While the extent of CM-DiI-labelling of skeletal derivatives is always greatly reduced, relative to the labelling of progenitor cells at the time of injection due to dilution of the CM-DiI label over several weeks of growth , positively-labelled cells are nevertheless unequivocally recognizable within the skeleton, due to the persistent brightness of the label.
To add an additional level of stringency to our analysis, we only scored contributions to the skeleton consisting of clusters of two or more labelled cells, and contributions that were located in the centre of a skeletal element to avoid inadvertently scoring CM-DiI-labelled connective tissue abutting the cartilage.
As embryonic cartilage is a homogeneous tissue, consisting of a single cell type the chondrocyte , we can therefore trace, with great certainty, the contributions of labelled progenitors to the differentiated cartilaginous endoskeleton.
Using the approach outlined above, we readily observed clusters of NC-derived chondrocytes, for example, in the cartilage of the palatoquadrate Figure 2C and the epibranchial and branchial ray cartilages of the first gill arch Figure 2D.
Overall, our analysis recovered NC contributions to major paired elements of the pharyngeal skeleton i. These findings are consistent with previous assessments of NC contribution to the pharyngeal and paired fin skeleton of zebrafish using genetic lineage tracing Kague et al.
We next sought to complement our NC fate map with a test for mesodermal contributions to the pharyngeal and paired fin skeleton in the skate. D, D i in the branchial rays of gill arch 2.
F, F i Mesodermally-derived chondrocytes were also recovered in the ceratobranchial of gill arch 5, in close proximity to the label-retaining pectoral girdle and surrounding connective tissue. G Schematic summary of pharyngeal and paired fin skeletal elements in the S32 skate embryo, with elements receiving any mesodermal contributions HM, HM-LPM or LPM coloured red, and a plot showing the number of embryos observed with mesoderm contributions to the pharyngeal arch and pectoral fin skeleton.
In D , E and F , cartilaginous elements are false-coloured yellow. Mesodermally-derived chondrocytes were recovered within the epi- or ceratobranchial cartilages and branchial rays of gill arches 1—4 e. Figure 3D,E , as well as in the ceratobranchial of gill arch 5, in close proximity to the label-retaining pectoral girdle and surrounding connective tissue Figure 3F — also, see Figure 3—figure supplement 2 for additional examples of mesoderm-derived label-retaining chondrocytes within the gill arch skeleton.
Overall, our analysis recovered no mesodermal contributions to the mandibular or hyoid arch skeleton, but substantial mesodermal contributions to the paired cartilages of gill arches 1—5, as well as to the pectoral girdle and fin skeleton Figure 3G ; Supplementary file 1.
When considered alongside lineage tracing data from bony fishes, our findings allow us to infer an ancestral mesodermal contribution to the jawed vertebrate gill arch skeleton Figure 4A , with the transition from neural crest-derived to mesodermally-derive skeletogenic mesenchyme occurring gradually, and spanning the region of the posterior i.
In light of the dual embryonic origin of the mammalian thyroid cartilage and exclusively mesodermal origin of the cricoid and arytenoid cartilages which are regarded as derivatives of the 4 th and 6 th pharyngeal arches , it is likely that boundaries of neural crest- and mesodermally-derived skeletogenic mesenchyme have shifted through vertebrate evolution.
A Mesodermal contributions red to the gill arch skeleton in skate, the basibranchial skeleton in axolotl and the laryngeal skeleton of chick and mouse points to an ancestral mesodermal contribution to the pharyngeal arch skeleton of jawed vertebrates. B Schematic representation of neural crest- blue and mesoderm-derived red skeletogenic mesenchyme in the skate pharyngeal arches and pectoral fin bud, in relation to epithelial and mesenchymal Shh expression, respectively.
C We propose that the mandibular and hyoid arch skeleton are neural crest-derived and the pectoral fin skeleton mesodermal derived, while the gill arch skeletal elements are of dual neural crest and mesodermal origin. Our findings also have important implications for understanding the evolutionary origin of paired appendages. This hypothesis purports that paired fins originated from a continuous epithelial fold that flanked the trunk of the embryo, and that was subsequently segmented into distinct appendages at the pectoral and pelvic levels reminiscent of the origin of the 1 st and 2 nd dorsal fins from a continuous median fin fold in sharks.
