Ion regulation at gills precedes gas exchange and the origin of vertebrates.

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1-4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6-9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchange that is unrelated to vertebrate size and activity-perhaps at the very inception of pharyngeal pores in stem deuterostomes.

Fgf signalling is required for gill slit formation in the skate, Leucoraja erinacea.

The gill slits of fishes develop from an iterative series of pharyngeal endodermal pouches that contact and fuse with surface ectoderm on either side of the embryonic head. We find in the skate (Leucoraja erinacea) that all gill slits form via a stereotypical sequence of epithelial interactions: 1) endodermal pouches approach overlying surface ectoderm, with 2) focal degradation of ectodermal basement membranes preceding endoderm-ectoderm contact; 3) endodermal pouches contact and intercalate with overlying surface ectoderm, and finally 4) perforation of a gill slit occurs by epithelial remodelling, without programmed cell death, at the site of endoderm-ectoderm intercalation. Skate embryos express Fgf8 and Fgf3 within developing pharyngeal epithelia during gill slit formation. When we inhibit Fgf signalling by treating skate embryos with the Fgf receptor inhibitor SU5402 we find that endodermal pouch formation, basement membrane degradation and endodermal-ectodermal intercalation are unaffected, but that epithelial remodelling and gill slit perforation fail to occur. These findings point to a role for Fgf signalling in epithelial remodelling during gill slit formation in the skate and, more broadly, to an ancestral role for Fgf signalling during pharyngeal pouch epithelial morphogenesis in vertebrate embryos.

The pseudobranch of jawed vertebrates is a mandibular arch-derived gill.

The pseudobranch is a gill-like epithelial elaboration that sits behind the jaw of most fishes. This structure was classically regarded as a vestige of the ancestral gill arch-like condition of the gnathostome jaw. However, more recently, hypotheses of jaw evolution by transformation of a gill arch have been challenged, and the pseudobranch has alternatively been considered a specialised derivative of the second (hyoid) pharyngeal arch. Here, we demonstrate in the skate (Leucoraja erinacea) that the pseudobranch does, in fact, derive from the mandibular arch, and that it shares gene expression features and cell types with gills. We also show that the skate mandibular arch pseudobranch is supported by a spiracular cartilage that is patterned by a shh-expressing epithelial signalling centre. This closely parallels the condition seen in the gill arches, where cartilaginous appendages called branchial rays, which support the respiratory lamellae of the gills, are patterned by a shh-expressing gill arch epithelial ridge. Together with similar discoveries in zebrafish, our findings support serial homology of the pseudobranch and gills, and an ancestral origin of gill arch-like anatomical features from the gnathostome mandibular arch.

Evolution of the new head by gradual acquisition of neural crest regulatory circuits.

The neural crest, an embryonic stem-cell population, is a vertebrate innovation that has been proposed to be a key component of the 'new head', which imbued vertebrates with predatory behaviour1,2. Here, to investigate how the evolution of neural crest cells affected the vertebrate body plan, we examined the molecular circuits that control neural crest development along the anteroposterior axis of a jawless vertebrate, the sea lamprey. Gene expression analysis showed that the cranial subpopulation of the neural crest of the lamprey lacks most components of a transcriptional circuit that is specific to the cranial neural crest in amniotes and confers the ability to form craniofacial cartilage onto non-cranial neural crest subpopulations3. Consistent with this, hierarchical clustering analysis revealed that the transcriptional profile of the lamprey cranial neural crest is more similar to the trunk neural crest of amniotes. Notably, analysis of the cranial neural crest in little skate and zebrafish embryos demonstrated that the transcriptional circuit that is specific to the cranial neural crest emerged via the gradual addition of network components to the neural crest of gnathostomes, which subsequently became restricted to the cephalic region. Our results indicate that the ancestral neural crest at the base of the vertebrate lineage possessed a trunk-like identity. We propose that the emergence of the cranial neural crest, by progressive assembly of an axial-specific regulatory circuit, allowed the elaboration of the new head during vertebrate evolution.

hox gene expression predicts tetrapod-like axial regionalization in the skate, Leucoraja erinacea.

