Co-option of neck muscles supported the vertebrate water-to-land transition

Domain: earth_science
Published: 2024-12-04
Editorial outcome: minor_revision

Abstract
A major event in vertebrate evolution was the separation of the skull from the pectoral girdle and the acquisition of a functional neck, transitions that required profound developmental rearrangements of the musculoskeletal system. The neck is a hallmark of the tetrapod body plan and allows for complex head movements on land. While head and trunk muscles arise from distinct embryonic mesoderm populations, the origins of neck muscles remain elusive. Here, we combine comparative embryology and anatomy to reconstruct the mesodermal contribution to neck evolution. We demonstrate that head/trunk-connecting muscle groups have conserved mesodermal origins in fishes and tetrapods and that the neck evolved from muscle groups present in fishes. We propose that expansions of mesodermal populations into head and trunk domains during embryonic development underpinned the emergence and adaptation of the tetrapod neck. Our results provide evidence for the exaptation of archetypal muscle groups in ancestral fishes, which were co-opted to acquire novel functions adapted to a terrestrial lifestyle.

Introduction
The conquest of the terrestrial realm during the Carboniferous (360 MYA) represented a critical step in the evolution of vertebrates1. This major transition was accompanied by the emergence of key morphological novelties, including the transformation of fins into complex limbs and the emergence of a mobile neck, traits that are specific to tetrapods2,3. The transition from an immobile connection between the head and pectoral girdle in fishes to a free-moving head in tetrapods involved profound musculoskeletal rearrangements at the head/trunk transition during vertebrate evolution3.

A functional neck allows orientation of the head independently from the trunk towards sensory cues and it plays a critical role in efficient feeding, respiration, and vocalisation by housing the hyoid, pharynx, and larynx components4. Extant tetrapods possess a complex network of four main evolutionarily conserved neck muscle groups: (1) pharyngeal and laryngeal muscles located deep in the neck; (2) hypobranchial muscles that connect to the hyoid bone ventrally; (3) dorsal epaxial and hypaxial muscles that surround the cervical vertebrae; and (4) superficial cucullaris-derived muscles that overlay the neck musculature dorso-laterally4. Pharyngeal and laryngeal muscles are mainly involved in food intake, respiration, and vocalisation together with hypobranchial muscles, whereas dorsal epaxial and hypaxial musculature as well as cucullaris-derived muscles predominantly coordinate head mobility and locomotion5,6.

Deciphering the developmental origins of neck muscles is central to understanding the mechanisms that underpinned the emergence and adaptation of a functional neck in tetrapods. However, the embryonic origins of the different components composing the neck musculoskeletal system remained unclear, being at the interface of the head and trunk domains3,4,7–15.

Head and trunk muscles arise from different mesodermal progenitors in vertebrates, and their development is governed by distinct myogenic programs16,17. At the cranial level, a mesodermal progenitor field termed the cardiopharyngeal mesoderm (CPM) gives rise to head muscles and the second heart field17. CPM specification is initiated by the activation of a set of genes including Mesp1, Islet1, and Tbx14,16,17. Loss of Tbx1 in zebrafish and mouse embryos causes hypoplasia of branchial arch muscles and heart defects consistent with a role of Tbx1 in CPM specification4,14,18–20. Posterior to the CPM, Pax3 is expressed in the somitic mesoderm that gives rise to trunk and fin/limb muscles21–23. Pax3 mutant mice have epaxial and hypaxial (including limb) muscle defects21,23. In zebrafish, pax3 and pax7 genes are redundantly required for myogenesis of migrating muscle progenitors that form fin and ventral hypaxial muscles24. After the distinct specification of cranial and trunk muscle progenitors, both lineages activate myogenic regulatory factors (MRFs) and myofiber markers such as different myosin heavy chains (MyHC) for myogenic commitment and differentiation16.

In the neck, the somitic origin of the epaxial and hypaxial (including hypobranchial) musculature has been well documented by expression, grafting, and functional experiments11,21,23,25. More recently, laryngeal muscles were demonstrated to be of CPM origin by lineage tracing and functional analyses in the mouse4,26. In contrast, the embryological origin of cucullaris-derived muscles has remained controversial due to the location of its embryonic precursor at the interface of cardiopharyngeal, somitic, and lateral plate mesoderm populations3,4,6–15,27–29. The cucullaris is a generic term defining putative homologous muscles, innervated by the vagal nerve (cranial nerve CN X), that connect the skull to the pectoral girdle in fishes (also known as the protractor pectoralis5) and amphibians13,29. In amniotes, the cucullaris represents the embryonic anlage that gives rise to the trapezius and sternocleidomastoid muscles innervated by the accessory nerve (CN XI) derived from the vagus nerve (CN X)4,6,29. Depending on the species analysed and the experimental approach, the cucullaris derivatives were reported to originate either from CPM, somites, or trunk lateral plate mesoderm9,12–14. However, lineage and functional analyses in mouse indicate that the trapezius and sternocleidomastoid muscles originate from the CPM4,13,15.

In fishes, ontogenetic morphological analyses in teleost and non-teleost actinopterygians also suggest that the putative cucullaris homologue (protractor pectoralis) is of CPM origin5,10,30. However, the embryonic genetic origin of the fish putative cucullaris has not been assessed, despite its importance for reconstructing the ancestral neck myogenic programs in bony vertebrates (actinopterygians + sarcopterygians) and deciphering the origin the tetrapod neck.

Here, we perform transgenic lineage tracing in zebrafish embryos to map the relative contributions of the cardiopharyngeal and somitic mesoderm to the head-trunk transition. We show that muscle groups connecting the head and trunk in teleosts and tetrapods share conserved mesodermal lineage origins in the embryo. Based on this ancestral developmental blueprint of jawed vertebrates, we then use 3D high-resolution scans for comparative anatomical analyses to infer the relative contribution of cardiopharyngeal and somitic mesoderm to neck musculature in living taxa spanning the fish-tetrapod transition including ray-finned fishes, lobe-finned fishes, salamanders, and lizards. Our combined data demonstrate that neck muscle groups predated the emergence of tetrapods. We propose that archetypal muscle groups in ancestral fishes were co-opted for new functions that facilitated the adaptation to land in early tetrapods.

Results
Conserved mesodermal origins of muscle groups at the head/trunk transition
The identity of skeletal muscles is generally defined by three factors: the embryonic mesodermal origin, the innervation, and the attachments to skeletal structures. We used these criteria to determine which and how different mesodermal populations contributed to the emergence and adaptation of the neck.

We first sought to assess the homology of muscles that connect the head and trunk between zebrafish and tetrapods by studying their embryonic origins and innervation. We applied transgenic pax3a:EGFP and tbx1:creERT2 reporter zebrafish as complementary markers to map the contribution of the somitic and cardiopharyngeal mesoderm respectively, to the head/trunk-connecting muscles (HTM).