While palaeontological and embryological evidence for the existence of a lateral fin fold in phylogeny or ontogeny remains scant, there is evidence of shared molecular patterning mechanisms between dorsal median fins and paired appendages Freitas et al. From these observations, a scenario has emerged in which an established appendage patterning developmental module was co-opted, bilaterally, from the dorsal midline to the flank, giving rise to paired pectoral and pelvic appendages.
We previously discovered shared, biphasic roles for Shh signalling in anteroposterior axis establishment and proliferative expansion of skeletal progenitors in the skate hyoid and gill arches and the tetrapod limb bud Gillis et al. We propose that shared responses of hyoid, gill arch and limb skeletal elements to perturbations in Shh signalling — despite differences in the source of Shh in these organs i.
Indeed, reports of a fossil jawless vertebrate with gill arches extending down the length of the trunk Janvier et al. It has been proposed that the neural crest acquired its skeletogenic potential by co-opting a chondrogenic gene regulatory network that arose, ancestrally, within mesoderm Meulemans and Bronner-Fraser, ; Cattell et al.
It is therefore to be expected that neural crest and mesodermal mesenchyme share fundamental molecular mechanisms of skeletogenesis. However, there is nevertheless a heterogeneity across mesenchymal subpopulations in their competence to respond to particular patterning cues. For example, in birds, specific regions of foregut endoderm are both necessary and sufficient for the specification of mandibular arch skeletal elements, but can only induce these elements to form from the neural crest mesenchyme that populates the mandibular arch and not from the mesenchyme of the more caudal pharyngeal arches — Couly et al.
Conversely, quail-chick heterotopic transplantation experiments have shown that midbrain-derived neural crest mesenchyme is competent to give rise to the pleurosphenoid of the lateral braincase wall, even though this element typically derives exclusively from paraxial mesoderm Schneider, Examples such as these point to more cryptic domains of skeletogenic mesenchyme, with distinct competencies, that do not necessarily align with germ layer boundaries.
While the molecular basis of this mesenchymal regionalization may not be known, such regions of shared competence may be operationally defined using cell lineage tracing or transplantation experiments, and may be further tested for shared transcriptional features i.
We also propose that, on an evolutionary time scale, these regions of competence may be predisposed to the iterative deployment of developmental mechanisms, resulting in serial homology. This, in turn, precludes the straightforward inference of homology of anatomical structures based on shared molecular patterning mechanisms Dickinson, Homology is a hierarchical concept, and two complex features e.
While reconstructing the evolutionary history homology of individual genes or gene regulatory network nodes is becoming increasingly straightforward, meaningfully testing the homology of putatively distantly-related structures at the anatomical level — whether historical homologues across taxa, or serial homologues within a taxon — has, in many cases, lingered as problematic. Developmental competence, or the cell-autonomous property that imparts on tissues an ability to respond to external stimuli e.
In light of the demonstrated lability of germ layer fates within the vertebrate skeleton Teng et al. Labelled embryos to be analysed by paraffin histology were embedded and sectioned as previously described O'Neill et al. All data generated or analysed during this study are included in the manuscript and supporting files.
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. Developmental data sets from cartilaginous fish are critical for understanding the early evolution of the vertebrate body plan. This paper provides clear lineage mapping evidence that in Little Skate, the gill arches have a dual embryonic origin, receiving contributions from both neural crest and lateral plate mesoderm.
These observations provide insight into how distinct embryonic cell populations segregate at the head-trunk interface in a representative cartilaginous fish and fuel new hypotheses for the origin of the vertebrate appendicular system. Thank you for submitting your article "Embryonic origin and serial homology of gill arches and paired fins in the skate Leucoraja erinacea " for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor.
The following individuals involved in review of your submission have agreed to reveal their identity: Elizabeth M Sefton Reviewer 2 ; Robert Cerny Reviewer 3. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation. In this manuscript, the authors characterize neural crest and mesoderm contributions to the pharyngeal region and pectoral fins of the Little Skate as a representative Chondrichthyan.
They use these new findings, together with their previously published data sets on arch patterning, to argue for the serial homology of gill arch and paired fin skeletons. The authors' experimental strategy involved first using molecular markers to verify the position of pre-migratory neural crest, head mesoderm, and lateral plate mesoderm populations in early stage skate embryos, followed by a series of targeted injections with lipophilic vital dyes DiI or SpDiOC18 to test if these populations contribute to the pharyngeal skeleton and pectoral fin.
