the two transgenic lines based on technical needs:
229 TgBAC<sup>n3-GFP</sup> NSCs are less crowded with trafficking vesicles, facilitating quantifications, while TgKI<sup>n3-AG</sup> can be crossed into GFP reporter lines.

231 First, we analyzed the expression of Notch ligands. The zebrafish genome contains four *delta* (*dl*) genes
232 (*dla*, *dlb*, *dlc* and *dld*) and three *jagged* genes (*jag1a*, *jag1b* and *jag2*), and scRNAseq<sup>25</sup> predicts detectable
233 expression of *dla* and *jag1b* in adult pallial NSCs (Fig.S5A-G). We validated these findings using
234 sensitive single molecule fluorescent ISH (smFISH) with RNAscope probes on whole-mount adult
235 pallia. This highlighted *dla* and *jag1b* in NSCs while *jag1a* was not expressed and *jag2* was restricted to
236 neurons, below the NSC layer (Fig.S5H), confirming and extending previous work<sup>23</sup>.

237 Strikingly, the expression of *dla* and *jag1b* appeared different both in intensity and distribution. *dla* is
238 expressed with a broad range of intensities, measured by the number of RNAscope dots (interpreted
239 to reveal individual RNA molecules), with an exponential decrease from null/low levels to very high
240 levels (Fig.4A,B). In situ, *dla*<sup>high</sup> cells displayed a salt-and-peppery distribution similar to the pattern
241 previously described in Tg(*dla*:gfp) fish (Fig.4A,S5A)<sup>27,44</sup>. In contrast, *jag1b* expression levels followed a
242 normal distribution over a narrower range of levels, with most NSCs expressing some *jag1b*
243 transcripts (Fig.4A,B,S5C). Overall, there was no correlation between *dla* and *jag1b* expression levels at
244 the individual cell level (Fig.4C). Like for GFP in the adult pallium of Tg(*dla*:gfp) fish<sup>44</sup>, we confirmed
245 significantly higher *dla* transcript levels in Pcn<sup>pos</sup> compared to Pcn<sup>neg</sup> cells (Fig.4D). The relationship
246 between *jag1b* expression levels and cell proliferation was opposite, quiescent cells expressing
247 significantly higher levels of *jag1b* (Fig.4E).

248

249 Nuclear N3ICD levels cell-autonomously correlate with *jagged1b* but not *deltaA* expression, and
250 sign NSC position along lineage and quiescence progression

251 To elaborate hypotheses on Notch3 signaling regulation, we directly combined smFISH for *jag1b* or *dla*
252 with N3ICD quantifications in TgBAC<sup>n3-GFP/+</sup>. We found a clear positive correlation between *jag1b*
253 expression and nuclear N3ICD levels in individual cells (Fig.4F,G), while there was no correlation
254 between *dla* and N3ICD (in fact, most cells with high *dla* expression are N3ICD<sup>low</sup>) (Fig.4H,I). For
255 comparison with previous work<sup>27,44</sup>, we also exploited TgKI<sup>n3-AG</sup> crossed into Tg(*dla*:gfp)<sup>45</sup> to quantify
256 N3ICD-AG vs GFP (Fig.4J). We confirmed that *dla*:GFP<sup>neg</sup>;nlsRFP<sup>pos</sup> cells on average display
257 significantly higher N3ICD levels than *dla*:GFP<sup>pos</sup>;nlsRFP<sup>pos</sup> cells (Fig.4K).

258 Among NSCs, *dla*:GFP expression differentiates between subpopulations endowed with self-renewal
259 (*dla*:GFP<sup>neg</sup>) versus neurogenic (*dla*:GFP<sup>pos</sup>) potential, the latter showing increased GFP levels as they
260 progress along the lineage towards neurogenesis commitment<sup>44</sup>. We found that, within *dla*:GFP<sup>pos</sup>;
261 nlsRFP<sup>pos</sup> cells, N3ICD levels decreased as *dla*:GFP levels increased (Fig.4L). Within each *dla*:GFP<sup>pos</sup>
262 and *dla*:GFP<sup>neg</sup> subpopulation, NSCs with larger apical area are statistically closer to their next
263 division<sup>44</sup>. Within each of these subpopulations in nlsRFP<sup>pos</sup> cells, N3ICD levels generally increased
264 with apical area size -but stopped increasing linearly as apical areas reached high values- (Fig.4M,N).
265 Together, these results also connect N3ICD levels with ligands expression and the position of
266 individual NSCs along their lineage and quiescence progression (Fig.S5I).

267

268 Experimental downregulation of Notch signaling highlights paradoxical responses of *jag1b/hey1* vs
269 *dla* expression

270 Notch3 signaling in NSCs was proposed to involve lateral inhibition originating from Dla<sup>pos</sup> NP
271 neighbors<sup>27</sup>. Modeling lateral inhibition in a cell lattice also suggested that signaling intensity depends
272 on the length of Notch/Delta-contacting membranes<sup>46</sup>. To test whether such interactions could suffice
273 to explain the quantitative N3ICD pattern, we quantified nuclear N3ICD-AG in TgKI<sup>n3-AG/+</sup>;Tg(*dla*:gfp)
274 for each NSPC relative to its number of *dla*:gfp<sup>pos</sup> direct neighbors (i.e., sharing a membrane segment at
275 their apicobasal interface, revealed with Zo1 IHC)<sup>27,27</sup> or its total number of neighbors. We found that
276 there was no correlation between N3ICD levels and these parameters (Fig.S6A,B). Thus, the Notch3
277 activity pattern in adult pallial NSPCs likely involves more than a simple lateral inhibition model
278 predicted by the length of Dlapos cell contacts.

279 The similar distribution of Notch3 activity levels and *jag1b* expression is suggestive of lateral
280 induction. Reinforcing this possibility, we identified several RBPj binding sites in the second exon of
281 *jag1b*, including 2 in a head-to-head configuration reported to efficiently bind RBPj47 (Fig.S6C). To test
282 this hypothesis, we first assessed *in situ* the response of *dla* and *jag1b* to lowering Notch signaling by
283 gamma-secretase inhibition. In the case of lateral inhibition, lowering Notch should increase ligand
284 transcription; conversely, for lateral induction, decreased ligand transcription is expected (Fig.S6D).
285 We treated TgBACn3-GFP/+ adults with 10 µM LY511475 for 24 hours and assessed *dla* and *jag1b*
286 expression using smFISH together with Zo1 revealed by IHC (Fig.5A). This treatment decreases
287 N3ICD-GFP to almost half (as in Fig.2I), but NSCs do not yet enter the cell cycle48 (Fig.5B), avoiding
288 confounding measurements. We observed that the average levels of *dla* increased, while expression
289 levels of *jag1b* dropped dramatically (Fig.5C,D). These observations support Dla vs Jag1b being
290 engaged in lateral inhibition vs lateral induction with Notch3, respectively.

291

292 Jagged sustains Notch3 signaling and the progenitor state in adult NSCs

293 Next, to assess the role of Jag1b in Notch3 signaling in adult pallial NSCs, we tested whether Jag1b
294 knock-down affects N3ICD levels. We used first a lissamine-tagged version of the *jag1b* morpholino
295 (MO) validated in previous studies⁴⁹, using electroporation to transduce NSCs in vivo⁵⁰. We failed to
296 observe reproducible effects (not shown); however, this approach reduces *jag1b* in isolated cells, and
297 phenotypes might be confounded by the complex feedback interactions on N3ICD due to the presence
298 of several cell neighbors and ligands. We therefore used the cell-permeable “vivo-MO” version of
299 *jag1b*MO, injected intracranially into the brain ventricle of TgKI<sup>n3-AG/+</sup> adults, to target all cells with
300 ventricular contact<sup>51,52</sup> (Fig.5E). At 1-day post-injection (dpi), we quantified nuclear N3ICD-AG levels
301 in Sox2<sup>pos</sup> cells (Fig.5F), comparing *jag1b*MO to a standard control vivo-MO (ctrlMO). N3ICD-AG was
302 significantly reduced upon *jag1b* knock-down (Fig.5F,G), indicating that Jag1b is necessary for
303 maintaining global Notch3 signaling in adult pallial NSCs.

304 Notch3 signaling has two reported functions in adult pallial NSCs: the promotion of quiescence and
305 the promotion of stemness²⁶. We assessed the effects of *jag1b* knock-down on these activities using
306 whole mount IHC for readouts of cell activation (Pcna) and the progenitor state (Sox2) at 1dpi. Global
307 proliferation levels (Pcna<sup>pos</sup>) remained unchanged (Fig.5H). In contrast, the intensity of Sox2
308 expression was massively decreased -although ventricular cells remained Sox2<sup>pos</sup> - (Fig.5I). There was
309 also a small but not statistically significant trend increase in *dla* transcription, more prominent in NSCs
310 than in NPs (as assessed using apical size enrichment cut-offs)⁴⁴ (Fig.5J,K). This *dla* phenotype differed
311 in two respects with the increased *dla* transcription observed with LY treatment: it was quantitatively
312 much reduced (Fig.5D,K), and the *dla* salt-and-peppery expression pattern was preserved (Fig.5J).
313 These differences are striking given that N3ICD levels are divided by approximately half in both
314 cases.

315 Together, Jag1b contributes significantly to Notch3 signaling in situ, with an impact on stemness but
316 not on the salt-and-peppery pattern of *dla* nor on NSC proliferation rate.

317

318 *hey1* expression responds to the Jag1b-Notch3 signaling axis in adult pallial NSCs

319 Effectors of canonical Notch signaling include Hairy/Enhancer-of-Split transcription factor genes.
320 Several are expressed in pallial NSCs, among which *her4* genes and *hey1*, which are downregulated
321 upon gamma-secretase inhibition at adult stage, and in *notch3*<sup>fh332/fh332</sup> larvae<sup>25-27</sup>. Of particular interest
322 here was *hey1*, associated with Notch activation by Jagged in other contexts<sup>53-56,55-57</sup>. *hey1* proved
323 expressed at variable levels in individual NSPCs (Fig.S7A,B). To compare it with Jag1b/Notch3
324 signaling, we combined smFISH for *hey1* and *jag1b* in WT and TgBAC<sup>n3-GFP/+</sup> fish. *hey1* expression levels
325 positively correlated with *jag1b* (Fig.S7C,D) and with N3ICD levels -albeit weakly- (Fig.S7E). Further, 326 like *jag1b*, *hey1* was virtually expressed in all quiescent cells and was absent from proliferating cells 327 (Fig.S7F), and was dramatically reduced by LY treatment (Fig.S7G,H).

328 The similar behavior of *hey1* and *jag1b* expression suggesting a functional relationship, we tested the 329 effect of blocking *jag1b* on *hey1* expression, quantifying *hey1* expression in situ after intracranial 330 injection of *jag1b*MO (Fig.S7I). There was no effect at 1dpi (not shown), but at 3dpi *hey1* expression 331 was weakly decreased compared to fish treated with the *ctrlMO* (Fig.S7J). These results position *hey1* 332 directly or indirectly downstream of Jag1b/Notch3 signaling in adult pallial NSCs, although *hey1* 333 expression also responds to other inputs.