Zebrafish harbour two pax3 paralogous genes, pax3a and pax3b with differential expression within somitic mesoderm. During early development, pax3a is expressed in the neural tube and in the somites along the entire axis while pax3b is restricted to the hypaxial region of the anterior somitic mesoderm31. As observed for Pax3 in the mouse embryo, zebrafish pax3a is expressed during muscle progenitor specification, then decreased in differentiating myofibers. The transgenic pax3a-EGFP zebrafish reporter exhibits perdurance of GFP in myofibers for several days, rendering it a valuable tool for temporally monitoring the somitic lineage22. We analyzed the spatiotemporal expression of pax3a-EGFP from 1 to 5 days post-fertilization (dpf) to track HTM development until the emergence of the putative cucullaris, which is the last muscle to differentiate by activating MyHC at the head/trunk transition5 (Fig. 1a–o).

Throughout all stages analysed, we observed strong GFP labelling in the neural tube and in hypaxial muscles of migratory origin including the ventral hypaxial, the pectoral fin, and the hypobranchial sternohyoid muscles (Fig. 1, green arrowheads). In contrast, no GFP was detected in the branchial arch muscles (Fig. 1a–l), the cucullaris (Fig. 1m–o), and the coracobranchial muscles (Fig. 1p–r), the latter connecting the branchial series to the cleithrum (a part of the pectoral girdle) in fishes32 and being absent in tetrapods.

To map the muscles emerging from the tbx1-expressing CPM in zebrafish, we performed genetic lineage tracing using inducible Cre driver tbx1:creERT219,33 embryos (Fig. 2a–I, Supplementary Fig. 1). tbx1:creERT2;hsp70l:Switch embryos were induced with 4-OH-Tamoxifen (4-OHT) at shield stage (6 h post-fertilization, hpf) to 30 hpf, and the resulting hsp70l:Switch-dependent GFP-lineage labelling were analysed at 5 dpf. Consistent with previous work19, anterior lateral plate mesoderm and CPM derivatives were labelled, including cardiomyocytes (Fig. 2c, blue arrowhead) and branchial arch muscles (Fig. 2c, green arrowheads). The cucullaris myofibers showed reproducible positive tbx1:creERT2 lineage labelling, along with the posterior branchial levator and coracobranchial muscles (Fig. 2a–i, Supplementary Fig. 1, green arrowheads). In contrast, and consistent with specific CPM-focused muscle lineage labelling by tbx1:creERT2, GFP expression was not detected in adjacent somite-derived hypaxial musculature of migratory origin (Fig. 2g–i, Supplementary Fig. 1a–c, note the vhp/pfm/sh muscles). Combined with the pax3a-GFP reporter analysis, we conclude that the cucullaris muscle in zebrafish does not originate from somitic mesoderm, but originates from CPM as reported for mice4,15.

To further test this homology, we analysed the innervation pattern of these muscles in zebrafish larvae and axolotl larvae as a tetrapod reference (Fig. 2j–l, Supplementary Figs. 2, 3, Supplementary Movies 1–3). The developing cucullaris of 5 dpf zebrafish larvae resided alongside the vagus nerve (CN X) (Fig. 2j, k, Supplementary Fig. 2a–d, Supplementary Movie 1), that later connects the muscle during neuromuscular differentiation at 7 dpf, as also observed at larval stage in axolotl (Supplementary Fig. 2e–l, Supplementary Fig. 3, Supplementary Movies 2, 3). The vagus nerve also innervates the ventrally located coracobranchial muscles, while the sternohyoid muscle is innervated by the hypoglossal nerve (CN XII)3,6,29 (Fig. 2j, l, Supplementary Fig. 2, Supplementary Movie 1). Of note, the embryonic origin of fish coracobranchial muscle, also positioned at the head/trunk transition akin to the cucullaris muscle, was previously unclear; both somitic and cardiopharyngeal mesoderm origins were suggested6,32,34. Our combined lineage and innervation data are consistent with a cardiopharyngeal origin of coracobranchial muscles (Fig. 1p–r, Fig. 2g–i, l, o).

Altogether, our genetic lineage tracing and anatomical observations support the presence of a cucullaris homologue in zebrafish (Figs. 1, 2, Supplementary Figs. 1–3, Supplementary Movies 1–3). Our results indicate that HTM likely share conserved mesodermal origins among bony vertebrates, with dorsal epaxial/hypaxial and hypobranchial musculature of somitic origin and a cucullaris muscle of cardiopharyngeal origin. However, the evolutionary trajectory of neck muscles remains to be reconstructed (Fig. 2p).

Evolution of head/trunk-connecting muscles across vertebrates
The conserved embryonic origin of HTM between teleost fishes and tetrapods allowed us to infer the relative contribution of cardiopharyngeal and somitic mesoderm to HTM in extant species spanning the fish-tetrapod transition. We therefore selected key species for a comparative anatomical study to obtain a wide overview of HTM rearrangements characterising jawed vertebrate evolution. The analysis was made on one specimen for each species of interest after validation of soft tissue integrity on virtual micro-CT sections. We included the representative teleost zebrafish (Danio rerio), for which we collected data in the embryo, and the basal actinopterygian bichir (Polypterus senegalus) (Fig. 3a, b, Supplementary Figs. 4, 5). The bichir shows skull-pectoral girdle separation, uses aerial lung respiration, and can perform tetrapod-like terrestrial locomotion using its pectoral fins, making this extant fish particularly pertinent to study vertebrate terrestrialisation35. We also analysed the African coelacanth (Latimeria chalumnae) and African lungfish (Protopterus dolloi) as representatives of the two known extant lineages of lobe-finned fishes that also show skull-pectoral girdle separation (Fig. 3c, Supplementary Figs. 6, 7). In contrast to the living coelacanth that has lost the capacity of lung ventilation, the African lungfish is an obligate air-breather36. In addition, we investigated two salamander species showing a short neck with limited mobility: the paedomorphic axolotl (Ambystoma mexicanum) that does not undergo metamorphosis and is bound to an aquatic habitat, and the emperor newt (Tylototriton shanjing) that becomes terrestrial after metamorphosis (Fig. 3d, e, Supplementary Figs. 8, 9). Finally, we included the Green Anole lizard (Anolis carolinensis) as an amniote representative with a flexible long neck (Fig. 3f, Supplementary Fig. 10).

Ray-finned and lobe-finned fishes exhibited limited hypobranchial and cucullaris musculature compared to tetrapods (Fig. 3). In fishes, the hypobranchial musculature is composed of the sternohyoid and coracomandibular muscles that connect the pectoral girdle to the ceratohyal (hyoid arch) and the mandible (mandibular arch), respectively5. In agreement with a previous report5, the hypobranchial coracomandibular component was absent in zebrafish and lungfish (Fig. 3a, c). The coracobranchial and cucullaris-derived muscles were present in all fish species sampled but not in the coelacanth, as previously reported37 (Fig. 3a–c, Supplementary Figs. 4–7). We also noted that a laryngeal musculature, posterior to the branchial series and connecting the entrance of the oesophagus, was present in the bichir, coelacanth, and lungfish (Fig. 3b, c, Supplementary Figs. 5–7).

Aquatic and terrestrial salamanders showed differences in hypobranchial, laryngeal, and cucullaris-derived musculature (Fig. 3d, e, Supplementary Figs. 8, 9). In the axolotl, the shape of these muscles was akin those of lobe-finned fishes. However, the coracobranchial muscles were absent and laryngeal muscles were associated with laryngeal cartilages located anterior to the oesophagus. The metamorphic newt showed a dorso-ventral extension of the cucullaris-derived muscle onto the scapula, the laryngeal muscles and cartilages were more developed compared to the paedomorphic axolotl, and the hypobranchial musculature extended into the oral cavity to form the tongue. Changes in the musculature between paedomorphic and metamorphic salamanders was associated with a reduction of the branchial skeleton.

