J. Andrew Gillis

Shared developmental mechanisms pattern the vertebrate gill arch and paired fin skeletons.

Here, we describe the molecular patterning of chondrichthyan branchial rays (gill rays) and reveal profound developmental similarities between gill rays and vertebrate appendages. Sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8) regulate the outgrowth and patterning of the chondrichthyan gill arch skeleton, in an interdependent manner similar to their roles in gnathostome paired appendages. Additionally, we demonstrate that paired appendages and branchial rays share other conserved developmental features, including Shh-mediated mirror-image duplications of the endoskeleton after exposure to retinoic acid, and Fgf8 expression by a pseudostratified distal epithelial ridge directing endoskeletal outgrowth. These data suggest that the skeletal patterning role of the retinoic acid/Shh/Fgf8 regulatory circuit has a deep evolutionary origin predating vertebrate paired appendages and may have functioned initially in patterning pharyngeal structures in a deuterostome ancestor of vertebrates.

A Natural Deletion of the HoxC Cluster in Elasmobranch Fishes

Sequence analysis shows that sharks and batoids lack one of four Hox gene clusters once thought necessary for development. Hox proteins are a metazoan-specific family of transcription factors that are required for developmental patterning. The genomic arrangement of Hox genes into four paralogous clusters is a primitive feature of jawed vertebrates. By using high-throughput sequencing, we demonstrate the absence of all HoxC transcripts from embryos of the shark Scyliorhinus canicula and the skate Leucoraja erinacea and the absence of all HoxC genes and two HoxC-associated microRNAs from the genome of L. erinacea. These data suggest a loss of the entire HoxC cluster in elasmobranch fishes and represent evidence for the natural deletion of an entire Hox cluster in vertebrates.

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.

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.

The evolution and development of vertebrate lateral line electroreceptors

Summary 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.

Chondrogenesis and homology of the visceral skeleton in the little skate, Leucoraja erinacea (Chondrichthyes: Batoidea)

Chondrichthyan fishes possess visceral skeletons that differ considerably, morphologically, from those of their sister taxon, the osteichthyans. Here, we use histological techniques and whole‐mount skeletal preparations to visualize and describe the sequence of visceral skeletal condensation and chondrogenesis in a chondrichthyan, the little skate (Leucoraja erinacea). We demonstrate that visceral skeletal condensation begins rostrally, with the mandibular arch, and progresses caudally with the hyoid arch and posterior branchial arches condensing soon after. We provide a detailed account of the condensation and chondrogenesis of all major components of the L. erinacea visceral skeleton and discuss these data in the context of what is known from classical descriptions of chondrichthyan visceral skeletal development. Significant differences exist between the hypobranchial and basibranchial skeleton of L. erinacea and other chondrichthyan species, and the possible evolutionary and developmental significance of this is considered. We discuss the homology of the chondrichthyan hyoid arch and, based on patterns of mesenchymal condensation, we propose a model of condensation splitting and diversification that may account for the morphological diversification of gnathostome branchial arch derivatives. Finally, we suggest that the unique presence of certain visceral skeletal elements in chondrichthyans make oviparous chondrichthyans an ideal system for addressing questions of endoskeletal axial patterning during development. J. Morphol., 2009.

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 a sonic 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 Gegenbaurs gill arch hypothesis. Summary: Shh signalling polarises skate gill arches and maintains proliferative expansion of gill arch appendage endoskeletal progenitors, mirroring the function of Shh signalling in the tetrapod limb.