334

335 Jagged-mediated Notch3 signaling potentiates lateral inhibition

336 The pattern of N3ICD signaling in physical space (Fig.3D), and the observed coincidence of low 337 N3ICD<sup>low</sup> and *dla*:GFP<sup>high</sup> cells (Fig.4L), suggest that this spatial pattern results from (Dla-mediated) 338 lateral inhibition. In addition, the Jag1b data presented above are compatible with Jag1b-mediated 339 lateral induction. Previous modeling work predicts that, when lateral induction is superimposed to 340 lateral inhibition, more robust pattern formation ensues where the Notch-Delta signaling difference 341 between neighboring cells can become reinforced<sup>58,59</sup>. *jag1b*MO experiments, which decrease Jag1b at 342 the population level, provide a context to address this prediction.

343 We first addressed whether the spatial pattern of N3ICD signaling levels was altered upon *jag1b* 344 knock-down. We injected TgKI<sup>n3-AG/+</sup> adults intracranially with the *ctrlMO* or the *jag1b*MO (Fig.6A) and 345 recorded N3ICD levels and the spatial location of each individual cell. We then applied the marked L- 346 function as in Fig.3D to measure deviation of N3ICD levels compared to an uncorrelated pattern in the 347 vicinity of N3ICD<sup>low</sup> cells. *ctrlMO*-treated brains behaved as described in untreated fish (Fig.6C), with 348 N3ICD<sup>low</sup> cells surrounded by higher N3ICD levels than expected. We found that this effect was not 349 modified in *jag1b*MO brains (Fig.6C). However, the distribution range of N3ICD levels was 350 significantly decreased upon *jag1b*MO knock-down (variance under *ctrlMO* conditions: 5.0 x 10<sup>5</sup>; 351 under *jag1b*MO conditions: 2.5 x 10<sup>5</sup>, p-value=3.4 x 10<sup>-16</sup>) (Fig.5G). Thus, lowering Jag1b-mediated 352 signaling preserves the structure of the Notch3 signaling pattern, but the difference between sender 353 (N3ICD<sup>low</sup>) and receiver (N3ICD<sup>high</sup>) cells is decreased. These results are in fitting with predictions that 354 superimpose lateral inhibition and lateral induction, and compatible with a role for Jag1b-mediated 355 signaling in reinforcing lateral inhibition<sup>58,59</sup>.

356

357 Discussion

358 Together, we propose that N3ICD levels and their spatiotemporal distribution pattern among the 359 NSPC population result from the differential but synergistic engagement of Notch3 with two ligands, 360 together controlling different NSPC properties, their spatial distribution, and their temporal 361 unfolding.

362

363 Quantification of Notch3 signaling, allelic expression and dosage buffering in adult NSCs in situ

364 TgBAC<sup>n3-GFP</sup> and TgKI<sup>n3-AG</sup> permit to selectively readout Notch3 expression, subcellular localization and 365 signaling in situ. In the absence of a reliable antibody, this is necessary to report the selective 366 engagement of Notch3 vs Notch1 in NSPCs. Reporter lines based on RBPj binding elements do not 367 distinguish between different Notch receptors. Further, major transcriptional changes occur in NSCs 368 within 24 hours or less of Notch signaling blockade<sup>48</sup>, stressing the importance of a fast and dynamic 369 readout. We cannot ascertain that N3ICD-GFP and N3ICD-AG exactly mimic the half-life of 370 endogenous N3ICD; in particular, these fusions exclude the PEST domain normally promoting N3ICD 371 degradation. However, their functionality is at least close to that of WT N3ICD, suggesting preserved 372 dynamics (Fig.1, S2, S3).
373 Our work brings forward interesting observations comparing gene dosage, signaling levels and function in adult NSCs. The fact that NSCs in TgKI<sup>n3-AG/+</sup> adults overall harbor a half-dose of nuclear N3ICD-AG compared to TgKI<sup>n3-AG/n3-AG</sup> (Fig.1J) indicates that both *notch3* alleles are expressed equally to contribute to signaling. Tracking N3ICD-GFP in contexts expressing one, two or three functional gene copies of *notch3* further supports that Notch3 signaling levels are buffered by ligand availability. This likely also explains why three functional gene copies do not lead to modified NSC properties in situ (Fig.1K). Together, our findings stress the "dose/cell state" relationship of Notch3 signaling levels in adult NSCs, and its tight regulation by limiting ligands.

381

382 N3ICD levels correlate with NSC decisions and combine differential input from DeltaA and Jagged1b
383

384 Notch3 signaling levels vary from cell to cell along two NSC decision axes: (i) the quiescence/activation balance, and (ii) NSC lineage progression. Indeed, N3ICD levels (i) are significantly lower during activation (Fig.2G), in agreement with Notch3 signaling promoting quiescence²⁴, and (ii) negatively correlate with *dlaA*:GFP levels (Fig.4L), which globally read lineage progression⁴⁴. These results support that N3ICD levels quantify "transcriptional signaling" in situ, in agreement with previous work in Drosophila and C. elegans where nuclear NICD levels quantitatively correlate with target genes activation³⁶⁻³⁸.

391 *dla* and *jag1b* are the main ligands expressed within the pallial germinal population²³. We reveal that their expression patterns strikingly differ, from each other and in their relationship to N3ICD levels: *dla* transcription is salt-and-peppery and *dla*<sup>high</sup> NSCs are generally N3ICD<sup>low</sup>, while *jag1b* is expressed broadly and positively correlates with N3ICD at the single cell level (*jag1b*<sup>high</sup> NSCs are N3ICD<sup>high</sup>) (Fig.4). Although consolidation by analyses of the Dla and Jag1b proteins is needed, these patterns suggest different mechanisms of Notch3 signaling. The role of Dla-Notch3 signaling can be inferred combining previous work and this work: (i) at any given time, *dla*<sup>pos</sup> cells (some NSCs and all NPs) are in majority activated<sup>27</sup>, (ii) activated NPs promote quiescence in their immediate neighbors and this is mediated by Notch<sup>27</sup>, (iii) N3ICD and *dla* levels are inversely correlated (Fig.4L), (iv) N3ICD<sup>low</sup> cells are surrounded by higher Notch3 levels than expected (Fig.3D), and (v) experimentally decreasing *dlaA* in individual cells *in situ* induces their quiescence<sup>44</sup>. These observations converge towards proposing that Dla is engaged in lateral inhibition with Notch3 to promote NSC quiescence by the induction of high Notch3 signaling levels in contacting cells. *dla*<sup>pos</sup> NSCs do themselves receive a Notch signal, although weaker than their *dla*<sup>neg</sup> counterparts, as indicated by their reactivation frequency upon Notch blockade<sup>44</sup>. This final observation further suggests that Dla is also engaged in cis-inhibition.

406 Although Jag1 is expressed in neurogenic niches in mouse<sup>60,61</sup>, the role of Jag1b-Notch3 signaling is addressed here for the first time. Collectively, we show that (i) *jag1b* expression is widespread in NSPCs, (ii) Notch3 signaling and *jag1b* in individual NSPCs positively correlate (Fig.4G), and (iii) are necessary for each other's expression or activity (Fig.5C,G). Finally, (iv) lowering Jag1b decreases Sox2 levels. Sox2 is a direct Notch signaling target in hippocampal NSCs<sup>31</sup>. It promotes the NSPC state in a dose-dependent manner<sup>62,63</sup>, an effect already visible when Sox2 dosage is genetically reduced by 3-fold in the adult mouse hippocampus<sup>64</sup>. Together, these results converge to suggest an engagement of Jag1b/Notch3 in an alternative Notch signaling mechanism: lateral induction, the output of which would be to promote stemness. Hey1 may be involved downstream of Jag1 (Fig.5K) (and itself maintains Sox2<sup>26</sup>). The delayed and weak effect of *jag1b* knock-down on *hey1* may suggest additional mechanisms, or that the stability of *hey1* transcripts<sup>65</sup> buffers the effect of a transient *jag1b* knock-down. Formally confirming lateral induction would require assessing N3ICD levels cell-autonomously after down-regulating *jag1b*, for example after mosaic *jag1b* knock-down. The presence of both Dla and Jag1b ligands triggering Notch3 in our system led however to weak results in such experiments.

421 We previously demonstrated that Notch3 controls quiescence and stemness via distinct downstream effectors<sup>24,26</sup>. We show now that N3ICD levels integrate the combined action of two ligands and
signaling modes, to control these NSC decision axes. Jagged ligands are generally considered to
trigger lower Notch signaling levels than Delta<sup>34,56,66-68</sup>, an effect that may result in the activation of
different target genes. Here, *jag1b* knock-down reduces Notch3 signaling by 1.6-fold at population
scale (Fig.5G), close to what is observed upon LY treatment (Fig.2I), but it is difficult to formally
conclude as these interventions are likely partial and, as discussed below, Jag1b/Notch3 signaling also
impacts Dla/Notch3 signaling. How individual NSCs decode these two signals and what their
respective tipping points are to release NSCs from quiescence or permit lineage progression remain
important aspects to be studied. These NSC decisions in vivo are partially linked: for example, self-
renewing NSCs activate less frequently than neurogenic NSCs, which are further downstream along
lineage progression<sup>44,69</sup>. For self-renewing NSCs to divide yet maintain stemness, it is possible that the
quiescence/activation balance is regulated by a different (higher) N3ICD threshold than lineage
progression. This would add to the emerging list of examples where graded, i.e., level-based, Notch
signaling is used to encode cell (state) diversity<sup>66,70-72</sup>. The tipping points are however unlikely to be
only quantitative as N3ICD levels in Pcn<sup>pos</sup> cells remain broadly distributed (Fig.2G) and overlap
with their values along lineage progression (Fig.4L).

438

Dla- and Jag1b-mediated Notch3 signaling pattern NSC decisions in physical space

439

The current work also reveals the distribution of N3ICD levels in physical space. As discussed above,
the spatial surrounding of N3ICD<sup>low</sup>, *dla*:GFP<sup>high</sup> cells by highest N3ICD levels likely reads Dla-
mediated lateral inhibition. The broad expression of *jag1b*, and its positive regulatory loop with
N3ICD levels suggest, in contrast, that it propagates a minimal level of Notch3 signaling encoding the
progenitor cell state across the germinal population. A similar situation was described in the
developing inner ear, where Jag-Notch-mediated lateral induction encodes the progenitor state across
a prosensory field where Delta-mediated lateral inhibition then selects individual cells for
neurogenesis commitment<sup>55-57,73-75,76,77</sup>. The superimposition of lateral induction to lateral inhibition
was previously modeled, predicting in addition that intermediate levels of Jagged-mediated lateral
induction reinforce lateral inhibition<sup>58,59,78</sup>. Our spatial analyses of N3ICD signaling upon Jag1b
invalidation interestingly fit these predictions: the difference between highest and lowest N3ICD
levels is decreased (Fig.5G) while a pattern structured by lateral inhibition is retained (Fig.5J, Fig.6).
Several interpretations for this phenomenon have been proposed, such as cis-inhibition of Notch by
Jagged (which when released would free Notch to interact with Delta presented in trans)<sup>58</sup>, or
blockade of cis-inhibition of Notch by Delta via the formation of Jagged-Delta dimers<sup>79,80</sup>.