The lizard presented a laryngeal musculoskeletal system associated to a robust hypobranchial, including tongue, musculature. Moreover, the cucullaris derivatives had an additional attachment site ventrally and differentiated into the trapezius and sternocleidomastoid muscles, which connect the scapula and sternum to the skull, respectively (Fig. 3f, Supplementary Fig. 10)5. In mammals, the trapezius differentiates into anterior acromiotrapezius and posterior spinotrapezius muscle processes, the latter expanding over the entire thoracic region4.

Methods
Animals
Transgenic zebrafish. Zebrafish were maintained and staged as described48 in agreement with recommendations of the national authorities of Switzerland (Animal Protection Ordinance) and following the regulation of the 2010/63 UE European directives. Protocols were approved by the cantonal veterinary office of the Canton Zurich (Kantonales Veterinäramt, permit no. 150) and by the veterinary office of the IACUC of the University of Colorado School of Medicine (protocol no. 00979), Aurora, Colorado. All zebrafish embryos were raised in temperature-controlled incubators without light cycle at 28 °C as described48. Established transgenic lines used in this study include tbx1:creERT2zh703 (Tg(–3.2tbx1:creERT2, cryaa:Venus)), hsp70l:Switchzh701 (Tg(–1.5hsp70l:loxP-STOP–loxP-EGFP, cryaa:Venus))19,33, and pax3a-GFP (Tg(pax3a:eGFP))22. tbx1:creERT2 lineage tracing experiments were performed by crossing female hsp70l:Switch carriers with male CreERT2 driver transgenics33. For CreERT2 recombinase activation, embryos were treated at shield stage (6 hours post-fertilization (hpf)) in preheated 4-OHT-containing E3 medium (Cat# H7904: Sigma) as per our established protocols33. 4-OHT was washed off at 30 hpf and replaced by E3 medium containing 0.003% 1-phenyl-2-thiourea (PTU, Cat# P7629; Sigma) to prevent the formation of pigmentation. Heat-shock to trigger hsp70l:Switch reporter expression was performed at 5 days post-fertilization (dpf) for 60 min at 37 °C in glass tubes in a water bath and larvae were raised 4-5 hours after heat shock. pax3a:EGFP expression analyses were performed by crossing wild-type males and pax3a:EGFP females and embryos were raised in temperature-controlled incubators until the desired stage. Embryos were anesthetised in tricaine 1X (MS222, Sigma-Aldrich, A5040), equivalent to a final concentration of 160 μg/ml. EGFP-positive embryos and negative controls were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight and subsequently washed and stored in PBS. Embryos from 3 independently crossed pairs were used.

Axolotl. Axolotls were maintained and staged as described49 in accordance with the 2010/63 UE European directives. Axolotl larvae were generated by crossing male and female albino specimens. Larvae were maintained in water at 18 °C and fed daily. Larvae at the desired developmental stage were anesthetised in MS222, fixed in 4% PFA overnight at 4 °C and subsequently washed in PBS, dehydrated, and stored in absolute methanol.

The axolotl juvenile used for micro-computed tomography (micro-CT) scanning was euthanised four months after hatching with an overdose of MS222 in a water tank for 30 min. The specimen was then directly fixed in 4% PFA for 2 days at room temperature to preserve tissue integrity, washed in PBS and stored in 70% ethanol before contrast agent treatment.

Other species. The other juvenile specimens for tomography scanning were obtained from public natural history collections, no specimens were collected in the field for this project. Details on the juvenile coelacanth specimen were reported50. The juvenile coelacanth (ZSM 28409 (CCC 162.21)) was housed in the Bavarian State Collection for Zoology, Germany. The specimen was fixed in formalin and preserved in this fixative until 2010 before being transferred to a solution of ethanol (75%)50. The bichir and lungfish were obtained from the local collection at the National Museum of Natural History in Paris (MNHN, FR) and a deceased newt was obtained from our MNHN colony. Both specimens were fixed into 4% formalin solution, and then preserved in 70% ethanol solution. The precise timing of collection, fixation and storage procedures were not available for bichir, coelacanth, lungfish, newt and lizard specimens. Tissue damages may have occurred prior to analyses, but the overall soft tissue quality was validated on virtual micro-CT sections before specimen selection and structure segmentation.

Sex and gender considerations were not relevant to the biological process analysed in larval and juvenile specimens included in the study.

Immunofluorescence staining
Samples in absolute methanol were incubated overnight at 4 °C in Dent’s fixative (20% DMSO–80% methanol) for permeabilization and rehydrated in methanol-PBS grade series. Rehydrated samples in PBS were then blocked for 2 h in 10% normal goat serum, 3% BSA, 0.5% Triton X-100 in PBS. Primary antibodies against the green fluorescent protein (GFP, dilution 1/1000, ab13970 from Abcam), Myosin Heavy Chain (MyHC, dilution 1/20, A4.1025 from DHSB) and acetylated tubulin (ac-Tub, dilution 1/500, T7451 from Sigma-Aldrich) were diluted in blocking solution and incubated 3 days at 4 °C. After several washes in PBS with 0.1% Tween 20 (PBST), goat anti-chick Alexa 488 and goat anti-mouse Alexa 555-IgG2a and Alexa 647-IgG2b secondary antibodies (dilution 1/500, Invitrogen A-11039, A-21137, A-21242) were diluted in blocking solution and incubated 2 days at 4 °C. After whole-mount immunostaining, zebrafish larvae were washed in PBST, post-fixed in PFA 4% overnight at 4 °C and store in PBST. For confocal and light-sheet acquisitions, larvae were clarified according to the ECi protocol as described51.

Immunofluorescence acquisitions
Confocal images were acquired using Zeiss LSM 700 and LSM 980 laser-scanning upright confocal microscopes with dry 10x NA 0.25 and water immersion 20x NA 0.75 Zeiss objectives and ZEN software (Carl Zeiss, Germany). Light-sheet images were acquired with the Alpha3 system (PhaseView, France) with a pixel size of 650 nm and a z-step size of 1 µm. The microscope was equipped with two dry 10x NA 0.25 Zeiss objectives for a simultaneous illumination on the right and left side, the detection was performed with an 10x NA 0.60 Olympus objective with a correction ring for refractive index matching. For the blue, red and far-red channels, the 405 nm, 561 nm, and 633 nm laser lines were used for the excitation respectively. To have the same light-sheet thickness all along the x-axis, the system was equipped with the “real time focus sweeping” option. For 3D rendering, acquired Z-stacks were 3D reconstructed using the Arivis software. All images were assembled in Adobe Photoshop (Adobe Systems, USA).