The axial skeleton of tetrapods is organized into distinct anteroposterior regions of the vertebral column (cervical, trunk, sacral, and caudal), and transitions between these regions are determined by colinear anterior expression boundaries of Hox5/6, -9, -10, and -11 paralogy group genes within embryonic paraxial mesoderm. Fishes, conversely, exhibit little in the way of discrete axial regionalization, and this has led to scenarios of an origin of Hox-mediated axial skeletal complexity with the evolutionary transition to land in tetrapods. Here, combining geometric morphometric analysis of vertebral column morphology with cell lineage tracing of hox gene expression boundaries in developing embryos, we recover evidence of at least five distinct regions in the vertebral skeleton of a cartilaginous fish, the little skate (Leucoraja erinacea). We find that skate embryos exhibit tetrapod-like anteroposterior nesting of hox gene expression in their paraxial mesoderm, and we show that anterior expression boundaries of hox5/6, hox9, hox10, and hox11 paralogy group genes predict regional transitions in the differentiated skate axial skeleton. Our findings suggest that hox-based axial skeletal regionalization did not originate with tetrapods but rather has a much deeper evolutionary history than was previously appreciated.

Distinct proliferative and middle ear skeletal-patterning functions for SHH-expressing epithelia in the chick hyoid arch.

During chick craniofacial development, the second (hyoid) pharyngeal arch expands to close the neck and gives rise to skeletal elements, including the columella of the middle ear (a homologue of the mammalian stapes). Sonic hedgehog (SHH) signalling has been implicated in hyoid arch expansion and columella formation, but spatial and temporal aspects of these signalling interactions within the hyoid arch remain poorly understood. Here, we show that SHH is initially expressed in the posterior endoderm of the hyoid arch, and that this domain subsequently splits into a distal domain at the site of arch expansion (the posterior epithelial margin, PEM), and a proximal domain that lines the foregut (the proximal hyoid epithelium, PHE). Pharmacological manipulations and heterotopic grafting experiments demonstrate that SHH signalling is required for hyoid arch expansion and skeletogenesis, and reveal distinct roles for the PEM and PHE in these processes. The PEM promotes mesenchymal cell proliferation during arch expansion but is not sufficient to repattern the columella. Conversely, the PHE promotes mesenchymal cell survival, and PHE grafts induce partial duplication of the columella. This work demonstrates crucial and distinct roles for endodermal SHH signalling in hyoid arch morphogenesis and patterning of the middle ear skeleton.

Evolution of vertebrate gill covers via shifts in an ancient Pou3f3 enhancer.

Whereas the gill chambers of jawless vertebrates open directly into the environment, jawed vertebrates evolved skeletal appendages that drive oxygenated water unidirectionally over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single large gill cover in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here, we find that these divergent patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs. We identify a deeply conserved Pou3f3 arch enhancer present in humans through sharks but undetectable in jawless fish. Minor differences between the bony and cartilaginous fish enhancers account for their restricted versus pan-arch expression patterns. In zebrafish, mutation of Pou3f3 or the conserved enhancer disrupts gill cover formation, whereas ectopic pan-arch Pou3f3b expression generates ectopic skeletal elements resembling the multimeric covers of cartilaginous fishes. Emergence of this Pou3f3 arch enhancer >430 Mya and subsequent modifications may thus have contributed to the acquisition and diversification of gill covers and respiratory strategies during gnathostome evolution.

Conserved and unique transcriptional features of pharyngeal arches in the skate (Leucoraja erinacea) and evolution of the jaw.

The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral (DV) patterning mechanisms within the developing mandibular, hyoid, and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralizing BMP and endothelin signaling pathways and their effectors, the joint markers nkx3.2 and gdf5 and prochondrogenic transcription factor barx1, and the dorsal territory marker pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, whereas differences in notch signaling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the DV axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

Trunk neural crest origin of dermal denticles in a cartilaginous fish.