440

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448

449

450

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458

459

460

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Together, we propose that the different expression and signaling modes of the two ligands
superimpose and refine local and global information, patterning Notch3 signaling at cell- and tissue-
scale. Because N3ICD levels read the position of individual NSC along their two main axes of
temporal progression, these cell-cell interactions may coordinate the individual trajectories of
otherwise asynchronous NSPCs. In particular, Dla<sup>pos</sup> signaling cells are generated by lineage
progression and are transient<sup>44</sup>; thus, the Dla<sup>pos</sup> pattern is spatially dynamic and includes an intrinsic
temporal value. We speculate that the combined spatial vs spatiotemporal activities of Jag1b and Dla,
respectively, encode the dynamic homeostasis of the pallial NSPC population, maintaining individual
cell trajectories in spatiotemporal equilibrium.

465

Resource availability:

466

467

468

Lead contact: laure.bally-cuif@pasteur.fr. Materials availability: all materials and transgenic lines generated in this study are available upon request to laure.bally-cuif@pasteur.fr. Data and code availability: all raw data are presented in Table S1. There were no codes generated for this study.

469

Acknowledgments:
470 We are greatly indebted to François Schweisguth for discussions and advice along this work, to Felix
471 Cheysson for advice on spatial statistics, to Neetu Gupta-Rossi for the gift of antibodies against
472 endocytic pathway components, to Sébastien Bedu and Nathan Guibert for expert fish care, to
473 members of the LBC team for support and discussions, and to M. Wappner for his critical reading of
474 the manuscript. This research was funded by the ANR (Labex Revive ANR-10-LABX-0073), La Ligue
475 Nationale Contre le Cancer (LNCC EL2019 BALLY-CUIF), the Fondation pour la Recherche Médicale
476 (EQU202203014636), the European Research Council (ERC SyG PEPS 101071786), CNRS, and Institut
477 Pasteur. L. Degroux was a recipient of an Institut Pasteur work-study contract.

478 Author Contributions:

479 Conceptualization: L.B-C., N.D., S.O.; Methodology: L.D., B.R., M.MH., S.O.; Writing original draft:
480 L.B-C.; Review and editing: of original draft: all authors; Supervision: L.B-C., N.D.; Project
481 administration: L.B-C.; Funding acquisition: L.B-C. All authors have read and agreed to the published
482 version of the manuscript.

483 Declaration of Interests:

484 The authors declare no competing interest. The funders had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to
485 publish the results.

487 Supplemental information titles and legends:

488 Document S1. Figures S1-S7 and supplemental references

489

490 Figure titles and legends

491 **Figure 1.** Notch3 fusion alleles are functional in adult NSCs. **A,B.** Transgenesis methods. **A.** 492 Recombination of *GFP-P2A-nlsRFP* in frame with *exon 33 of notch3* into *BAC CH211-69E21*, followed 493 by transposase-mediated recombination. **B.** CRISPR/Cas9-mediated knock-in into endogenous *notch3*. 494 A gRNA specific for the last *notch3* exon ("n3bait") was used to knock-in *AG-P2A-nlsRFP* in frame into 495 *exon 33* by Homology Directed Repair. **C,D.** Confocal dorsal views of dissected telencephala from 4 496 months post-fertilization (mpf) *TgBAC<sup>n3-GFP/+</sup>* and *TgKI<sup>n3AG/+</sup>* fish stained by whole-mount 497 immunohistochemistry (IHC) for *GFP*, *AG* or *nlsRFP* (color-coded). Cells expressing *notch3* show 498 nuclear *RFP* (magenta). Notch3 protein is visible in green on membranes (white arrows) and into 499 some nuclei (blue arrows). **E,F.** Similar images with IHC for *Zo1* and *nlsRFP* (color-coded; bottom: 500 cross sections). **G.** Quantification of cells positive for *Zo1* and/or *nlsRFP* in *TgBAC<sup>n3-GFP/+</sup>* and *TgKI<sup>n3AG/+</sup>* fish (*TgBAC<sup>n3-GFP/+</sup>*; n= 1666 cells, 4 fish; *TgKI<sup>n3AG/+</sup>*; n= 974 cells, 3 fish;). **H.** Proportion of *Gfap<sup>pos</sup>* (Zrf1 antibody, grey) and *nlsRFP<sup>pos</sup>* (magenta, high and low *nlsRFP* levels) cells among apical *Sox2<sup>pos</sup>* cells (cyan). **I.** Confocal whole-mount dorsal view of an adult *TgKI<sup>n3-AG/n3-AG</sup>* telencephalon with IHC for *Sox2*, *AG* and *Pcna* (color-coded). **J.** Percentage of *Pcna<sup>pos</sup>* cells among *Sox2<sup>pos</sup>* cells in adult *TgKI<sup>n3-AG/+</sup>* heterozygotes vs *TgKI<sup>n3-AG/n3-AG</sup>* homozygotes. **ns:** non-significant (Wilcoxon rank sum test, p-value=0.16) (*TgKI<sup>n3-AG/+</sup>*; n=1781 cells, 4 fish; *TgKI<sup>n3-AG/n3-AG</sup>*; n=1595 cells, 4 fish). **K-M.** Crossing *TgBAC<sup>n3-GFP/+</sup>* into the *notch3<sup>fl332</sup>* background generated genotypes carrying *notch3-GFP* and in total 1, 2 or 3 functional copies of *notch3* (color-coded, shades of gray) compared with WT (black). **K.** Percentages of *Pcna<sup>pos</sup>* cells among *Sox2<sup>pos</sup>* (ANOVA test, p-value=0.32, ns between genotypes) (1 copy: n=760 cells, 2 fish; 2 copies: n=1507 cells, 4 fish; 3 copies: n=1243 cells, 3 fish; WT: n=558 cells, 1 fish). **L.** Confocal whole-mount dorsal views of adult telencephala of the different genotypes with IHC for *Sox2*, *Zo1* and *Pcna* (color-coded). Scale bars: 20µm.

513

514 **Figure 2.** Notch3 fusion alleles are dynamic and quantitative reporters of Notch3 nuclear signaling
515 in adult NSCs. **A.** Schematic of Notch3 signaling in *TgBAC* and *TgKI*. *nlsRFP* is separately translated
and translocated into the nucleus. Notch3-fluo is membrane-bound in the signal-receiving cell. Ligand interaction and cleavage by γ-secretase lead to translocation of the tagged Notch3 intracellular domain (N3ICD-Fluo) into the nucleus. γ-secretase is inhibited by LY-411575 (LY). B. Scheme of the expected Notch3-fluo pattern in the adult pallial progenitor population: each cell expressing *notch3* has a red nucleus (nlsRFP, magenta) and a green plasma membrane (membrane-bound Notch3-fluo), and a yellow nucleus (green and red) after Notch3 activation. C. Segmentation method to quantify nuclear N3ICD-fluo using the Imaris software (Oxford Instruments). A mask is created to remove strong non-specific red dots, then 3D nuclear surfaces are automatically generated and manually corrected. 100 to 200 nuclei in the Dm region of the pallium are usually segmented for each hemisphere. N3ICD-fluo is quantified in each nucleus in situ (right: nuclei color-coded according to green intensity). D, E. N3ICD-fluo concentration (conc°, total pixels intensity normalized to nuclear volume) in pallial NSPCs in adult TgKI<sup>n3-AG/n3-AG</sup> (D) and TgBAC<sup>n3-GFP/+</sup> (E), without ("fresh") or after ("IHC") antibody staining for AG or GFP, respectively (TgKI<sup>n3-AG/n3-AG</sup>: n=317 cells for "fresh" and n=428 cells for "IHC"; TgBAC<sup>n3-GFP/+</sup>: n=422 cells for "fresh" and n=431 cells for "IHC"; 3 brains / condition / genotype). Beeswarm plots: each dot is a nucleus. Histograms: frequency distribution of N3ICD conc° intensities among all *notch3*<sup>pos</sup> cells. X test on frequencies, ns, p-value=0.95 for TgKI<sup>n3-AG/n3-AG</sup> and 0.65 for TgBAC<sup>n3-GFP/+</sup>. F. N3ICD-AG conc° in TgKI<sup>n3-AG/+</sup> vs TgKI<sup>n3-AG/n3-AG</sup> in pallial NSPCs imaged using the same confocal parameters without or after IHC. Wilcoxon rank sum test, p-value < 2.2e-16 (TgKI<sup>n3-AG/+</sup>: n=368 cells « fresh », n= 571 cells « IHC »; TgKI<sup>n3-AG/n3-AG</sup>: n=501 cells « fresh », n=725 cells « IHC »; n=3 and 4 brains / genotype, respectively). G. Left: Nuclear N3ICD-GFP conc° in non-proliferative (Pcna<sup>neg</sup>, n=565 cells) vs proliferative (Pcna<sup>pos</sup>, n=103 cells) pallial NSPCs in TgBAC<sup>n3-GFP/+</sup> (n=3 brains). Wilcoxon rank sum test, p-value < 2.2e-16. Right: N3ICD-GFP conc° (Z-score values) in "RFP<sup>neg</sup> and RFP<sup>low</sup>" cells (n=55 cells), compared to values in RFP<sup>pos</sup> cells (n=782 cells), allow to set a range of intensities for background noise (from 1 to 3 standard deviations under the mean) (n=3 brains from TgBAC<sup>n3-GFP/+</sup> fish, segmentation on Sox2<sup>pos</sup> nuclei). H, I. TgBAC<sup>n3-GFP/+</sup> fish were treated with 10 mM LY411575 (LY) or carrier (DMSO) for 24h. H. Whole-mount brains (3 per condition) imaged « fresh » using the same confocal parameters (scale bars: 20µm). Membrane accumulation of GFP signal at the membrane can be seen in LY samples. I. Quantification of nuclear N3ICD-GFP conc° (segmentation on nlsRFP signal) (DMSO: n=474 cells, LY: n=383 cells). Wilcoxon rank sum test, p-value < 2.2e-16. J, K. Evaluation of ligand dose-dependency in Notch3 signaling. TgBAC<sup>n3-GFP/+</sup> fish were combined with *notch3*<sup>fl332</sup> mutants to generate TgBAC<sup>n3-GFP/+</sup> fish with 1, 2 or 3 functional *notch3* copies. J. Theoretical predictions on the amount of nuclear N3ICD-GFP for each genotype. Top: limiting ligands: signaling (total amount of N3ICD, tagged or not) is constant while the absolute nuclear level of N3ICD-GFP, and its ratio over total nuclear N3ICD (N3ICD-GFP/[N3ICD + N3ICD-GFP]), decrease as *notch3* copies increase (bar codes on the right). Bottom: excess ligands: signaling increases as *notch3* copies increase, while nuclear N3ICD-GFP levels remain constant and the ratio of N3ICD-GFP over total nuclear N3ICD decreases. Ligands, Notch3 (N3), Notch3-GFP (N3-GFP) and Notch3<sup>fl332</sup> (N3<sup>fl332</sup>) proteins are color-coded; mb: cell plasma membrane, nuc: nucleus. K. Nuclear N3ICD-GFP levels ("N3ICD-GFP" in J, measured as conc°, i.e. normalized over nuclear volume) in the three genotypes (1 copy: n=1344 cells, 4 brains; 2 copies: n=544 cells, 3 brains; 3 copies: n=751 cells, 3 brains; segmentation performed on Sox2 expression) (Welch's ANOVA test, p-value < 2.2e-16).