Tomography scan acquisitions and analysis
The synchrotron CT scan of the zebrafish juvenile (33 dpf) treated with phosphotungstic acid (PTA) was publicly available from the Chen Laboratory repository52. After fixation, Anolis, Ambystoma and Protopterus specimens were treated with 2.5 to 5% PMA (phosphomolybdic acid) contrast agent and micro-CT scanning was performed at the XTM Facility (Palaeobiology Research Group, University of Bristol) following the parameters reported in (Supplementary Table 1). Tylototriton, Polypterus and Latimeria specimens were scanned using long distance phase-contrast X-ray synchrotron tomography on BM19 at the European Synchrotron Facility following the parameters reported in (Supplementary Table 2). Segmentation was performed using Mimics Innovation Suite 19 software (Materialise SV). After segmentation, STL files were imported in Blender (Blender Foundation) for rendering.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript Heude et al. provide a very comprehensive yet concise analysis and synthesis of the evolutionary trajectory of muscles at the head-trunk interface associated with the evolution of a neck during the fish to tetrapod transition. Two sets of data are presented. The first set (Fig. 1 and 2) uses genetic lineage tracing in zebrafish to visualize the respective contributions of trunk mesoderm (pax3 expressing) and cardiopharyngeal mesoderm (CPM) (tbx1 expressing) to the head trunk interface muscles. This is further complemented by visualising the innervation pattern of cranial nerves. This strategy identifies the cucullaris muscle (the main muscle group to connect head and trunk) and shows a lineage tracing pattern consistent with CPM and not trunk muscle origin, while hypobranchial muscles are identified as being trunk derived. This confirms that these muscle groups have similar developmental origins in zebrafish as in tetrapods.\nThe second set of data (Fig 3. and supplement) is a comprehensive analysis of uCT data in zebrafish, bichir, lungfish, coelacanth salamanders and Anolis. This data set goes to show the presence or absence of hypobranchial, cucullaris, coracobranchial and laryngeal muscles in a cogently selected taxon sample relevant to the osteichthyes. This analysis confirms an ancestral origin of the various muscle groups and shows that absence of the laryngeal muscles in zebrafish is secondarily derived, presumably due to loss of air breathing, thereby strongly supporting an original function of the laryngeal muscles in a respiratory capacity. Combined with the lineage tracing data the authors propose a model whereby during the fish to tetrapod transition, the CPM derived cucullaris expanded from head to trunk to encompass and stabilise the neck, and conversely trunk musculature expanded into the head to form the tongue.\nI find the manuscript extremely clean, interesting and well written. The final synthesis of the manuscript (Fig.4) certainly makes a text book worthy contribution.\nI also have some comments.\n- The Cre of particularly tbx1 does not seem to be extremely efficient and as I can judge by the images in Fig. 2 there appears to be quite some mosaicism. In the B panels where the cucullaris is to be identified, it struck me that in the merged image where yellow is presumably to indicate presence of both MyHC and GFP (also it would be good to better indicate this in the legend so that no confusion can arise to this point) exactly the cc is not yellow but mosaic in purple and green, suggesting that not the same cells could be labeled – or perhaps it is just an artefact of the image processing settings. Nevertheless, since this image critically supports one of the main claims of the paper, I think it is necessary to clarify this. Also, as a reader it is often very hard to assess whether the images shown are more or less randomly chosen out many very similar experiments, or that they are the very best out of a few times that things finally worked. In this context it is relevant to report how often this was reproduced and perhaps introduce a figure with reproduced results in the supplement. (I apologize if I overlooked the numbers somewhere in an appendix, in that case it is better to make them prominently visible, for instance in the figure legend).\n- In the paper, the authors build onto a large body of comparative research detailing and speculating about the homology of muscle groups in fish that goes back to the 19th century. Inevitably therefore, there is overlap with the findings of the authors and findings/speculations/hypotheses that have been published before, which are I think also mostly cited in the manuscript. Still, as some places I however have a bit of difficulties is to understand what exactly is completely novel; what is simply confirming previous observations (and why the observations of the authors are not just repeating previous findings but actually provide a higher level of confidence in the final conclusions); if hypotheses are brought forward completely de novo or are built upon prior suggestions. An example I can give is for instance the paragraph of line 230-237, where the first sentence states “Interestingly, our data show that the bichir, coelacanth, and lungfish possess a laryngeal musculature.” Raising the impression with the reader that these are all novel findings. Followed by “a previous ontogenetic morphological analysis…supports a CPM origin of the laryngeal musculature in the early diverging Australian lungfish Neoceratodus” indicating that in fact the presence of laryngeal muscles in lungfish was uncontroversial. The paper is laudably concise, but perhaps some more space can be dedicated to elaborate in this context.\n- I find the yellow shade used to label the pectoral girdle in Fig. 2J/ Fig. 4 hard to distinguish. Please critically review/revise for better accessibility.\nJoost Woltering", "reviewer_2": "The separation of the skull from the pectoral girdle, which led to the development of a functional neck, required significant changes in the musculoskeletal system. Despite the clear origins of head and trunk muscles from distinct embryonic mesoderm populations, the evolutionary origins of neck muscles at the head-trunk junction remain unclear. In this study, the authors explored the evolution of the vertebrate neck by integrating experimental embryology with comparative anatomy. Based on their observations, the authors proposed the model in which muscle groups linking the head and trunk share conserved mesodermal origins in both fishes and tetrapods. Furthermore, they proposed that the expansion of mesodermal cells into the head and trunk domains during embryonic development was crucial for the emergence and adaptation of the tetrapod neck.\nIn this paper, the authors address a longstanding and significant issue concerning the origins of neck muscles in vertebrates. The hypothesis they propose is indeed intriguing; however, their experimental data fall short of providing a robust foundation for their hypothesis for the following reasons.\nThe most pivotal findings presented in this paper is the demonstration that cucullaris muscles (cc) and coracobranchial muscles (cbm) in the zebrafish tbx1-lineage GFP reporter line are GFP positive and innervated by the vagus nerve X (nX). However, the data provided do not convincingly support these findings. The muscle designated as cc in Figure 2 B’ is challenging to discern as GFP-positive in Figure 2 B”, and the cbm is only partially GFP positive in Figure 2C”. I suggest the authors to show clearer images showing GFP signals in cc and cbm, and utilize specimens where cc and bl are distinctly separated, as in Figure 1E. Enlarged pre-3D raw data images showing GFP signals in cc and cbm are also imperative. The discrepancy in the shape of cc between the 3D image in Figure 2E and Figure 2B raises questions; if different specimens were employed, ensuring consistency in the specimen used for 3D imaging is necessary. In addition, a finer depiction of nerves would enhance clarity in the neural innervations. If cc and cbm are indeed GFP-positive, 3D images of these GFP-positive regions should also be included. Pre-3D raw data on Ac Tu, MyHC and GFP as well as histological sections clearly depicting neural innervations to each muscle, are essential. If muscle definitions are based on neural innervations, proofs of such innervations via retrograde labeling or similar methods is necessary. Furthermore, the criteria for coloring nerves and muscles in 3D images should be explicitly stated.