Cartilaginous fishes (e.g., sharks and skates) possess a postcranial dermal skeleton consisting of tooth-like "denticles" embedded within their skin. As with teeth, the principal skeletal tissue of dermal denticles is dentine. In the head, cranial neural crest cells give rise to the dentine-producing cells (odontoblasts) of teeth. However, trunk neural crest cells are generally regarded as nonskeletogenic, and so the embryonic origin of trunk denticle odontoblasts remains unresolved. Here, we use expression of FoxD3 to pinpoint the specification and emigration of trunk neural crest cells in embryos of a cartilaginous fish, the little skate (Leucoraja erinacea). Using cell lineage tracing, we further demonstrate that trunk neural crest cells do, in fact, give rise to odontoblasts of trunk dermal denticles. These findings expand the repertoire of vertebrate trunk neural crest cell fates during normal development, highlight the likely primitive skeletogenic potential of this cell population, and point to a neural crest origin of dentine throughout the ancestral vertebrate dermal skeleton.

A shared role for sonic hedgehog signalling in patterning chondrichthyan gill arch appendages and tetrapod limbs.

Chondrichthyans (sharks, skates, rays and holocephalans) possess paired appendages that project laterally from their gill arches, known as branchial rays. This led Carl Gegenbaur to propose that paired fins (and hence tetrapod limbs) originally evolved via transformation of gill arches. Tetrapod limbs are patterned by asonic hedgehog(Shh)-expressing signalling centre known as the zone of polarising activity, which establishes the anteroposterior axis of the limb bud and maintains proliferative expansion of limb endoskeletal progenitors. Here, we use loss-of-function, label-retention and fate-mapping approaches in the little skate to demonstrate that Shh secretion from a signalling centre in the developing gill arches establishes gill arch anteroposterior polarity and maintains the proliferative expansion of branchial ray endoskeletal progenitor cells. These findings highlight striking parallels in the axial patterning mechanisms employed by chondrichthyan branchial rays and paired fins/limbs, and provide mechanistic insight into the anatomical foundation of Gegenbaur's gill arch hypothesis.

Embryonic origin of the gnathostome vertebral skeleton.

The vertebral column is a key component of the jawed vertebrate (gnathostome) body plan, but the primitive embryonic origin of this skeleton remains unclear. In tetrapods, all vertebral components (neural arches, haemal arches and centra) derive from paraxial mesoderm (somites). However, in teleost fishes, vertebrae have a dual embryonic origin, with arches derived from somites, but centra formed, in part, by secretion of bone matrix from the notochord. Here, we test the embryonic origin of the vertebral skeleton in a cartilaginous fish (the skate, Leucoraja erinacea) which serves as an outgroup to tetrapods and teleosts. We demonstrate, by cell lineage tracing, that both arches and centra are somite-derived. We find no evidence of cellular or matrix contribution from the notochord to the skate vertebral skeleton. These findings indicate that the earliest gnathostome vertebral skeleton was exclusively of somitic origin, with a notochord contribution arising secondarily in teleosts.

Electrosensory ampullary organs are derived from lateral line placodes in cartilaginous fishes.

Ampullary organ electroreceptors excited by weak cathodal electric fields are used for hunting by both cartilaginous and non-teleost bony fishes. Despite similarities of neurophysiology and innervation, their embryonic origins remain controversial: bony fish ampullary organs are derived from lateral line placodes, whereas a neural crest origin has been proposed for cartilaginous fish electroreceptors. This calls into question the homology of electroreceptors and ampullary organs in the two lineages of jawed vertebrates. Here, we test the hypothesis that lateral line placodes form electroreceptors in cartilaginous fishes by undertaking the first long-term in vivo fate-mapping study in any cartilaginous fish. Using DiI tracing for up to 70 days in the little skate, Leucoraja erinacea, we show that lateral line placodes form both ampullary electroreceptors and mechanosensory neuromasts. These data confirm the homology of electroreceptors and ampullary organs in cartilaginous and non-teleost bony fishes, and indicate that jawed vertebrates primitively possessed a lateral line placode-derived system of electrosensory ampullary organs and mechanosensory neuromasts.

Holocephalan embryos provide evidence for gill arch appendage reduction and opercular evolution in cartilaginous fishes.