**Figure 3.** N3ICD levels follow a non-random spatial distribution pattern across the NSC population. A, B. TgKI<sup>n3-AG/+</sup> adult pallia were processed for IHC for AG, Zo1 and Sox2 to classify cells according to their nuclear N3ICD-AG levels. A. Beeswarm plot showing the distribution of N3ICD-AG levels in two pallial hemispheres and its subdivision into cells of high (top 20% quantile, yellow), low (bottom 20% quantile, purple) and intermediate (green) Notch3 signaling. B. Same color code shown on the corresponding in situ images showing Sox2<sup>pos</sup> segmented nuclei (left: with Zo1; right: without). Scale bars: 20µm. C. Superposed neighborhoods of all N3ICD<sup>low</sup> cells. For each cell in this group, all the cells surrounding it at a maximal radius of 10µm are plotted at their position with respect to the original cell, color-coded as in A, B. D. Deviation from marked L-functions from an
uncorrelated model as a function of the distance (radius in micrometers, x axis) from cells of low N3ICD values (scheme on left). Identity of the mark from top to bottom: N3ICD conc° (i.e., sum normalized to nuclear volume) per nucleus, N3ICD sum (absolute value) per nucleus, apical area. n=401 cells from 2 hemispheres (173 + 228 cells) from 1 brain. Dashed lines: hypothesis rejection threshold, with significant deviations in red. p-values: N3ICD conc°: 0.01; N3ICD sum: 0.06; apical area: 0.89.

**Figure 4.** *jag1b* and *dla* are differentially expressed in relation with N3ICD levels along quiescence and lineage progression. A. Expression of *dla* and *jag1b* (RNAscope, green and magenta dots, respectively) on a whole-mount WT adult brain, with IHC for Z01 and Pcna (white). Some cells express only one ligand (green and magenta arrows), others show co-expression (white arrow). B,C. Quantification of ligand transcripts (number of dots) in each cell of (A) (n= 268 cells, 1 brain; Spearman's ρ=-0.12). D,E. Quantification of *dla* (Wilcoxon rank sum test, p-value < 2.2e-16) and *jag1b* (Wilcoxon rank sum test, p-value=0.0004) transcripts in proliferating (Pcna<sup>pos</sup>, n=229 cells) versus non-proliferating (Pcna<sup>neg</sup>, n=40 cells) cells of (A). F-I. RNAscope for *jag1b* (F) or *dla* (H) on whole-mount TgBAC<sup>n3-GFP/+</sup> brains, with IHC for Notch3-GFP, Sox2, Z01 and Pcna (color-coded, left panels). Nuclei are segmented on Sox2 staining (top right panels – color-code for N3ICD pixel sum intensity) and *jag1b* or *dla* dots are counted and manually assigned to each cell (bottom right panels, each color = one cell) for correlation analyses of N3ICD levels and *jag1b* (G) or *dla* (I). *jag1b* expression and N3ICD intensity in individual cells (G) show a correlation that plateaus after 10 dots (2 brains, 234 cells; Spearman's ρ=0.45); cells with high *dla* expression show low Notch3 activity (I) (2 brains, 244 cells; Spearman's ρ=0.01). J. Tg(*dla*:gfp);TgKI<sup>n3-AG/+</sup> brain with IHC for GFP, AG, Z01, Pcna and nlsRFP (color-coded). Left panel: whole-mount confocal view; right panel: segmented nlsRFP<sup>pos</sup> nuclei with N3ICD levels color-coded. K. Corresponding quantification of N3ICD in GFP<sup>pos</sup> vs GFP<sup>neg</sup> cells (Z-score of N3ICD pixel sum; 3 brains; n=416 and 67 cells, respectively). Wilcoxon rank sum test, p-value=3.045e-15. L. Quantification of N3ICD in *dla*<sup>pos</sup> cells relative to GFP (low, medium or high, color-coded) (n=3, 164 and 28 cells, respectively, 3 brains; Welch's ANOVA test on *dla*:GFP levels groups, p-value=0.058; Wilcoxon test on medium vs high *dla*:GFP levels: p-value=0.002). M,N. Corresponding quantification of N3ICD in GFP<sup>neg</sup> (M) vs GFP<sup>pos</sup> (N) cells relative to apical area (Z-score of N3ICD pixel sum; 3 brains; n=470 and 196 cells, respectively; Spearman's ρ=0.45 and 0.66, respectively). All scale bars: 10µm.

**Figure 5.** Jag1b and Notch3 positively interact and maintain the progenitor state. A. Expression of *dla* (top) and *jag1b* (bottom) (RNAscope, magenta dots), with IHC for Z01 and Pcna, Notch3-GFP and Sox2 (color-coded), in whole-mount TgBAC<sup>n3-GFP/+</sup> pallia after treatment with 10 mM LY-411575 or carrier (DMSO) for 24h. B-D. Corresponding quantifications in DMSO- vs LY-treated fish (8 hemispheres from 4 and 5 brains, respectively): percentage of Pcna<sup>pos</sup> cells (B) (Wilcoxon rank sum test, p-value=0.38), *jag1b* expression (C) (Wilcoxon rank sum test, p-value=0.002) and *dla* expression (D) (Wilcoxon rank sum test, p-value=0.001). In C and D, measurements are at population scale (total number of dots / number of apical surfaces). E. TgKI<sup>n3-AG/+</sup> fish were injected with a "vivo morpholino" against *jag1b* (*jag1b* MO) or a control *vivo*MO (*ctrl*MO) and analyzed after a one-day chase. F. Whole-mount confocal views of pallia from *jag1b*MO- or *ctrl*MO-injected fish immunostained for Notch3-AG, Sox2, Z01 and Pcna (color-coded). Left panels: whole-mount confocal views; right panels: nuclei segmented on the Sox2 signal and color-coded for their N3ICD-AG conc° (identical color scale in both conditions). G. Distribution of N3ICD-AG conc° in each cell of *ctrl*MO- vs *jag1b*MO-injected brains (n= 876 and 637 cells, respectively, in 6 hemispheres from 3 brains for each condition) (Wilcoxon rank sum test, p-value < 2.2e-16). Color range as in F. H,I. Proportion of proliferating (Pcna<sup>pos</sup>) cells (H - Wilcoxon rank sum test, p-value=0.11, ns) and Sox2 intensity (normalized to nuclear volume) (I - Wilcoxon rank sum test, p-value<2.2e-16) among Sox2<sup>pos</sup> cells in *ctrl*MO- vs *jag1b*MO-injected brains. J. Top: Whole-mount confocal views of pallia from *jag1b*MO- or *ctrl*MO-injected fish, showing expression of *dla* (RNAscope, magenta dots) and Z01 (IHC, white). Bottom: coloring, on the same
images, of the apical surfaces of the 10% cells with highest *dla* expression (pink) (over a total of n= 280 and 278 cells for *ctrlMO* and *jag1bMO* respectively). K. Number of *dla* dots per cell, in all cells (left) or in cells of large (middle) or small (right) apical surface area (aa) (60 and 71 cells of aa>130µm² and 57 and 45 cells of aa<30µm², respectively, in 2 hemispheres from 1 brain for each condition) (Wilcoxon rank sum tests: all cells: p-value < 2.2e-16, aa>130µm² p-value= 7.51e-10, aa<30µm² p-value=0.007)). Scale bars: A,F: 20µm; J: 10µm.

624

625 **Figure 6.** Downregulation of *Jag1b* preserves the spatial structure of the *Notch3* signaling pattern.

626 A. TgKI<sup>n3-AG/+</sup> fish were injected with *jag1bMO* or *ctrlMO*. After 1 day, nuclear N3ICD-AG levels were quantified and cell position recorded. B. Superposed neighborhoods of all N3ICD<sup>low</sup> cells, as in Fig.3C.

627 C. Deviation from marked L-functions from an uncorrelated model as a function of the distance from cells of low N3ICD values (as in Fig.3D, color-coded per condition: black: *ctrlMO*, gray: *jag1bMO*).

628 Identity of the mark from top to bottom: N3ICD conc° per nucleus, N3ICD sum per nucleus. *ctrlMO*: n=876 cells from 3 brains (6 hemispheres); *jag1bMO*: n=256 cells from 1 brain (2 hemispheres). Dashed lines: hypothesis rejection thresholds (color-coded as above), with significant deviations in red. All p-values <0.01.

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STAR Methods

636

KEY RESOURCES TABLE

<table>
  <thead>
    <tr>
      <th>REAGENT or RESOURCE</th>
      <th>SOURCE</th>
      <th>IDENTIFIER</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td><strong>Antibodies</strong></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Mouse monoclonal anti-Zrf1 (1:200)</td>
      <td>ZIRC</td>
      <td>zfin.org/ZDB-ATB-081002-46</td>
    </tr>
    <tr>
      <td>Chicken polyclonal anti-GFP (1:500)</td>
      <td>Aves Labs</td>
      <td>Cat#GFP-1020; RRID:AB_10000240</td>
    </tr>
    <tr>
      <td>Rat polyclonal anti-RFP (Chromotek clone 5f8-150) (1:250)</td>
      <td>ThermoFisher Scientific</td>
      <td>Cat#17609716; RRID:AB_2336064</td>
    </tr>
    <tr>
      <td>Rabbit polyclonal anti-Sox2 (1:250)</td>
      <td>Abcam</td>
      <td>Cat#ab97959; RRID:AB_2341193</td>
    </tr>
    <tr>
      <td>Mouse monoclonal anti-PCNA (1:250)</td>
      <td>Santa Cruz Biotechnology</td>
      <td>Cat# sc-56; RRID:AB_628110</td>
    </tr>
    <tr>
      <td>Mouse monoclonal anti-AzamiGreen (1:250)</td>
      <td>Clinisciences</td>
      <td>Cat#M102-3M; RRID:AB_590583</td>
    </tr>
    <tr>
      <td>Rabbit polyclonal anti-AzamiGreen (1:250)</td>
      <td>Clinisciences</td>
      <td>Cat#PM052M; RRID:AB_2827721</td>
    </tr>
    <tr>
      <td>Rat monoclonal anti-BrdU (1:200)</td>
      <td>Abcam</td>
      <td>Cat#ab6326; RRID:AB_305426</td>
    </tr>
    <tr>
      <td>Mouse monoclonal anti-HuC/D (clone 16A11) (1:250)</td>
      <td>ThermoFisher Scientific</td>
      <td>Cat#A-21271; RRID:AB_221448</td>
    </tr>
    <tr>
      <td>Rabbit polyclonal anti-Blbp (1:1000)</td>
      <td>Merck</td>
      <td>Cat#ABN14; RRID:AB_11210770</td>
    </tr>
    <tr>
      <td>Mouse monoclonal anti-ZO1, Alexa Fluor 647 (clone ZO1-1A12) (1:250)</td>
      <td>ThermoFisher Scientific</td>
      <td>Cat#MA3-39100-A647; RRID:AB_2663167</td>
    </tr>
    <tr>
      <td>Mouse monoclonal anti-ZO1, Unconjugated (clone ZO1-1A12) (1:250)</td>
      <td>ThermoFisher Scientific</td>
      <td>Cat# 33-9100; RRID:AB_87181</td>
    </tr>
  </tbody>
</table>
<table><tr><td>Mouse monoclonal anti-ZO1, Alexa Fluor 488 (clone ZO1-1A12) (1:250)</td><td>ThermoFisher Scientific</td><td>Cat# MA3-39100-A488; RRID:AB_2663169</td></tr><tr><td>Rabbit polyclonal anti-Rab5 (S-19) (1:100)</td><td>Santa Cruz Biotechnology</td><td>Cat#sc-309; RRID:AB_632295</td></tr><tr><td>Mouse monoclonal anti-Rab7 (1:100)</td><td>Merck</td><td>Cat#R8779; RRID:AB_609910</td></tr><tr><td>Rabbit polyclonal anti-Rab11 (1:200)</td><td>Gentex</td><td>Cat#GTX127328; RRID:AB_11171170</td></tr><tr><td>Rabbit polyclonal anti-Lamp1 (1:100)</td><td>Abcam</td><td>Cat#ab24170; RRID:AB_775978</td></tr><tr><td>Goat Anti-Chicken IgG(H+L), Alexa Fluor 488 Conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat#A-11039; RRID:AB_142924</td></tr><tr><td>Goat anti-Mouse IgG1, Alexa Fluor 488 Conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat# A-21121; RRID:AB_2535764</td></tr><tr><td>Goat anti-Mouse IgG1, Alexa Fluor 647 Conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat# A-21240; RRID:AB_2535809</td></tr><tr><td>Goat anti-Mouse IgG2a, Alexa Fluor 488 conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat#A-21131; RRID:AB_2535771</td></tr><tr><td>Goat anti-Mouse IgG2a, Alexa Fluor 633 conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat#A-21136; RRID:AB_2535775</td></tr><tr><td>Goat anti-Mouse IgG2b, Alexa Fluor 546 conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat# A-21143; RRID:AB_2535779</td></tr><tr><td>Goat Anti-Rabbit IgG(H+L), Alexa Fluor 405 Conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat#A-31556; RRID:AB_221605</td></tr><tr><td>Goat Anti-Rabbit IgG(H+L), Alexa Fluor 488 Conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat#A-11008; RRID:AB_143165</td></tr><tr><td>Goat Anti-Rabbit IgG(H+L), Alexa Fluor 546 Conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat#A-11010; RRID:AB_2534077</td></tr><tr><td>Goat anti-Rat IgG(H+L), Alexa Fluor 546 Conjugated (1:1000)</td><td>ThermoFisher Scientific</td><td>Cat#A-11081; RRID:AB_2534125</td></tr></table>