\nConcerning the anatomical data presented in Figure 3, the absence of pre-3D raw data or clear criteria for defining each muscle raises questions about the rationale behind the authors’ conclusions, reducing the figure to what may appear as arbitrarily colored illustrations. It is crucial to exhibit pre-3D raw data for the cucullaris musculature, coracobranchial musculature, laryngeal musculature, as well as nX across all specimens to substantiate the draw conclusions. The convincing images showing the exact innervation sites of nX are ambiguous; if nX innervation is used for defining each muscle, evidence (i.e. histological sections or retrograde analyses) of nX innervating the defined muscle across all samples is requisite. Furthermore, please also include the model name of the synchrotron CT scan used and the imaging conditions.\nFinally, given the extensive research on the muscle patterns of the vertebrate species under discussion, the authors are urged to elucidate why some of their findings diverge from previous studies.", "reviewer_4": "This is a great paper aiming to reveal the embryological origin of of head-neck transition muscles.\nAs cucullaris was in several studies already shown to be a branchial arch muscle it is (for me) not surprising that it is CPM derived, but it is great to see this in such clear data and figure plates.\nOverall there are mostly tiny things to remark as indicated in the PDF.\nMinor suggestions:\nOne thing I would like the authors to think about is the use of cucullaris throughout the investigated study. As soon as branchial arch levators are identifiable the remaining shoulder-girdle to head muscle is the protractor pectoralis (Ziermann et al. 2014: Zoological Journal of the Linnean Society, 172(4), 771-802). This protractor pectoralis gives then rise to trapezius and sternocleidomastoid. That would mean for this study that all animals have cucullaris-derived muscles. That is ZF already has protractor pectoralis, as well as the other fishes and salamanders mentioned in this paper and the amniotes have sternocleidomastoid and trapezius. I'm not against using cucullaris, but it would be more precise to call them cucullaris-derived muscles. This is true for the figure legends, too.\nAll figures are amazing. In figure 1 cc (cucullaris muscle) is mentioned in the figure legend but not marked any of the figure parts. Unfortunately, I wasn't able to watch the video (didn't start), but I'm sure it is also great."}, "author_response": "RESPONSE TO REVIEWERS\nMajor changes include:\n- concerns of Reviewers #1 and #2 with additional work to modify the content of Fig. 2a-i.\n- a new Supp. Fig. 1 that complements data in Fig. 2, and new panels in Supp. Fig. 2e-l\n- two Supp. Movies with associated legends:\n* Supplementary Movie 1. 3D interactive view of the neuromuscular system at the head/trunk\ntransition of a 5 dpf zebrafish larva to complete data shown in Fig. 2j-l and Supplementary\nFig. 2a-d.\n* Supplementary Movie 2. 3D interactive view of the neuromuscular system at the head/trunk\ntransition of a 7 dpf zebrafish larva to complete data shown in Supplementary Fig. 2e-l.\n- new raw data in Supp. Figs. 3-9b\n- all figures have been formatted according to the journal’s standard\nReviewer #1:\nIn this manuscript Heude et al. provide a very comprehensive yet concise analysis and synthesis of\nthe evolutionary trajectory of muscles at the head-trunk interface associated with the evolution of\na neck during the fish to tetrapod transition. Two sets of data are presented. The first set (Fig. 1 and\n2) uses genetic lineage tracing in zebrafish to visualize the respective contributions of trunk\nmesoderm (pax3 expressing) and cardiopharyngeal mesoderm (CPM) (tbx1 expressing) to the head\ntrunk interface muscles. This is further complemented by visualising the innervation pattern of\ncranial nerves. This strategy identifies the cucullaris muscle (the main muscle group to connect head\nand trunk) and shows a lineage tracing pattern consistent with CPM and not trunk muscle origin,\nwhile hypobranchial muscles are identified as being trunk derived. This confirms that these muscle\ngroups have similar developmental origins in zebrafish as in tetrapods.\nThe second set of data (Fig 3. and supplement) is a comprehensive analysis of uCT data in zebrafish,\nbichir, lungfish, coelacanth salamanders and Anolis. This data set goes to show the presence or\nabsence of hypobranchial, cucullaris, coracobranchial and laryngeal muscles in a cogently selected\ntaxon sample relevant to the osteichthyes. This analysis confirms an ancestral origin of the various\nmuscle groups and shows that absence of the laryngeal muscles in zebrafish is secondarily derived,\npresumably due to loss of air breathing, thereby strongly supporting an original function of the\nlaryngeal muscles in a respiratory capacity. Combined with the lineage tracing data the authors\npropose a model whereby during the fish to tetrapod transition, the CPM derived cucullaris\nexpanded from head to trunk to encompass and stabilise the neck, and conversely trunk\nmusculature expanded into the head to form the tongue.\nI find the manuscript extremely clean, interesting and well written. The final synthesis of the\nmanuscript (Fig.4) certainly makes a text book worthy contribution.\n- The Cre of particularly tbx1 does not seem to be extremely efficient and as I can judge by the\nimages in Fig. 2 there appears to be quite some mosaicism. In the B panels where the cucullaris is\nto be identified, it struck me that in the merged image where yellow is presumably to indicate\npresence of both MyHC and GFP (also it would be good to better indicate this in the legend so that\nno confusion can arise to this point) exactly the cc is not yellow but mosaic in purple and green,\nsuggesting that not the same cells could be labeled – or perhaps it is just an artefact of the image\nprocessing settings. Nevertheless, since this image critically supports one of the main claims of the\npaper, I think it is necessary to clarify this.\nAlso, as a reader it is often very hard to assess whether the images shown are more or less randomly\nchosen out many very similar experiments, or that they are the very best out of a few times that\nthings finally worked. In this context it is relevant to report how often this was reproduced and\nperhaps introduce a figure with reproduced results in the supplement. (I apologize if I overlooked\nthe numbers somewhere in an appendix, in that case it is better to make them prominently visible,\nfor instance in the figure legend).\nWe thank the Reviewer for their positive assessment of our work.\nThe Cre-mediated recombination of the tbx1-lineage GFP reporter with OHT treatment showed\nmosaicism in the first version of the study. Therefore, we have optimized the experimental approach\nwith longer OHT treatment from 6 hpf to 30 hpf (instead of 6 hpf to 16 hpf) and with extended\nrecovery after heat-shock GFP-switch induction (4-5 h instead of 2-3 h); we have modified the\nMethods section accordingly. This resulted in higher recombination efficiency and redundancy in\nbranchial arch derivatives including the cucullaris. We finally obtained at least 10 specimens with\nclear GFP signal in the cucullaris myofibers at 5 dpf; the number of positive specimens analysed is\nnow specified in Figure legends (Figs. 1-2). Moreover, we have added a Supplementary Fig. 1\nshowing control and several positive reporter specimens (#1 to #5) to demonstrate recombination\nspecificity, efficiency, and reproducibility.