The evolution of gnathostome development: Insight from chondrichthyan embryology

Chondrichthyans (cartilaginous fishes) represent one of the two lineages of gnathostomes, the other being the osteicthyans (bony fishes). Classical studies on chondrichthyan embryology have strongly impacted our views of vertebrate body plan evolution, while recent studies highlight oviparous chondrichthyans as emerging vertebrate model systems that are amenable to experimental embryological manipulation. Here, we review three particular areas of interest in the field of chondrichthyan developmental biology—gastrulation, neural development, and appendage patterning—and we discuss recentfindings within a broader chondrichthyan‐osteichthyan comparative framework. In some cases, comparative studies of chondrichthyan and osteichthyan development reveal conserved patterns of gene expression in common developmental contexts. Studies of chondrichthyan gastrulation reveal conserved patterns of developmental gene expression, despite highly divergent modes of mesendoderm internalization, while molecular characterization of chondrichthyan neurogenic placodes indicates a conservation of placode transcription factor expression across gnathostome phylogeny. In other cases, comparative studies of chondrichthyan and osteichthyan development yield evidence of shared patterning mechanisms functioning in different developmental contexts. This is exemplified by studies on the development of chondrichthyan appendages—paired fins, median fins, and gill rays. These have demonstrated that a retinoic acid‐responsive Shh‐expressing signaling center functions to pattern the endoskeleton of gnathostome paired fins and chondrichthyan gill rays, while expression patterns of Tbx18 and HoxD family members are shared by gnathostome paired fins and chondrichthyan median fins. These findings fuel novel hypotheses of developmental genetic homology, and demonstrate how comparative studies of gnathostome development can provide insight into the evolutionary processes that underlie morphological diversity. genesis 47:825–841, 2009.

Embryonic development of the axial column in the little skate, Leucoraja erinacea

The morphological patterns and molecular mechanisms of vertebral column development are well understood in bony fishes (osteichthyans). However, vertebral column morphology in elasmobranch chondrichthyans (e.g., sharks and skates) differs from that of osteichthyans, and its development has not been extensively studied. Here, we characterize vertebral development in an elasmobranch fish, the little skate, Leucoraja erinacea, using microCT, paraffin histology, and whole‐mount skeletal preparations. Vertebral development begins with the condensation of mesenchyme, first around the notochord, and subsequently around the neural tube and caudal artery and vein. Mesenchyme surrounding the notochord differentiates into a continuous sheath of spindle‐shaped cells, which forms the precursor to the mineralized areolar calcification of the centrum. Mesenchyme around the neural tube and caudal artery/vein becomes united by a population of mesenchymal cells that condenses lateral to the sheath of spindle‐shaped cells, with this mesenchymal complex eventually differentiating into the hyaline cartilage of the future neural arches, hemal arches, and outer centrum. The initially continuous layers of areolar tissue and outer hyaline cartilage eventually subdivide into discrete centra and arches, with the notochord constricted in the center of each vertebra by a late‐forming “inner layer” of hyaline cartilage, and by a ring of areolar calcification located medial to the outer vertebral cartilage. The vertebrae of elasmobranchs are distinct among vertebrates, both in terms of their composition (i.e., with centra consisting of up to three tissues layers—an inner cartilage layer, a calcified areolar ring, and an outer layer of hyaline cartilage), and their mode of development (i.e., the subdivision of arch and outer centrum cartilage from an initially continuous layer of hyaline cartilage). Given the evident variation in patterns of vertebral construction, broad taxon sampling, and comparative developmental analyses are required to understand the diversity of mechanisms at work in the developing axial skeleton of vertebrates. J. Morphol. 278:300–320, 2017.

The origin of vertebrate gills

Summary Pharyngeal gills are a fundamental feature of the vertebrate body plan [1]. However, the evolutionary history of vertebrate gills has been the subject of a long-standing controversy [2, 3, 4, 5, 6, 7, 8]. It is thought that gills evolved independently in cyclostomes (jawless vertebrates—lampreys and hagfish) and gnathostomes (jawed vertebrates—cartilaginous and bony fishes), based on their distinct embryonic origins: the gills of cyclostomes derive from endoderm [9, 10, 11, 12], while gnathostome gills were classically thought to derive from ectoderm [10, 13]. Here, we demonstrate by cell lineage tracing that the gills of a cartilaginous fish, the little skate (Leucoraja erinacea), are in fact endodermally derived. This finding supports the homology of gills in cyclostomes and gnathostomes, and a single origin of pharyngeal gills prior to the divergence of these two ancient vertebrate lineages.