Chemicals, peptides, and recombinant proteins

<table><tr><td>LY-411575</td><td>Merck</td><td>Cat#SML0506</td></tr><tr><td>5-Bromo-2-deoxyuridine (BrdU)</td><td>Merck</td><td>Cat#B5002</td></tr><tr><td>Paraformaldehyde Aqueous Solution 16%</td><td>ThermoFisher Scientific (Electron Microscopy Sciences)</td><td>Cat#15710</td></tr><tr><td>Antigen Retrevial (HistoVT One)</td><td>Nacalai Tesque</td><td>Cat#06380-05</td></tr><tr><td>PBS 10X</td><td>ThermoFisher Scientific</td><td>Cat#10649743</td></tr><tr><td>Goat Serum Donor Herd</td><td>Merck</td><td>Cat#G6767</td></tr><tr><td>TWEEN 20</td><td>Merck</td><td>Cat#P9416</td></tr><tr><td>Triton-X100</td><td>Merck</td><td>Cat#X100</td></tr></table>
<table><tr><td>Natural mouse serum</td><td>Abcam</td><td>Cat#ab7486</td></tr><tr><td>Petrolatum Jelly</td><td>Merck</td><td>Cat#16415</td></tr><tr><td>Lanolin</td><td>Merck</td><td>Cat#L7387</td></tr><tr><td>Paraffin wax</td><td>Merck</td><td>Cat# 76242</td></tr><tr><td>Aqua-Poly/Mount</td><td>Polysciences</td><td>Cat#18606</td></tr><tr><td>DIG RNA Labeling Mix</td><td>Merck</td><td>Cat#11277073910</td></tr><tr><td>T3 RNA polymerase</td><td>Promega</td><td>Cat#P2083</td></tr><tr><td>T7 RNA polymerase</td><td>Promega</td><td>Cat#P2075</td></tr><tr><td>Cas9 nuclease</td><td>Toolgen (Labomics)</td><td>Cat#Cas9-TOO-250</td></tr><tr><td>Green GoTaq G2 Hot Start Master Mix</td><td>Promega</td><td>Cat#M7422</td></tr><tr><td>DNase I recombinant</td><td>Merck</td><td>Cat#4716728001</td></tr><tr><td>RNase inhibitor</td><td>Takara Bio</td><td>Cat#2313A</td></tr><tr><td>Proteinase K recombinant</td><td>ThermoFisher Scientific</td><td>Cat#EO0491</td></tr><tr><td>T7 Endonuclease I</td><td>NEB</td><td>Cat#M0302S</td></tr><tr><td>Anti-Digoxygen-AP, Fab fragments</td><td>Merck</td><td>Cat#11093274910</td></tr><tr><td>Hydrogen peroxide solution 30 %</td><td>Merck</td><td>Cat#216763</td></tr><tr><td>Formamide</td><td>Merck</td><td>Cat#47671</td></tr><tr><td>Invitrogen SSC UltraPure, 20X</td><td>ThermoFisher Scientific</td><td>Cat#10515203</td></tr><tr><td>MS-222</td><td>ThermoFisher Scientific</td><td>Cat#A5040</td></tr><tr><td>DAPI</td><td>Merck</td><td>Cat#d8417</td></tr></table>

## Critical commercial assays

<table><tr><td>Phire Animal Tissue Direct PCR Kit</td><td>ThermoFisher Scientific</td><td>Cat#F140WH</td></tr><tr><td>StrataClone PCR Cloning Kit</td><td>Agilent Technologies</td><td>Cat#240205</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>Cat#28704</td></tr><tr><td>ProbeQuant G-50 Micro Columns</td><td>Merck</td><td>Cat#GE28-9034-08</td></tr><tr><td>RNAScope Multiplex Fluorescent Reagent Kit v2</td><td>Bio-Techne</td><td>Cat#323100</td></tr><tr><td>RNAScope Hiplex probe diluent</td><td>Bio-Techne</td><td>Cat#324301</td></tr><tr><td>OPAL 480 reagent pack</td><td>Akoya Biosciences</td><td>Cat#FP1500001KT</td></tr><tr><td>OPAL 570 reagent pack</td><td>Akoya Biosciences</td><td>Cat#FP1488001KT</td></tr><tr><td>OPAL 650 reagent pack</td><td>Akoya Biosciences</td><td>Cat#FP1496001KT</td></tr></table>

## Experimental models: Organisms/strains

<table><tr><td>D. rerio / AB WT strain</td><td>N/A</td><td>ZDB-GENO-960809-7</td></tr></table>
<table><tr><td>D. rerio / Casper (roy-/-;nacre-/-)</td><td>Zebrafish International ResourceCenter, Eugene, OR</td><td>ZDB-FISH-150901-6638</td></tr><tr><td>D. rerio / TgBAC(notch3:notch3-GFP-P2A-nlsRFP)<sup>ip13Tg</sup></td><td>This study</td><td>ZDB-ALT-230810-3</td></tr><tr><td>D. rerio / TgKI(notch3:notch3-mAG-P2A-nlsRFP)<sup>ip14Tg</sup></td><td>This study</td><td>ZDB-ALT-230814-2</td></tr><tr><td>D. rerio / Tg(dla:GFP)</td><td>Zebrafish International ResourceCenter, Eugene, OR</td><td>ZDB-TGCONSTRCT-100823-1</td></tr></table>

## Oligonucleotides

<table><tr><td>vivo-Morpholino Standard Control Oligo</td><td>GeneTools</td><td>https://store.gene-tools.com/node/334</td></tr><tr><td>MO1-jag1b</td><td>GeneTools</td><td>https://zfin.org/ZDB-MRPHLNO-041207-3</td></tr><tr><td>Alt-R CRISPR-Cas9 crRNA</td><td>IDT</td><td>Custom</td></tr><tr><td>Alt-R CRISPR-Cas9 tracrRNA</td><td>IDT</td><td>Cat#1072533</td></tr><tr><td>Dr-dla-C3 RNAscope probe (targeting 1568-2518 of NM_130954.2)</td><td>Bio-Techne</td><td>Ref#1133701-C3</td></tr><tr><td>Dr-jag1a-C2 RNAscope probe (targeting 1299-2372 of NM_131861.1)</td><td>Bio-Techne</td><td>Ref#1189971-C2</td></tr><tr><td>Dr-jag1b-C2 RNAscope probe (targeting 1129-2256 of NM_131863.2)</td><td>Bio-Techne</td><td>Ref#1189981-C2</td></tr><tr><td>Dr-jag2b-C1 RNAscope probe (targeting 3011-3955 of NM_131862.1)</td><td>Bio-Techne</td><td>Ref#1039801-C1</td></tr><tr><td>Dr-hey1-C1 RNAscope probe (targeting 2-1091 of the sequence NM_212561.1)</td><td>Bio-Techne</td><td>Ref#1234851-C1</td></tr></table>

## Recombinant DNA

<table><tr><td>BAC CH211-69E21</td><td>Zfin</td><td>ZDB-BAC-050218-541</td></tr><tr><td>Plasmid pCS2_GFP-P2A-nlsRFP</td><td>This study</td><td>N/A</td></tr><tr><td>Plasmid pCS2_AzamiGreen-P2A-nlsRFP</td><td>This study</td><td>N/A</td></tr><tr><td>pmAG1-MN1 (CoralHue monomeric AzamiGreen)</td><td>MBL</td><td>Cat#AM-V0033</td></tr></table>

## Software and algorithms

<table><tr><td>ZEN software</td><td>Carl Zeiss</td><td>RRID:SCR_018163 (Black edition); RRID:SCR_013672 (Blue edition)</td></tr><tr><td>Imaris</td><td>Bitplane</td><td>https://www.imaris.oxinst.com/packages; RRID: SCR_007370</td></tr></table>
<table><tr><td>Fiji</td><td>Fiji</td><td>https://fiji.sc; SCR_002285</td><td>RRID:</td></tr><tr><td>R Project for Statistical Computing</td><td>Posit Software</td><td>RRID:SCR_001905</td><td></td></tr><tr><td>Napari 0.4.18 (under Python 3.9)</td><td>In-house code based on Epyseg <sup>81</sup> and Big-FISH <sup>82</sup></td><td>RRID:SCR_022765</td><td></td></tr></table>

638

639 EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

640 **Zebrafish**

641 All the zebrafish (*Danio rerio*) used in this study have AB or Casper<sup>83</sup> (*roy-/-;nacre-/-*) genetic background. They are kept at 28.5°C in a pH 7.4 water, on a cycle of 14 hours light and 10 hours dark, in 3.5L tanks at a maximal density of 5 fish per liter. Feeding is performed three times a day with rotifers until 14 days post-fertilization and with standard commercial dry food (GEMMA Micro from Skretting) afterwards. Embryos and larvae were staged and raised according to<sup>84</sup>: developmental stage is indicated for each experiment. Adult fish were between 3 and 4 months old. Fish from the same parents were used in a same experiment. Both males and females were included in the analysis since no difference in brain development and NSCs homeostasis was observed in the past. Fish were euthanized in ice-cold water (temperature comprised between 1° and 2°C) for 10 minutes, according to a special dispensation and following the guidelines of the Ministry of superior education, research and innovation.