\n- In the paper, the authors build onto a large body of comparative research detailing and speculating\nabout the homology of muscle groups in fish that goes back to the 19th century. Inevitably\ntherefore, there is overlap with the findings of the authors and findings/speculations/hypotheses\nthat have been published before, which are I think also mostly cited in the manuscript. Still, as some\nplaces I however have a bit of difficulties is to understand what exactly is completely novel; what is\nsimply confirming previous observations (and why the observations of the authors are not just\nrepeating previous findings but actually provide a higher level of confidence in the final conclusions);\nif hypotheses are brought forward completely de novo or are built upon prior suggestions. An\nexample I can give is for instance the paragraph of line 230-237, where the first sentence states\n“Interestingly, our data show that the bichir, coelacanth, and lungfish possess a laryngeal\nmusculature.” Raising the impression with the reader that these are all novel findings. Followed by\n“a previous ontogenetic morphological analysis…supports a CPM origin of the laryngeal musculature\nin the early diverging Australian lungfish Neoceratodus” indicating that in fact the presence of\nlaryngeal muscles in lungfish was uncontroversial. The paper is laudably concise, but perhaps some\nmore space can be dedicated to elaborate in this context.\nnow modified and completed some paragraphs in the Discussion section to clarify the novelty of\nour work and the uncertainties in the literature.\n- I find the yellow shade used to label the pectoral girdle in Fig. 2J/ Fig. 4 hard to distinguish. Please\ncritically review/revise for better accessibility.\nThe color code of the pectoral girdle has been modified to dark grey instead of the yellow shade in\nFigures 2 and 4 for better visualisation.\nReviewer #2\nThe separation of the skull from the pectoral girdle, which led to the development of a functional\nneck, required significant changes in the musculoskeletal system. Despite the clear origins of head\nand trunk muscles from distinct embryonic mesoderm populations, the evolutionary origins of neck\nmuscles at the head-trunk junction remain unclear. In this study, the authors explored the evolution\nof the vertebrate neck by integrating experimental embryology with comparative anatomy. Based\non their observations, the authors proposed the model in which muscle groups linking the head and\ntrunk share conserved mesodermal origins in both fishes and tetrapods. Furthermore, they\nproposed that the expansion of mesodermal cells into the head and trunk domains during\nembryonic development was crucial for the emergence and adaptation of the tetrapod neck.\nIn this paper, the authors address a longstanding and significant issue concerning the origins of neck\nmuscles in vertebrates. The hypothesis they propose is indeed intriguing; however, their\nexperimental data fall short of providing a robust foundation for their hypothesis for the following\nreasons.\nThe most pivotal findings presented in this paper is the demonstration that cucullaris muscles (cc)\nand coracobranchial muscles (cbm) in the zebrafish tbx1-lineage GFP reporter line are GFP positive\nand innervated by the vagus nerve X (nX). However, the data provided do not convincingly support\nthese findings. The muscle designated as cc in Figure 2 B’ is challenging to discern as GFP-positive in\nFigure 2 B”, and the cbm is only partially GFP positive in Figure 2C”. I suggest the authors to show\nclearer images showing GFP signals in cc and cbm, and utilize specimens where cc and bl are\ndistinctly separated, as in Figure 1E. Enlarged pre-3D raw data images showing GFP signals in cc and\ncbm are also imperative. The discrepancy in the shape of cc between the 3D image in Figure 2E and\nFigure 2B raises questions; if different specimens were employed, ensuring consistency in the\nspecimen used for 3D imaging is necessary. In addition, a finer depiction of nerves would enhance\nclarity in the neural innervations. If cc and cbm are indeed GFP-positive, 3D images of these GFP-\npositive regions should also be included. Pre-3D raw data on Ac Tu, MyHC and GFP as well as\nhistological sections clearly depicting neural innervations to each muscle, are essential. If muscle\ndefinitions are based on neural innervations, proofs of such innervations via retrograde labeling or\nsimilar methods is necessary. Furthermore, the criteria for coloring nerves and muscles in 3D images\nshould be explicitly stated.\napproach to clarify the tbx1 lineage data. We now show reproducible GFP signal in the cucullaris\nand coracobranchial myofibers by confocal acquisitions and maximum intensity projections (Fig. 2a-\nI, Supplementary Fig. 1h-s) and 3D renderings (Supplementary Fig. 1a-c).\nIn the present study we decided to use lightsheet microscopy to study muscular innervation in the\nzebrafish larva. This approach permits access, at high 3D resolution (voxel 0,650µm), the relative\nconformation of the nervous and muscular microscopic components in the specimens analysed. The\ncucullaris and coracobranchial muscles of interest consist of only a few myofibers, thereby limiting\nproper histological analysis of neuromuscular connections on sections, and restricting retrograde\nlabelings due to technical and physical constraints. Thus, we believe that our experimental approach\nis the most relevant for analysis of the specimens.\nTo complete the data collected in 5 dpf control larvae (Fig. 2j-l, Supplementary Fig. 2a-d,\nSupplementary Movie 1), we have performed equivalent stainings, acquisitions and renderings later\nduring neuromuscular system differentiation at 7 dpf (Supplementary Fig. 2e-l, Supplementary\nMovie 2). Our data now show more clearly a branch of the vagus nerve connecting the cucullaris in\nzebrafish, adding experimental evidence and confirming its embryonic origin that we demonstrate\nby genetic expression and lineage analyses.\nConcerning the anatomical data presented in Figure 3, the absence of pre-3D raw data or clear\ncriteria for defining each muscle raises questions about the rationale behind the authors’\nconclusions, reducing the figure to what may appear as arbitrarily colored illustrations. It is crucial\nto exhibit pre-3D raw data for the cucullaris musculature, coracobranchial musculature, laryngeal\nmusculature, as well as nX across all specimens to substantiate the draw conclusions. The convincing\nimages showing the exact innervation sites of nX are ambiguous; if nX innervation is used for\ndefining each muscle, evidence (i.e. histological sections or retrograde analyses) of nX innervating\nthe defined muscle across all samples is requisite.\nWe have completed Supplementary Figs. 3-9 with CT scan raw data including pre- and post-otic\nvirtual frontal sections showing the structures of interest that we segmented for 3D reconstructions.\nAs stated at the beginning of the Results section, muscle identity is defined by the embryonic\nmesodermal origin, the innervation, and the attachments to skeletal structures. In the comparative\nanatomy analysis, the main factor and traditional way to define muscle identity involves muscle\nconnections to skeletal components (see for reference Dearden et al. 2021 J. Anat.). We have\nsegmented the nX in the bichir, coelacanth and lungfish as a second argument to confirm identity\nwhen embryological data or description in the literature are unclear or missing. The latter species\nanalysed in the present study constitute rare (bichir, lungfish juveniles) to unique (coelacanth\njuvenile) specimens worldwide, and histological sections and retrograde analyses are therefore not\nexperimentally feasible.\nconditions.\nWe have now added the model name in the Methods section and tables in Supplementary\nInformation reporting the parameters used for micro- and synchrotron CT scan imaging\n(Supplementary Tables 1-2).\nFinally, given the extensive research on the muscle patterns of the vertebrate species under\ndiscussion, the authors are urged to elucidate why some of their findings diverge from previous\nstudies.\nWe thank the Reviewer for raising this important point. The embryonic origin of neck muscles has\nbeen a subject of controversy due to the location of its embryonic precursors at the interface of\ncardiopharyngeal, somitic, and lateral plate mesoderm populations, aspects that we discuss in the\nIntroduction section of the manuscript. As described for Reviewer #1, we have now clarified our\nfindings in regards to previous studies in the Discussion section.