Chondrichthyans possess endoskeletal appendages called branchial rays that extend laterally from their hyoid and gill-bearing (branchial) arches. Branchial ray outgrowth, like tetrapod limb outgrowth, is maintained by Sonic hedgehog (Shh) signaling. In limbs, distal endoskeletal elements fail to form in the absence of normal Shh signaling, whereas shortened duration of Shh expression correlates with distal endoskeletal reduction in naturally variable populations. Chondrichthyans also exhibit natural variation with respect to branchial ray distribution--elasmobranchs (sharks and batoids) possess a series of ray-supported septa on their hyoid and gill arches, whereas holocephalans (chimaeras) possess a single hyoid arch ray-supported operculum. Here we show that the elongate hyoid rays of the holocephalan Callorhinchus milii grow in association with sustained Shh expression within an opercular epithelial fold, whereas Shh is only transiently expressed in the gill arches. Coincident with this transient Shh expression, branchial ray outgrowth is initiated in C. milii but is not maintained, yielding previously unrecognized vestigial gill arch branchial rays. This is in contrast to the condition seen in sharks, where sustained Shh expression corresponds to the presence of fully formed branchial rays on the hyoid and gill arches. Considered in light of current hypotheses of chondrichthyan phylogeny, our data suggest that the holocephalan operculum evolved in concert with gill arch appendage reduction by attenuation of Shh-mediated branchial ray outgrowth, and that chondrichthyan branchial rays and tetrapod limbs exhibit parallel developmental mechanisms of evolutionary reduction.

The origins of gas exchange and ion regulation in fish gills: evidence from structure and function.

Gill function in gas exchange and ion regulation has played key roles in the evolution of fishes. In this review, we summarize data from the fields of palaeontology, developmental biology and comparative physiology for when and how the gills first acquired these functions. Data from across disciplines strongly supports a stem vertebrate origin for gas exchange structures and function at the gills with the emergence of larger, more active fishes. However, the recent discovery of putative ionocytes in extant cephalochordates and hemichordates suggests that ion regulation at gills might have originated much earlier than gas exchange, perhaps in the ciliated pharyngeal arches in the last common ancestor of deuterostomes. We hypothesize that the ancestral form of ion regulation served a filter-feeding function in the ciliated pharyngeal arches, and was later coopted in vertebrates to regulate extracellular ion and acid-base balance. We propose that future research should explore ionocyte homology and function across extant deuterostomes to test this hypothesis and others in order to determine the ancestral origins of ion regulation in fish gills.

Identification of multiple transcription factor genes potentially involved in the development of electrosensory versus mechanosensory lateral line organs.

In electroreceptive jawed vertebrates, embryonic lateral line placodes give rise to electrosensory ampullary organs as well as mechanosensory neuromasts. Previous reports of shared gene expression suggest that conserved mechanisms underlie electroreceptor and mechanosensory hair cell development and that electroreceptors evolved as a transcriptionally related "sister cell type" to hair cells. We previously identified only one transcription factor gene, Neurod4, as ampullary organ-restricted in the developing lateral line system of a chondrostean ray-finned fish, the Mississippi paddlefish (Polyodon spathula). The other 16 transcription factor genes we previously validated in paddlefish were expressed in both ampullary organs and neuromasts. Here, we used our published lateral line organ-enriched gene-set (arising from differential bulk RNA-seq in late-larval paddlefish), together with a candidate gene approach, to identify 25 transcription factor genes expressed in the developing lateral line system of a more experimentally tractable chondrostean, the sterlet (Acipenser ruthenus, a small sturgeon), and/or that of paddlefish. Thirteen are expressed in both ampullary organs and neuromasts, consistent with conservation of molecular mechanisms. Seven are electrosensory-restricted on the head (Irx5, Irx3, Insm1, Sp5, Satb2, Mafa and Rorc), and five are the first-reported mechanosensory-restricted transcription factor genes (Foxg1, Sox8, Isl1, Hmx2 and Rorb). However, as previously reported, Sox8 is expressed in ampullary organs as well as neuromasts in a catshark (Scyliorhinus canicula), suggesting the existence of lineage-specific differences between cartilaginous and ray-finned fishes. Overall, our results support the hypothesis that ampullary organs and neuromasts develop via largely conserved transcriptional mechanisms, and identify multiple transcription factors potentially involved in the formation of electrosensory versus mechanosensory lateral line organs.

Incremental evolution of the neural crest, neural crest cells and neural crest-derived skeletal tissues.