652 The animal study protocol was approved by the Ethics Committee n°39 of Institut Pasteur (authorization #36936, April 26<sup>th</sup>, 2022) and DDPP-2021-021 of the Direction Départementale de la Protection des Populations de Paris.

655 The two transgenic lines generated for this study are detailed in the next section. Tg(dla:GFP)<sup>45</sup> and notch3<sup>fl332/+</sup> line<sup>24</sup> were used for crossings.

657 **METHOD DETAILS**

658 **Zebrafish TgBAC(notch3:notch3-GFP-P2A-nlsRFP)<sup>ip131g</sup> line generation**

659 Genomic zebrafish BAC CH211-69E21 was modified to insert a "Tol2L-AmpR-Tol2R-polyA-βcrystallin:eCFP" cassette at the loxP511 locus to enable transposase-mediated recombination and selection via the βcrystallin promoter. The BAC was further modified by homologous recombination to insert "linker-GFP-P2A-nlsRFP" sequence in position +6823 of notch3 coding sequence, in frame with the exon33. The construct "linker-GFP-P2A-nlsRFP" was obtained using the NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat#E5520S) to assemble a short linker (translating into "GVG" peptide), a GFP sequence (cloned by PCR from the plasmid p3E-IRES-EGFPpA - Tol2kit<sup>85</sup> #389), a ribosome-skipping P2A sequence<sup>86</sup> and a nlsRFP sequence (gift from Holley SA). This construct was then recloned using KpnI and SacI restriction sites into a modified pBluescript vector, along with two sequences in 5' and 3', homologous to the insertion site on notch3 sequence, to increase recombination efficiency. A Zeocin cassette oriented 3'-5' was inserted downstream the stop codon of nlsRFP, to facilitate selection of recombined clones. Recombined clones were selected, verified by PCR and sequencing of the insertion sites, and by EcoRI restriction profile (Agate bioservices, Bagard, France - see Fig.S1C for scheme).

673 The TgBAC(notch3:notch3-GFP-P2A-nlsRFP)<sup>ip13Tg</sup> line was obtained after microinjection of approximately 1 nl in one-cell stage zebrafish embryos of a solution containing the modified BAC (80 ng/ml) together with transposase capped RNA (40 ng/ml). Founder adults were screened by backcrossing in wild-type lines and monitoring of CFP expression in the eyes of F1 progeny. The TgBAC(notch3:notch3-GFP-P2A-nlsRFP)<sup>ip13Tg</sup> line was maintained in Casper double mutant
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679

(roy⁻/⁻; nacre⁻/⁻) background. Fish carrying the transgene were routinely selected at 48hpf based on fluorescence expression.

680

**Zebrafish TgKI(notch3:notch3-mAG-P2A-nlsRFP)ip14Tg generation**

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Cas9 target sequences were identified using CRISPOR tool⁸⁷,⁸⁸ for genome editing (see Fig.S1D). Custom crRNAs corresponding to each sequence and universal tracrRNA were ordered from IDT (Alt-R CRISPR-Cas9 system), annealed by heating equimolar amounts to 95°C for 5 minutes and then cooling to room temperature. Final guideRNA (gRNA) duplex concentration was 60 µM. Lyophilized Cas9 nuclease (Labomics, Cat#Cas9-TOO-250) was reconstituted to 30 µM in high-salt buffer⁸⁹ (750 mM KCl, 50% glycerol).

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Ribonucleoprotein complex (RNP) was assembled on injection day by mixing Cas9 (2 µL, 30 µM), gRNA duplex (1 µL, 60 µM), and nuclease-free water (2 µL). Complexes were incubated at 37°C for 5 minutes, then kept on ice. To test gRNAs efficiency, 1nl of RNP complex was injected into fertilized zebrafish eggs at the one-cell stage. At 48hpf, genomic DNA was extracted from 20 embryos per condition using Phire Animal Tissue Direct PCR Kit (ThermoFisher scientific, Cat#F140WH). Target region on *notch3* gene was PCR-amplified (forward primer: 5'-GGTGCACAGCAGTATCCTG-3' and reverse primer: 5'-GCGGATACCGGCAGATGG-3') and cutting efficiency assessed using T7 endonuclease I assay according to manufacturer's instructions (NEB, Cat#M0302S) (see Fig.S1E). gRNA4 sequence (5'-GGGGTAATCCTCTGGGCCTGCGG-3') was selected to generate the knock-in line.

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710

A donor vector was designed to insert a 1530 bp "linker-AzamiGreen-P2A-nlsRFP" cassette in frame with the exon 33 of the *notch3* gene. P2A and *nlsRFP* sequences were obtained as above; CoralHue monomeric *AzamiGreen* sequence was cloned by PCR from pmAG1-MN1 plasmid (MBL, Cat#AM-V0033). The pCS2P backbone was modified by PCR to remove the CMV promoter. Long homology arms (around 900 bp for the left arm and 800 bp for the right arm) were designed starting from the Cas9 genomic cutting site specific for gRNA4, to enhance recombination frequency. Homology arms were synthesized as gBlocks (IDT) and contained silent mutations in the Cas9 recognition sequence to prevent re-cutting after integration. Cas9 target sites specific for gRNA4 were positioned flanking both homology arms in opposite orientations to allow simultaneous linearization of donor and genomic DNA with the same guideRNA. A 17 amino acid linker sequence was included between Notch3 and AzamiGreen to prevent folding interference⁹⁰. The four fragments (modified vector backbone, left homology arm, "linker-AzamiGreen-P2A-nlsRFP" insert, right homology arm) were assembled at equimolar ratios (0.05 pmol each) using NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat#E5520S).

711
712
713
714
715
716
717
718
719
720
721
722
723
724
725

On injection day, ribonucleoprotein complex was assembled and injected as above, with donor vector (2 µL, 500 ng/µL) replacing water. At 48hpf, individual embryos were screened for successful integration efficiency (see Fig. S1F). Genomic DNA extraction was performed using Phire Animal Tissue Direct PCR Kit (ThermoFisher scientific, Cat#F140WH). Target regions spanning the integration sites were PCR-amplified (for 5' integration: forward primer: 5'-CATGGACAGACTCCCTCGAG-3' and reverse primer: 5'-GGTGCGCCTTCAGTGACG-3'; for 3' integration: forward primer: 5'-GCCTACAAGACCGACATCAAG-3' and reverse primer: 5'-CAGCTCTGGCCTTTGTAAGTC-3'), purified, A-tailed, and TA-cloned (StrataClone PCR cloning kit, Agilent Technologies, Cat# 240205) for sequence verification to confirm precise integration and absence of indels. Fish batches showing correct integrations were screened at 3mpf for germline transmission: this involved fluorescence observation of offspring between 48hpf and 72hpf, followed by sequencing validation of integration sites at 3mpf using tail-extracted DNA and the methodology described above. Once established, the TgKI(notch3:notch3-mAG-P2A-nlsRFP)ip14Tg was maintained in Casper double mutant (roy⁻/⁻; nacre⁻/⁻) background. Fish carrying the transgene were routinely selected at 48hpf based on fluorescence expression.
726 **Genotyping**

727 Adult fish genotyping was performed using tail samples. Before tissue collection, fish were
728 anesthetized by immersion for 1 to 2 minutes in water containing 0.01% MS222 (Sigma-Aldrich, Cat#
729 A5040). Tail clips were collected and DNA was extracted using the Phire Animal Tissue Direct PCR
730 Kit (ThermoFisher scientific, Cat#F140WH) according to manufacturer's instructions.

731 TgKI<sup>n3-AG/+</sup> versus TgKI<sup>n3-AG/n3-AG</sup> genotyping was performed using primers positioned outside the
732 insertion site (forward primer: 5'-CATGGACAGACTCCCTCGAG-3' and reverse primer: 5'-
733 GCGGATACCGGCAGATGG-3'). GoTaq- mediated PCR (GoTaq G2 Hot Start Master Mix - Promega,
734 Cat#M7422) was conducted on a pre-heated thermocycler according to the following protocol: 95°C x
735 2', [95°C x 30" - 54°C x 30" - 72°C x 3'] repeated for 35 cycles, 72°C x 5'. PCR products were analyzed
736 by electrophoresis on 1% agarose gel to discriminate the alleles according to their size: 1.5 Kb for the
737 WT allele and 3 Kb for the knocked-in allele.

738 TgBAC<sup>n3-GFP/+</sup> crossed with <i>notch3</i><sup>fh332/+</sup> offspring genotyping was performed as previously published<sup>24</sup>,
739 on fish selected for BAC expression at embryonic stage. After PCR product digestion and
740 electrophoresis on 3% agarose gel, different genotypes were discriminated according to band
741 intensity: presence of only upper band indicated 3 functional <i>notch3</i> copies, two bands of the same
742 intensity indicate 2 copies of <i>notch3</i><sup>fh332</sup> and 1 <i>notch3</i> WT copy. An upper band of high intensity and a
743 fainter lower band indicated 1 copy of <i>notch3</i><sup>fh332</sup> and 2 WT <i>notch3</i> copies.

744 **Chromogenic in-situ hybridization (ISH)**

745 DIG-labeled probes for *notch3*<sup>23</sup>, *gfp* (obtained from an in-house plasmid) and *AzamiGreen* (PCR cloned
746 from the KI donor vector described above [forward primer: 5'-
747 ATGGGTGAGTGTGATTAAACCAGAG-3' and reverse primer: 5'-CTTGGCCTGACTCGGCAGC-3']
748 and inserted into the pSCA vector using the StrataClone PCR cloning kit) were produced incubating 5
749 µL of linearized DNA (1 µg), 2 µL DIG-NTP labeling mix (Merck, Cat#11277073910), 1 µL RNase
750 inhibitor (Takara Bio, Cat#2313A), 4 µL 5X transcription buffer, 2 µL RNA polymerase (T3 or T7,
751 Promega, Cat#P2083/2075), and 6 µL nuclease-free water at 37°C for 3 hours. Following transcription,
752 1 µL DNase I (Merck, Cat#4716728001) was added and incubated for 15 minutes at 37°C to remove
753 template DNA. The probe was purified using G-50 Micro Columns (Merck, Cat#GE28-9034-08).

754 28hpf embryos from WT, TgKI<sup>n3-AG/+</sup> or TgBAC<sup>n3-GFP/+</sup> lines (sorted based on fluorescence) were
755 dechorionated using pronase and fixed overnight in 4% PFA (Paraformaldehyde, ThermoFisher
756 Scientific, Cat#15710) at 4°C, then dehydrated through a graded methanol series (25%, 50%, 75%, 100%
757 in PBT) and stored at -20°C. On Day 1, samples were treated with 3% H<sub>2</sub>O<sub>2</sub> diluted in methanol for 20
758 minutes at Room Temperature (RT), rehydrated through the reverse methanol/PBT (PBS [Phosphate-
759 Buffered saline, ThermoFisher, Cat#10649743] with 0.1% TWEEN-20, Merck, Cat#P9416) series and
760 treated with proteinase K (ThermoFihser Scientific, Cat#EO0491) at 10 µg/ml for 4 minutes, then post-
761 fixed in PFA 4% for 20 minutes at RT. Hybridization and revelation of the whole embryos were
762 performed as previously published <sup>23</sup>. Another post-fixation step in PFA 4% for 20 minutes to 1 hour
763 was performed before imaging with an Axio Zoom V16 stereo microscope (Zeiss).