\nReviewer #4\nThis is a great paper aiming to reveal the embryological origin of head-neck transition muscles.\nAs cucullaris was in several studies already shown to be a branchial arch muscle it is (for me) not\nsurprising that it is CPM derived, but it is great to see this in such clear data and figure plates. Overall\nthere are mostly tiny things to remark as indicated in the PDF.\nWe thank the Reviewer for their positive assessment of our work.\nWe have considered the comments and suggestions indicated in the PDF and modified the text\naccordingly.\nMinor suggestions:\nOne thing I would like the authors to think about is the use of cucullaris throughout the investigated\nstudy. As soon as branchial arch levators are identifiable the remaining shoulder-girdle to head\nmuscle is the protractor pectoralis (Ziermann et al. 2014: Zoological Journal of the Linnean Society,\n172(4), 771-802). This protractor pectoralis gives then rise to trapezius and sternocleidomastoid.\nThat would mean for this study that all animals have cucullaris-derived muscles. That is ZF already\nhas protractor pectoralis, as well as the other fishes and salamanders mentioned in this paper and\nthe amniotes have sternocleidomastoid and trapezius. I'm not against using cucullaris, but it would\nbe more precise to call them cucullaris-derived muscles. This is true for the figure legends, too.\nWe believe that there might be some confusion in the terminology. We used the cucullaris name to\nfacilitate comparison between the vertebrate species analysed. We agree that the use of cucullaris\nderivatives or cucullaris-derived muscles is more accurate when comparing species from different\nclades; we have modified the text accordingly. However, we have kept the term cucullaris when\nreferring to the embryonic anlagen or to the overall muscle group cucullaris musculature (as for\n5\nother muscle groups such as laryngeal or hypobranchial musculature) in the color code panels\npresented in Figs 3-4 and Supplementary Figs 3-9.\nAll figures are amazing. In figure 1 cc (cucullaris muscle) is mentioned in the figure legend but not\nmarked any of the figure parts.\nIn Figure 1, the cucullaris is indicated in panel (m).\nUnfortunately, I wasn't able to watch the video (didn't start), but I'm sure it is also great.\nAs indicated for Reviewers #2, we have now added a new movie (Supplementary movie 2) showing\nthe connection of the vagal nerve to the cucullaris at 7 dpf to complete data previously presented\nonly at 5 dpf (Supplementary movie 1). We hope that the current mp4 versions of the videos will\nwork correctly and if not, we can provide another coded format.\n6"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my previous concerns, in particular I find the new tbx1-Cre lineage tracing data very convincing.", "reviewer_2": "Thank you for addressing several of the comments and concerns raised during the initial review process. However, I find that some crucial aspects remain unsolved. I would like to highlight a few key points that I believe are crucial for the integrity of the conclusions.\n1. Data for zebrafish: In my previous review, I emphasized the importance of clearly depicting neural innervations in the 3D images, particularly given that the definition of the cucullaris muscles (cc) hinges on their innervation by the vagus nerve (nX). Specifically, I stated, \"Pre-3D raw data on Ac Tu, MyHC, and GFP as well as histological sections clearly depicting neural innervations to each muscle, are essential. If muscle definitions are based on neural innervations, proofs of such innervations via retrograde labeling or similar methods is necessary.\" Despite authors’ response that current methods are optimal, the images provided, including Figure 2k and the corresponding movie (0:11~0:12), do not convincingly demonstrate the nX innervations in the cucullaris muscles. The cc appears to be merely adjacent to the nX, rather than being innervated by it. This ambiguity raises concerns about the accuracy of the muscle definitions. To address this, I strongly recommend including pre-3D raw data or histological sections that explicitly show the nX innervating the cc. I expect authors to provide images of similar quality to those in Figure 3F of (Higashijima et al., 2016, J. Morph.). Without such evidence, the conclusions drawn regarding muscle identity remain speculative.\n2. Data for the Bichir, Lungfish, Coelacanth, Axolotl, Newt, and Lizard: The authors’ response indicates that obtaining additional samples is not feasible due to limitations in the availability of bichir, coelacanth, lungfish. While I understand these constraints for coelacanth and lungfish, other species (bichir, axolotl, newt, and lizard) are readily available through various suppliers, including pet shops and aquariums. Therefore, it is entirely feasible to obtain these specimens for further analysis. The availability of these samples negates the justification for not performing additional experiments, particularly when such data could significantly strengthen the conclusions of the study. As I stated in my previous review, it is critical to provide more robust evidence for nX innervation if this is a central criterion for muscle definition. For instance, in the supplementary figures, the images provided do not clearly delineate the cc and bone boundaries (Supplementary Fig. 6b) or show definitive innervation by nX (Supplementary Fig. 3b, 4b, 6b, 7b, 8b). Additionally, synchrotron imaging and the reconstruction of these images are time-consuming and that the authors have analyzed only a single specimen for each sample. Given that images were obtained from specimens that were fixed post-mortem, it is possible that the cucullaris muscle may have been compromised, especially considering that these muscles consist of only a few myofibers. Although the trapezius muscle in lizard is anatomically clear and additional experiment may not be required, given the availability of other samples (bichir, axolotl, and newt), as well as antibodies for muscle and nerve markers, I believe that further efforts should be made to present supporting data that validate the muscle definitions. If histological sections are indeed inadequate, as authors have suggested, whole-body immunolabeling in fish and amphibian juveniles (Pende et al., 2020 Sci Adv 6, aba0365) should be considered as a feasible alternative. This would significantly strengthen the credibility of the study.\nIn conclusion, while this study addresses a significant and longstanding question in vertebrate evolutionary biology, the robustness of the findings hinges on the clear depiction and verification of neural innervations. I urge authors to consider the suggestions outlined above to enhance the rigor and reliability of the conclusions.", "reviewer_4": "The authors have addressed all my previous comments. They now highlight clearer novel insights based on their research. They clarified the text by adding ‘cucullaris-derived’ where applicable for muscles. The new included figures (in main figures and supplementary) support the reproducibility of the research. This time, I was also able to watch both videos.\nI have only some minor suggestions:\n- Line 64: ‘muscles that connect to the hyoid bone ventrally’ – add ‘to’\n- Line 66: laterally\n- Line 94: you wrote ‘the vagal nerve (cranial nerve CN X)’ and then in line 97 ‘accessory nerve (CN XI) derived from the vagus nerve 4,6,29’. I would suggest to add (CN X) after nerve in line 97 for readers that are not familiar with cranial nerve terminology\n- Line 110, 124: ‘connecting the head to the trunk’ – exchange ‘to’ with ‘and’\n- Line 121: ‘innervation, the attachments to skeletal’ – add ‘and’ after comma\n- Line 164: ‘The vagus nerve also innervates the coracobranchial muscles ventrally” suggestion to change to ‘The vagus nerve also innervates the ventrally located coracobranchial muscles’ – Reason: the original sentence could be misunderstood in the way that the nerve approaches the muscle from ventral\n- Lines 199-200: ‘ceratohyal (hyoid arch) and the mandible, respectively’ – suggestion to add ‘(mandibular arch)’ after mandible\n- Line 207: ‘hypobranchial, laryngeal and cucullaris-derived’ – add comma after ‘laryngeal’\n- Line 297: add comma after ))\n- Line 571: add comma after coracobranchial"}, "author_response": "RESPONSE TO REVIEWERS\nMajor changes include:\n- concerns of Reviewer #2 with addiConal panels in Supp. Fig. 2m-o, a new Supp. Fig. 3 with associated\nvideo (Supp. Movie 3) and corresponding capCons\n- Some text modificaCons following comments from Reviewer #2 and Reviewer #4 and from the Editor\nReviewer #1:\nThe authors have addressed all my previous concerns, in particular I find the new tbx1-Cre lineage tracing\ndata very convincing.