Injected embryos were raised eight to ten weeks, sectioned, and the distribution of labeled cells examined. To minimize the risk of false positives, labeled cells were scored as contributing to a skeletal element only if the cells were centrally located within the element and present in a cluster of at least two.
The results demonstrate neural crest contributions to the jaw, hyoid, and gill arches, consistent with NC lineage analyses in other vertebrates. Injections of the head mesoderm, lateral mesoderm at the head-trunk interface, and trunk lateral plate mesoderm were also performed. The central finding of these mapping experiments was that lateral plate mesoderm contributed to both the gill arches, and the pectoral girdle and fin skeleton. These data, together with the authors' neural crest maps, provide evidence for a dual embryonic origin of the gill arches in a chondrichthyan.
Although dilution by cell division limits the resolution of vital dye fate maps, the consensus among reviewers is that the figures and results are clear and convincing. Further, the use of the Little Skate to address how embryonic cell populations segregate during skeletal formation at the head trunk interface fills an important phylogenetic gap in our knowledge of vertebrate development, one that has broader implications for understanding the evolution of the vertebrate body plan.
The findings are therefore of general interest to the field. The Discussion is well written, but revision is necessary to address several points raised by reviewers. However, as the authors point out, modern developmental genetics has shown that germ layer origin as a criterion for assigning homology can be suspect since cell identity and functionality are due to gene regulatory network activity.
These experiments demonstrated that Shh-responsive gill arch mesenchyme does contribute to branchial rays Fig. Finally, to test the function of Shh signaling during skate branchial ray development, we conducted a series of in ovo pharmacological treatments. Skate embryos develop in large, leathery egg shells, and are amenable to bath treatment by in ovo injection of small molecules. We used cyclopamine — a small molecular inhibitor of the hedgehog signaling pathway — to inhibit Shh signaling at different stages of gill arch development, and to test for stage-specific roles for Shh signaling in bronchial ray patterning.
We chose three stages for treatment: stage 22 gill arches have still formed, and Shh is expressed in posterior arch epithelium , stage 27 gill arches are undergoing lateral expansion, with Shh signaling resolved to an epithelial stripe along the leading edge of the expanding arch and stage 29 just prior to the condensation of the gill arch endoskeleton.
Interestingly, upon manipulation of Shh signaling at these different stages of gill arch development, we observed branchial ray defects that were broadly reminiscent of the skeletal defects observed upon manipulation of Shh signaling during limb development.
For example, we observed that progressively earlier inhibition of Shh signaling resulted in a progressively greater deletion of branchial rays i. We also observed that cyclopamine treatment at stage 22 resulted in loss of anterior-posterior axis specification i.
It therefore appears that, as in the limb bud, Shh signaling functions initially in skate gill arches to establish the anterior-posterior axis, and subsequently to maintain proliferative expansion of branchial ray endoskeletal progenitors. So what does this shared role for Shh signaling in gill arch and limb bud patterning mean? It is possible that limbs share a patterning mechanism with gill arches because these structures are, indeed, transformational homologues i.
However, it is now clear that some of the anatomical parallels that led Gegenabur to propose his gill arch hypothesis of fin origins reflect common underlying patterning mechanisms, and further investigation of the molecular basis of branchial ray patterning in cartilaginous fishes will allow us to determine whether these common mechanisms are the result of parallel evolution or convergence.
I think that this study sets out an exciting path forward to address the origin and evolution of paired appendages in vertebrates, and highlights how complementary palaeontological and developmental approaches are needed in order to truly address the big, unanswered questions in vertebrate body plan evolution.
Gegenbaur, C Elements of Comparative Anatomy. London, UK: Macmillan. Gillis, J. Development , Owen, R. Fishes and Reptiles. London, U. Riddle, R. Cell 75 , Roth, V. Saunders, J. Ectodermal and mesenchymal interactions in the origin of limb symmetry. In Epithelial Mesenchymal Interactions Ed. Fleischmajer and R. Baltimore, William and Wilkins, pp. Stopper, G. Towers, M. Yin, Y. Nature , Van Valen, L. Wagner, G. Zhu, J. Tags: evo devo , skate , woods hole Categories: Research.
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