764 Immunohistochemistry

765 Fish between 3 and 4 months old were euthanized as described above, and whole brains were
766 dissected in cold PBS, fixed in 4% PFA at RT for 2 hours and dehydrated through sequential washes in
767 25%, 50%, 75% and 100% methanol (diluted in PBT). Brains were stored at -20°C for at least one night.
768 After rehydration in reverse methanol series, antigen retrieval was performed using HistoVT One
769 solution (Nacalai Tesque, Cat#06380-05) at 65°C for 1 hour (for anti-BrdU antibody in Fig.S3A, this
770 step was replaced by a HCl 2M treatment for 20 minutes at RT). Samples were soaked in blocking
771 solution (5 % Goat Serum Donor Herd [Merck, Cat#G6767], 0.1 % Triton-X100 [Merck, Cat#X100], 0.1
772 % DMSO [Merck, Cat#D8418] in PBS) for at least 1 hour at RT. Primary and secondary antibodies were
773 diluted in the blocking solution and incubated overnight at 4°C (except for Alexa Fluor-directly
774 conjugated anti-ZO1antibodies, that were usually incubated between 1 and 2 hours at RT). Samples
775 were washed several times in PBDT at RT after each antibody incubation. When needed, goat serum
776 was replaced with 4% of natural mouse serum (Abcam, Cat#ab7486) in the blocking solution.
777 Dissected telencephala were mounted in PBS on slides using 0.5-mm-thick holders (iSpacer, Nikon,
778 Cat#2SUN1016). The slides were sealed using Valap, a mixture of petrolatum jelly (Merck, Cat#16415),
779 paraffin wax (Merck, Cat#76242), and lanolin (Merck, Cat#L7387).

780 For embryos or larvae, brains were previously cut either in 10 mm-thick sections with a Leica CM3000
781 cryostat (for 29-30hpf embryos, beforehand included in 7.5% gelatin-15% sucrose solution and frozen
782 by immersion for 30 seconds in a bucket containing isopentane and maintained in liquid nitrogen to
783 reach a temperature between -60°C and -80°C) or in 50 mm-thick sections with a Leica VT1000 S
784 vibratome (for 8dpf larvae). Sections were then processed as above and mounted on slides sealed with
785 Aqua-Poly/Mount (Polysciences, Cat#18606).

786 RNAscope

787 The protocol for RNAscope fluorescent in situ hybridization (FISH) (Advanced Cell Diagnostics,
788 which is part of BioTechne, Cat#323100) was adapted to whole-mount fish brains⁵⁰, and all steps were
789 performed in 1.5 ml tubes. Adult brains were dissected, fixed and dehydrates as described in the
790 immunohistochemistry protocol. On Day 1, after rehydration a pigment removal step was performed
791 by incubating tissues in a water solution containing 3% H₂O₂ (Merck, Cat#216763), 5% formamide
792 (Merck, Cat#47671) and 0.5X SSC (ThermoFisher Scientific, Cat#10515203) and exposing them to direct
793 light for 10 minutes. After washing, tissues were incubated in RNAscope's proprietary diluent buffer
794 from Bio-Techne (Cat#324301) at 40°C for at least 2 hours. Primary probes diluted 1:50 in diluent
795 buffer and prewarmed for 10 minutes at 40°C were then incubated with the brains overnight at 40°C
796 with gentle shaking. On Day 2, after a series of washes and amplification steps with Amp1, Amp2 and
797 Amp3 buffers, samples were treated with the appropriate HCR reagent (-C1, -C2 or -C3 in accordance
798 with the probe used) at 40°C for 15 minutes, followed by incubation with OPAL dyes (480, 570 or 650)
799 at 40°C for 30'. The reaction was blocked with HRP-blocker at 40°C for 15 minutes. Sequential
800 detection with new HRP-dye combinations was performed if required, allowing visualization of up to
801 three different colors. To assess specificity and exclude background noise, negative control probes in
802 each channel were tested prior to experiments. When necessary, immunohistochemistry was then
803 performed on the same samples, following the protocol described above except for the antigen
804 retrieval step in HistoVT One that was omitted.

805 Image acquisition

806 Image acquisition of brains was obtained on a LSM700 or LSM710 confocal microscope (Zeiss) using a
807 40X oil objective (Plan-Apochromat 40×/1.3 Oil M27 – Effective NA between 1.3 and 1.4) and
808 processed with the ZEN software (version Blue or Black, Zeiss). For adult brains, the dorso-medial
809 part (Dm)⁹¹ was imaged for each telencephalon. Laser power (from 1% to 6%), detector gain (from 600
810 to 900) and Z-steps were optimized for each experiment and maintained constant for all the samples
811 of a same experiment. Bit depth for all the images acquired was 16 bit, and size 1024x1024 pixels
812 (except for Fig.S2D [where Rab5 was 1894x1894 pixels] and S2E [where Rab7 was 1893x1918 pixels
813 and Lamp1 was 1894x1898 pixels]). Scan zoom was set between 0.8X and 1.2X, except for Fig.S2E
814 where the scan zoom was 2.5X. 3D renderings were generated using Imaris software (versions 8 and 9,
815 Bitplane). Vertical plane images were extracted when needed.

816 Fig.S2C was recorded on a living embryo (dechorionated, anesthetized as above and maintained in
817 1.2% low melting agarose) with the LSM700 confocal microscope, focusing on one Z plane and leaving
818 the objective open for around 1 minute.
819

820

821

Fig.S2B was obtained on a living embryo (dechorionated, anesthetized as above and maintained in 1.2% low melting agarose) with a Z1 Lightsheet microscope (Zeiss), a 40x W Plan-Apochromat objective with water immersion and a numerical aperture of 1.0.

822

### BrdU pulse

823

824

825

Dechorionated embryos were put in a solution containing 10 mM BrdU (Merck, Cat#B5002), 15% DMSO and embryo medium, left on ice for 20 minutes, then transferred at 28°C for 20 minutes to recover. Embryos were euthanized, fixed and immunostained as described above.

826

### LY-411575 treatment

827

828

829

830

Inhibition of Notch signaling was performed using the LY-411575 γ-secretase inhibitor (Merck, Cat#SML0506), prepared and stored as 100 µM stock solution in DMSO and applied in the fish swimming water at a final concentration of 10 µM for 24 hours. Control fish were treated with the same final concentration of DMSO solvent.

831

### Morpholino (MO) injection

832

833

834

835

836

837

838

To selectively knock down *jag1b* in the whole NSPC population, a vivo-MO construct provided by the Gene Tools company was injected in the adult pallium *in vivo*. The ATG-directed *jag1bMO* sequence (5'-CTGAACTCCGTCGCAGAATCATGCC-3') was previously validated⁹², and a generic *controlMO* sequence (5'-CCTCTTACCTCAGTTACAATTTATA-3') was used to compare the effects. vivo-MO were injected at a final concentration of 0.125 mM directly in the cerebrospinal fluid of fish anesthetized by immersion for 1 to 2 minutes in water containing 0.01% MS222. Brains were dissected and fixed as above 1 to 3 days after injection.

839

### Identification of RBPj sites on intron 1 and 2 of *jag1b* gene

840

841

842

The intron 1-2 and 2-3 sequences of *jag1b* gene (ZDB-GENE-011128-4) were used as input to scan for transcription factor binding sites motifs in the Animal Transcription Factor Database v4.0 (https://guolab.wchscu.cn/AnimalTFDB4/#/).

843

## QUANTIFICATION AND STATISTICAL ANALYSIS

844

### Nuclear signal quantification

845

846

847

848

849

A semi-automatic image analysis pipeline was developed for this study using Imaris (Bitplane) software to quantify Notch3 activity in zebrafish transgenic lines. Individual imaging fields (100-200 cells each) were independently segmented and treated as discrete datasets to ensure homogeneity. Edge effects were minimized by excluding cells positioned at image periphery. The pipeline employed two distinct approaches for nuclear segmentation depending on the marker used.

850

851

852

853

854

855

856

857

858

859

When using nlsRFP signal for nuclear identification, a preprocessing step was required to avoid strong red background noise that could impact segmentation. This involved: using the Surfaces module on the red channel including all the strong and not specific red dots (settings: smooth enabled with surface grain size= 0.2 mm; method = background subtraction with manual threshold; diameter of the largest sphere = 0.5 mm), placing signal intensity within these surfaces to zero, and generating a new cleaned channel where these surfaces are masked. The Surfaces module was then applied again on the cleaned red channel (settings: smooth enabled with surface grain size= 0.45 mm; method= background subtraction with manual threshold; diameter of the largest sphere= 1.29 mm; seed points diameter= 3 mm, intensity based) to identify and segment 3D nuclear objects. If needed, manual correction of individual nuclei was conducted to avoid errors.

860

861

When using Sox2 signal for nuclear segmentation, the first cleaning step was unnecessary due to the high specificity and clarity of the anti-Sox2 antibody signal (settings: smooth enabled with surface
862 grain size= 0.45 mm; method = background subtraction with manual threshold; diameter of the largest
863 sphere= 1.29 mm; seed points diameter= 3 mm, intensity based).

864 After segmentation, the following quantitative parameters were extracted from each nucleus across all
865 channels: spatial coordinates (position of the center of each surface for spatial statistical analysis),
866 nuclear volume (3D volume measurement of segmented nuclei), sum (total pixel intensities within
867 each nuclear surface) and concentration (conc°) (average pixel intensity normalized by nuclear
868 volume). For Notch3 activity assessment, sum intensity represents the absolute amount of nuclear
869 Notch3 receptor, while conc° represents the concentration of Notch3 within the nucleus. Since the
870 most biologically meaningful parameter for Notch3 activity quantification remains undetermined,
871 both measurements were systematically analyzed to provide comprehensive assessment of receptor
872 nuclear levels.

873 mRNA quantification

874 mRNA dots revealed after RNAscope were quantified using Imaris (Bitplane) software. The Spots
875 module was used to identify dots in the chosen channel (settings: 0.5 mm estimated diameter,
876 background subtraction, quality above 1000), which were then manually assigned to individual cells,
877 except for Fig.5C,D and Fig.S7I, where the total number of dots on the image was divided by the
878 number of cells with an apical area.

879 Apical area and cytoplasmic signal quantification

880 Apical area was automatically calculated using Napari software (running on Python 3.9) and the
881 Fishfeat plugin (in-house code based on Epyseg⁸¹ and big-FISH⁸², which will be published elsewhere).
882 Zo1 signal was selected to identify apical membranes. Segmentation of individual cell areas was
883 corrected by hand if needed. After apical area segmentation, each cell was manually assigned to its
884 identity as determined on Imaris software, to enable correlations between Napari and Imaris
885 quantification datasets.

886 Cytoplasmic GFP signal in Fig.4L was also quantified using Napari software, after apical area
887 segmentation: the mean value of GFP on a Z thickness of 1 under the apical surface was extracted for
888 each cell, and separated in three categories (low, medium and high).