\nWe thank the Reviewer for the positive assessment of our work.\nReviewer #2\nThank you for addressing several of the comments and concerns raised during the initial review process.\nHowever, I find that some crucial aspects remain unsolved. I would like to highlight a few key points that I\nbelieve are crucial for the integrity of the conclusions.\n1. Data for zebrafish: In my previous review, I emphasized the importance of clearly depicting neural\ninnervations in the 3D images, particularly given that the definition of the cucullaris muscles (cc) hinges on\ntheir innervation by the vagus nerve (nX). Specifically, I stated, \"Pre-3D raw data on Ac Tu, MyHC, and GFP\nas well as histological sections clearly depicting neural innervations to each muscle, are essential. If muscle\ndefinitions are based on neural innervations, proofs of such innervations via retrograde labeling or similar\nmethods is necessary.\" Despite authors’ response that current methods are optimal, the images provided,\nincluding Figure 2k and the corresponding movie (0:11~0:12), do not convincingly demonstrate the nX\ninnervations in the cucullaris muscles. The cc appears to be merely adjacent to the nX, rather than being\ninnervated by it. This ambiguity raises concerns about the accuracy of the muscle definitions. To address this,\nI strongly recommend including pre-3D raw data or histological sections that explicitly show the nX\ninnervating the cc. I expect authors to provide images of similar quality to those in Figure 3F of (Higashijima\net al., 2016, J. Morph.). Without such evidence, the conclusions drawn regarding muscle identity remain\nspeculative.\nWe thank the Reviewer for raising these important points.\nTo address the concerns and according to Editor’s suggestions we have completed Supp. Fig. 2 with orthoslice\nviews of raw images (panels m-o) that now clearly show on a single plane one of the branchial branches of\nthe vagus nerve connecting to the cucullaris. We hope that these new perspectives combined with our data\non the embryonic origin and skeletal connections will fully convince the community on the cucullaris identity\nand homology in zebrafish.\n2. Data for the Bichir, Lungfish, Coelacanth, Axolotl, Newt, and Lizard: The authors’ response indicates that\nobtaining additional samples is not feasible due to limitations in the availability of bichir, coelacanth, lungfish.\nWhile I understand these constraints for coelacanth and lungfish, other species (bichir, axolotl, newt, and\nlizard) are readily available through various suppliers, including pet shops and aquariums. Therefore, it is\nentirely feasible to obtain these specimens for further analysis. The availability of these samples negates the\njustification for not performing additional experiments, particularly when such data could significantly\n1\nstrengthen the conclusions of the study. As I stated in my previous review, it is critical to provide more robust\nevidence for nX innervation if this is a central criterion for muscle definition. For instance, in the\nsupplementary figures, the images provided do not clearly delineate the cc and bone boundaries\n(Supplementary Fig. 6b) or show definitive innervation by nX (Supplementary Fig. 3b, 4b, 6b, 7b, 8b).\nAdditionally, synchrotron imaging and the reconstruction of these images are time-consuming and that the\nauthors have analyzed only a single specimen for each sample. Given that images were obtained from\nspecimens that were fixed post-mortem, it is possible that the cucullaris muscle may have been\ncompromised, especially considering that these muscles consist of only a few myofibers. Although the\ntrapezius muscle in lizard is anatomically clear and additional experiment may not be required, given the\navailability of other samples (bichir, axolotl, and newt), as well as antibodies for muscle and nerve markers,\nI believe that further efforts should be made to present supporting data that validate the muscle definitions.\nIf histological sections are indeed inadequate, as authors have suggested, whole-body immunolabeling in fish\nand amphibian juveniles (Pende et al., 2020 Sci Adv 6, aba0365) should be considered as a feasible\nalternative. This would significantly strengthen the credibility of the study.\nIn conclusion, while this study addresses a significant and longstanding question in vertebrate evolutionary\nbiology, the robustness of the findings hinges on the clear depiction and verification of neural innervations.\nI urge authors to consider the suggestions outlined above to enhance the rigor and reliability of the\nconclusions.\nTo address the concerns of Reviewer #2 about sample size and tissue integrity, and following the Editor’s\nsuggestions, we have clarified these points in the results and method sections for more transparency.\nRegarding the comparative anatomy analysis, the main proxy for defining muscle identity is the muscle\nconnections to skeletal components (see for reference Dearden et al. 2021 J. Anat.). We have segmented the\nnX in the Bichir, Coelacanth and Lungfish as a second argument to confirm identity when embryological data\nor description in the literature were unclear or missing.\nIn contrast, the innervation and identity of cucullaris-derived muscles in the tetrapod species analyzed is\nalready well documented in the literature and is not under debate.\nHowever, as a tetrapod reference to complete data shown during zebrafish development, we have\nperformed whole-mount immunolabelling of the neuromuscular system of an axolotl larva presented in a\nnew Supp. Fig. 3 and Supp. Movie 3 to highlight the connection of the vagus nerve to the cucullaris as\nobserved in fish.\nReviewer #4\nThe authors have addressed all my previous comments. They now highlight clearer novel insights based on\ntheir research. They clarified the text by adding ‘cucullaris-derived’ where applicable for muscles. The new\nincluded figures (in main figures and supplementary) support the reproducibility of the research. This time, I\nwas also able to watch both videos.\nI have only some minor suggestions:\n- Line 64: ‘muscles that connect to the hyoid bone ventrally’ – add ‘to’\n2\n- Line 66: laterally\n- Line 94: you wrote ‘the vagal nerve (cranial nerve CN X)’ and then in line 97 ‘accessory nerve (CN XI) derived\nfrom the vagus nerve 4,6,29’. I would suggest to add (CN X) after nerve in line 97 for readers that are not\nfamiliar with cranial nerve terminology\n- Line 110, 124: ‘connecting the head to the trunk’ – exchange ‘to’ with ‘and’\n- Line 121: ‘innervation, the attachments to skeletal’ – add ‘and’ after comma\n- Line 164: ‘The vagus nerve also innervates the coracobranchial muscles ventrally” suggestion to change to\n‘The vagus nerve also innervates the ventrally located coracobranchial muscles’ – Reason: the original\nsentence could be misunderstood in the way that the nerve approaches the muscle from ventral\n- Lines 199-200: ‘ceratohyal (hyoid arch) and the mandible, respectively’ – suggestion to add ‘(mandibular\narch)’ after mandible\n- Line 207: ‘hypobranchial, laryngeal and cucullaris-derived’ – add comma after ‘laryngeal’\n- Line 297: add comma after ))\n- Line 571: add comma after coracobranchial\nWe are grateful to the Reviewer for the positive assessment of our work and careful reading of our\nmanuscript.\nWe have considered the comments and suggestions and modified the text accordingly.\nWe hope that this revised version fulfills all comments we received from the Reviewers and Editor and is now\nsuitable for publication.\n3"}]

Decision letter