Urochordates (ascidians) have recently supplanted cephalochordates (amphioxus) as the extant sister taxon of vertebrates. Given that urochordates possess migratory cells that have been classified as 'neural crest-like'- and that cephalochordates lack such cells--this phylogenetic hypothesis may have significant implications with respect to the origin of the neural crest and neural crest-derived skeletal tissues in vertebrates. We present an overview of the genes and gene regulatory network associated with specification of the neural crest in vertebrates. We then use these molecular data--alongside cell behaviour, cell fate and embryonic context--to assess putative antecedents (latent homologues) of the neural crest or neural crest cells in ascidians and cephalochordates. Ascidian migratory mesenchymal cells--non-pigment-forming trunk lateral line cells and pigment-forming 'neural crest-like cells' (NCLC)--are unlikely latent neural crest cell homologues. Rather, Snail-expressing cells at the neural plate of border of urochordates and cephalochordates likely represent the extent of neural crest elaboration in non-vertebrate chordates. We also review evidence for the evolutionary origin of two neural crest-derived skeletal tissues--cartilage and dentine. Dentine is a bona fide vertebrate novelty, and dentine-secreting odontoblasts represent a cell type that is exclusively derived from the neural crest. Cartilage, on the other hand, likely has a much deeper origin within the Metazoa. The mesodermally derived cellular cartilages of some protostome invertebrates are much more similar to vertebrate cartilage than is the acellular 'cartilage-like' tissue in cephalochordate pharyngeal arches. Cartilage, therefore, is not a vertebrate novelty, and a well-developed chondrogenic program was most likely co-opted from mesoderm to the neural crest along the vertebrate stem. We conclude that the neural crest is a vertebrate novelty, but that neural crest cells and their derivatives evolved and diversified in a step-wise fashion--first by elaboration of neural plate border cells, then by the innovation or co-option of new or ancient metazoan cell fates.

The evolution and development of vertebrate lateral line electroreceptors.

Electroreception is an ancient vertebrate sense with a fascinating evolutionary history involving multiple losses as well as independent evolution at least twice within teleosts. We review the phylogenetic distribution of electroreception and the morphology and innervation of electroreceptors in different vertebrate groups. We summarise recent work from our laboratory that has confirmed the homology of ampullary electroreceptors in non-teleost jawed vertebrates by showing, in conjunction with previously published work, that these are derived embryonically from lateral line placodes. Finally, we review hypotheses to explain the distribution of electroreception within teleosts, including the hypothesis that teleost ampullary and tuberous electroreceptors evolved via the modification of mechanosensory hair cells in lateral line neuromasts. We conclude that further experimental work on teleost electroreceptor development is needed to test such hypotheses.

Ectodermal Wnt signaling, cell fate determination, and polarity of the skate gill arch skeleton.

The gill skeleton of cartilaginous fishes (sharks, skates, rays, and holocephalans) exhibits a striking anterior-posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Sonic hedgehog (Shh) signaling from a distal gill arch epithelial ridge (GAER) signaling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here, we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.

A stem-deuterostome origin of the vertebrate pharyngeal transcriptional network.

Hemichordate worms possess ciliated gills on their trunk, and the homology of these structures with the pharyngeal gill slits of chordates has long been a topic of debate in the fields of evolutionary biology and comparative anatomy. Here, we show conservation of transcription factor expression between the developing pharyngeal gill pores of the hemichordate Saccoglossus kowalevskii and the pharyngeal gill slit precursors (i.e. pharyngeal endodermal outpockets) of vertebrates. Transcription factors that are expressed in the pharyngeal endoderm, ectoderm and mesenchyme of vertebrates are expressed exclusively in the pharyngeal endoderm of S. kowalevskii. The pharyngeal arches and tongue bars of S. kowalevskii lack Tbx1-expressing mesoderm, and are supported solely by an acellular collagenous endoskeleton and by compartments of the trunk coelom. Our findings suggest that hemichordate and vertebrate gills are homologous as simple endodermal outpockets from the foregut, and that much vertebrate pharyngeal complexity arose coincident with the incorporation of cranial paraxial mesoderm and neural crest-derived mesenchyme within pharyngeal arches along the chordate and vertebrate stems, respectively.