889 Statistical analysis of quantifications

890 All statistical analysis were performed using R or the Excel add-in Analysis ToolPak. All the tests used
891 are included in the figure legends, along with p-values and n (number of cells per condition, collected
892 from 1 to 5 fish, or number of hemispheres analyzed, as indicated). Experimental data are presented
893 using different visualization methods depending on the purpose: beeswarm plots and scatter
894 diagrams (individual dots representing single cells), histograms, bar plots (mean ± sd) or boxplot
895 (median and interquartile range). Z-scores (Standard scores = [(value - mean of the
896 population)/standard deviation]) were calculated when needed, to be able to normalize and achieve
897 uniform inter-samples comparisons. For correlations of Z-score values and other parameters as apical
898 area or mRNA dots (as in Figs.4G, 4I, 4M, 4N, and S7F), range similarity of apical areas or dot counts
899 across samples was confirmed prior to analysis. Shapiro-Wilk test was used to verify normality. When
900 data were not normally distributed, the variance was not homogeneous and/or the sample size was
901 too small, non-parametric tests were used: Wilcoxon rank sum test to compare two samples (as in
902 Figs.1J, 2F, 2G, 2I, 4D, 1E, 4K, S7G and Fig.5) and Spearman's rank correlation to measure statistical
903 dependence between two variables (as in Figs.4C, 4G, 4I, 4M, 4N, S6A, S6B, S7E, S7F). When
904 parametric tests were appropriate, Student's t-test was used to compare 2 samples (Fig.S3G) and
905 ANOVA or Welch's ANOVA tests were employed to compare three or more groups with (Fig. 1K) or
906 without equal variances (Figs.2K, 4L, S3D and S3E). Chi-Square test of independence (X² test) was
907 used to compare the frequency distribution across different groups. Statistical significance was
908 defined as p<0.05. Data presented with asterisks indicate significance levels: \*p<0.05, \*\*p<0.01, and
909 \*\*\*p<0.001.

910 Spatial statistics

911 Summary statistics: marked L-function

912 The main goal is to study the correlation between the spatial structure of the adult pallial ventricular
913 cells and their levels of N3ICD. We make use of second-order summary statistics which are used for
914 describing correlation patterns between points in spatial settings.

915 For this, we model this tissue as a *marked* point process: we represent the spatial positions of the nuclei
916 of the cells by the coordinates $(x_i)$, and to each point in space, there is an associated *mark* noted $(m_i)_i$
917 (for instance, N3ICD sum, N3ICD mean, or Apical Area). The mark represents any numerical value
918 that is related to a physical position.

919 To describe the correlation between marks by taking into consideration the relative position of points,
920 it is common practice to use the *marked K-function* denoted $K_m^{93}$. Adapted from Ripley's *K-function*<sup>94</sup>,
921 for any given radius $r$, this function counts the pair of points that are within a distance of $r$ weighted
922 by a function $f$ of their corresponding marks. In order to compare the *K-function* between different
923 point processes, this quantity is normalised by both the average number of points per unit squared
924 and by the expected value of the marks.

925 In our study, we focus on the marked version of Bessag's *L-function*, noted $L_m = \sqrt{K_m}/\pi$ (Figs.3D,
926 6C and S4D). This corresponds to a variance-stabilized version of $K_m$ which tends to display better
927 statistical properties.

928 To avoid edge issues when computing the empirical *L-function* $\hat{L}_m$ from our data, we add an edge
929 correction term to this quantity through translation correction as described in Section 7.4.5 in
930 Baddeley et al. <sup>95</sup>. Technical details on theoretical guarantees, statistical inference and numerical
931 computation of the $K_m$ and the $L_m$ functions can be found in <sup>96</sup> and <sup>95</sup>.

932 Benchmarking spatial randomness

933 The function $\hat{L}_m$ alone can help understanding specific spatial patterns linked to the studied marks,
934 such as clustering or repulsiveness, but its exact interpretation can be difficult without comparing it to
935 a reference model. Therefore, we decide to compare the observed function $\hat{L}_m$ to a benchmark $L_0$
936 representing a model without correlation between marks and spatial locations. Then, if for a certain
937 radius $r$, we observe that $\hat{L}_m < L_0$ (respectively $\hat{L}_m > L_0$), then this suggests that the marks of all points
938 in a radius of $r$ from a typical point in space are "lower" (resp. "higher") than expected if there was no
939 correlation between space and marks.

940 The first approach would be to define a mathematical model able to simulate the spatial location of
941 cells in the adult pallium along with theoretical N3ICD values that would be biologically close (in a
942 sense) to what would be expected of a tissue where no correlation exists between these two quantities.
943 Such a model would need to take into account the particular structure of an epithelial tissue
944 (minimum distances between cells, fully populating the space in terms of apical area) and the
945 mechanism generating the different N3ICD values of each cell. This is the first hurdle. Second, it
946 would be necessary to either have a closed-form expression of its theoretical function $L_0$ or to be able
947 to simulate such a process to obtain an empirical version of the function. In practice, this is a very
948 difficult challenge.

949 To define this benchmark model of spatial uncorrelation, we turn to permutation methods for
950 approximation. The goal is to generate an empirical version $\hat{L}_0$ of what would be this $L_0$ function using
951 only the observed data. For this, we simulate 100 "random" scenarios where the locations and marks
952 are uncorrelated. For each simulation, we compute its empirical marked *L* function, and by averaging
953 them we obtain $\hat{L}_0$.
954 It remains to define how to simulate such scenarios. Remember that we observe a set of locations ($x_i$)
955 and associated marks ($m_i$). We simulate "random" scenarios by permuting the marks associated to
956 each location as follows:

957 1. We fix the spatial locations, so the nuclei of the cells are assumed to be always in the same
958 place.

959 2. We randomly exchange the order of the marks ($m_i$)$_i$ with a permutation $\sigma$ to obtain ($m_{\sigma(i)}$)$_i$.

960 3. We then associate to each point $x_i$ its "new" mark $m_{\sigma(i)}$.

961 Note that if the observed marks truly come from this "random" scenario, then the observed pattern is
962 exchangeable with the simulated pattern.

963 Averaging 100 different permutations allows to obtain the "expected" value of the marked L function,
964 that can be then used for comparison.

965 Hypothesis testing and simulation envelopes

966 To account for the inherent randomness of any probabilistic model, we carry out a hypothesis testing
967 procedure to compare $\hat{L}_m$ and $\hat{L}_0$ and to make any interpretations. The null hypothesis we test is:

968 $\mathcal{H}_0: L_m = L_0,$

969 If $\mathcal{H}_0$ is true, this suggests that there is no correlation between the spatial structure of nuclei and their
970 corresponding N3ICD values. The chosen procedure is a deviation test, as it was first proposed in ⁹⁷. A
971 well-detailed explanation is also present in Myllymäki et al. ⁹⁸ that we summarize here:

972 1. Let $R > 0$, we compute the maximal deviation of our observation with respect to the
973 theoretical value $\hat{L}_0$:

$$u = \max_{r \in [0, R]} |\hat{L}_m(r) - \hat{L}_0(r)|.$$

974 2. For a set of $s$ simulated realisations of the benchmark model, we estimate their respective
975 marked L functions $\hat{L}_i$.

976 3. We compute the deviations:

$$u_i = \max_r |\hat{L}_i(r) - \hat{L}_0(r)|.$$

977 4. The estimated $p$-value of our test is:

$$\hat{p} = 1 - \frac{1}{s+1} \sum_{i=1}^{s} \mathbf{1}(u_i < u).$$

978 In practice, we obtained the simulations in step 2 by considering the same simulation procedure as
979 before.

980 A limitation with this deviation test is that while it allows to test hypothesis $\mathcal{H}_0$, it does not inform
981 about the specific radius at which rejection occurs, nor if the rejection is due to $L_m > L_0$ or $L_m < L_0$ or a
982 mix of both effects. For this, we add a visualisation of the global envelope tests as originally proposed in
983 Ripley ⁹⁴, which correspond to Fig. 3D, 6C, S4D. We represent the "deviation" $\hat{L}_m - \hat{L}_0$ of the observed
984 value from the expected "uncorrelated" function (as a continuous line). Then we compute the maximal
985 simulated deviation:

$$D_{max} = \max_i u_i,$$

986 and we represent the rejection thresholds $y = D_{max}$ and $y = -D_{max}$. If the "deviation" curve goes
987 above $D_{max}$ (resp. below $-D_{max}$) at a radius $r$, it suggests that the marks of all points within a radius $r$
988 of a typical point is higher (resp. lower) than expected if marks and space structure were uncorrelated.
989 This way, any rejection of hypothesis $\mathcal{H}_0$ can be interpreted as the presence of correlation between
990 cells' N3ICD values at a distance smaller or equal to $r$.

991 In our simulations, we choose $s = 99$ in order to have a confidence level of 99% on any hypothesis
992 rejection.
993 **Practical considerations**

994 In this section, we specify the different choices made for our data.

995 • For the 3 studies carried out, we have different numbers of samples available (2 hemispheres for TgKI<sup>n3AG/+</sup>, 1 hemisphere for TgBAC<sup>n3-GFP/+</sup>, 6 hemispheres for each *ctrlMO* and *jag1bMO*). As the marked *L*-functions are normalised by the local density of points and mark values, all functions can be directly compared. Therefore, to obtain the final function *Î*<sub>m</sub> of an experiment, we pool the estimations of each sample before implementing the hypothesis testing and envelope curves.

1001 • As our study focuses on describing the correlations surrounding N3ICD<sup>low</sup> cells, and as these values may be highly variable in distribution between individual fish, the groups are defined locally. The N3ICD<sup>low</sup> cells correspond to the 20% of cells with lower N3ICD activity for the given sample.

1005 Once the group N3ICD<sup>low</sup> is defined, all the marked *L*-functions described before are computed by considering as "center" points these cells. In practice, for each position *x*<sub>i</sub> of an N3ICD<sup>low</sup> cell, we compute the mark-weighted sum of **all** surrounding cell at a given distance *r*.

1009 When permuting marks to approximate the benchmark model, the marks of all points (including from N3ICD<sup>low</sup> cells) are randomly exchanged.

1011 • As mentioned in the description of the marked *K*-function, it is important to choose a function *f* that defines the *weight* of the marks. More precisely, if two points *x*<sub>*i*</sub> and *x*<sub>*j*</sub> are at a distance smaller than a studied radius *r*, their contribution to the sum is equal to *f*(*m*<sub>*i*</sub>, *m*<sub>*j*</sub>). Different choices of *f* are used in the literature, many of which are described in Section 5.3.3 in Illian et al.⁹⁶. Although we consider different options in our study, the presented results correspond to the function *f*(*m*<sub>*i*</sub>, *m*<sub>*j*</sub>) = *m*<sub>*j*</sub>. This function allows to represent the total mark value surrounding a cell at a position *x*<sub>*i*</sub> for a radius *r*.

1018 This choice is justified because we decided to focus on studying N3ICD<sup>low</sup> cells, and so we want to focus on their surrounding signal.

1020 Code availability

1021 The spatial statistics analysis was coded in Python. Computing the *K*<sub>*m*</sub> and *L*<sub>*m*</sub> functions along with the hypothesis testing procedures were entirely implemented by leveraging classic functions from pandas, NumPy and Scipy libraries and parallelisation was performed using the multiprocessing package. All codes and a Readme to compute the quantities and to generate all images in Figs.3, 6 and S4 can be found at https://github.com/migmtz/spatialN3ICD.

1026

1027

1028 References

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