EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE DURING THE EMERGENCE OF DIGITS AND GENITALS.

The transition from fins to limbs has been a rich source of discussion for more than a century. One open and important issue is understanding how the mechanisms that pattern digits arose during vertebrate evolution. In this context, the analysis of Hox gene expression and functions to infer evolutionary scenarios has been a productive approach to explain the changes in organ formation, particularly in limbs. In tetrapods, the transcription of Hoxd genes in developing digits depends on a well-characterized set of enhancers forming a large regulatory landscape1,2. This control system has a syntenic counterpart in zebrafish, even though they lack bona fide digits, suggestive of deep homology3 between distal fin and limb developmental mechanisms. We tested the global function of this landscape to assess ancestry and source of limb and fin variation. In contrast to results in mice, we show here that the deletion of the homologous control region in zebrafish has a limited effect on the transcription of hoxd genes during fin development. However, it fully abrogates hoxd expression within the developing cloaca, an ancestral structure related to the mammalian urogenital sinus. We show that similar to the limb, Hoxd gene function in the urogenital sinus of the mouse also depends on enhancers located in this same genomic domain. Thus, we conclude that the current regulation underlying Hoxd gene expression in distal limbs was co-opted in tetrapods from a preexisting cloacal program. The orthologous chromatin domain in fishes may illustrate a rudimentary or partial step in this evolutionary co-option.

The axial skeleton of Tiktaalik roseae.

The axial columns of the earliest limbed vertebrates show distinct patterns of regionalization as compared to early tetrapodomorphs. Included among their novel features are sacral ribs, which provide linkage between the vertebral column and pelvis, contributing to body support and propulsion by the hindlimb. Data on the axial skeletons of the closest relatives of limbed vertebrates are sparce, with key features of specimens potentially covered by matrix. Therefore, it is unclear in what sequence and under what functional context specializations in the axial skeletons of tetrapods arose. Here, we describe the axial skeleton of the elpistostegalian Tiktaalik roseae and show that transformations to the axial column for head mobility, body support, and pelvic fin buttressing evolved in finned vertebrates prior to the origin of limbs. No atlas-axis complex is observed; however, an independent basioccipital-exoccipital complex suggests increased mobility at the occipital vertebral junction. While the construction of vertebrae in Tiktaalik is similar to early tetrapodomorphs, its ribs possess a specialized sacral domain. Sacral ribs are expanded and ventrally curved, indicating likely attachment to the expanded iliac blade of the pelvis by ligamentous connection. Thus, the origin of novel rib types preceded major alterations to trunk vertebrae, and linkage between pelvic fins and axial column preceded the origin of limbs. These data reveal an unexpected combination of post-cranial skeletal characters, informing hypotheses of body posture and movement in the closest relatives of limbed vertebrates.

Identification of ancestral gnathostome Gli3 enhancers with activity in mammals.

Abnormal expression of the transcriptional regulator and hedgehog (Hh) signaling pathway effector Gli3 is known to trigger congenital disease, most frequently affecting the central nervous system (CNS) and the limbs. Accurate delineation of the genomic cis-regulatory landscape controlling Gli3 transcription during embryonic development is critical for the interpretation of noncoding variants associated with congenital defects. Here, we employed a comparative genomic analysis on fish species with a slow rate of molecular evolution to identify seven previously unknown conserved noncoding elements (CNEs) in Gli3 intronic intervals (CNE15-21). Transgenic assays in zebrafish revealed that most of these elements drive activities in Gli3 expressing tissues, predominantly the fins, CNS, and the heart. Intersection of these CNEs with human disease associated SNPs identified CNE15 as a putative mammalian craniofacial enhancer, with conserved activity in vertebrates and potentially affected by mutation associated with human craniofacial morphology. Finally, comparative functional dissection of an appendage-specific CNE conserved in slowly evolving fish (elephant shark), but not in teleost (CNE14/hs1586) indicates co-option of limb specificity from other tissues prior to the divergence of amniotes and lobe-finned fish. These results uncover a novel subset of intronic Gli3 enhancers that arose in the common ancestor of gnathostomes and whose sequence components were likely gradually modified in other species during the process of evolutionary diversification.

The origin of blinking in both mudskippers and tetrapods is linked to life on land.

Blinking, the transient occlusion of the eye by one or more membranes, serves several functions including wetting, protecting, and cleaning the eye. This behavior is seen in nearly all living tetrapods and absent in other extant sarcopterygian lineages suggesting that it might have arisen during the water-to-land transition. Unfortunately, our understanding of the origin of blinking has been limited by a lack of known anatomical correlates of the behavior in the fossil record and a paucity of comparative functional studies. To understand how and why blinking originates, we leverage mudskippers (Oxudercinae), a clade of amphibious fishes that have convergently evolved blinking. Using microcomputed tomography and histology, we analyzed two mudskipper species, Periophthalmus barbarus and Periophthalmodon septemradiatus, and compared them to the fully aquatic round goby, Neogobius melanostomus. Study of gross anatomy and epithelial microstructure shows that mudskippers have not evolved novel musculature or glands to blink. Behavioral analyses show the blinks of mudskippers are functionally convergent with those of tetrapods: P. barbarus blinks more often under high-evaporation conditions to wet the eye, a blink reflex protects the eye from physical insult, and a single blink can fully clean the cornea of particulates. Thus, eye retraction in concert with a passive occlusal membrane can achieve functions associated with life on land. Osteological correlates of eye retraction are present in the earliest limbed vertebrates, suggesting blinking capability. In both mudskippers and tetrapods, therefore, the origin of this multifunctional innovation is likely explained by selection for increasingly terrestrial lifestyles.

The little skate genome and the evolutionary emergence of wing-like fins.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins-including gene expression, chromatin occupancy and three-dimensional conformation-we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Evolutionary relevance of single nucleotide variants within the forebrain exclusive human accelerated enhancer regions.

BACKGROUND: Human accelerated regions (HARs) are short conserved genomic sequences that have acquired significantly more nucleotide substitutions than expected in the human lineage after divergence from chimpanzees. The fast evolution of HARs may reflect their roles in the origin of human-specific traits. A recent study has reported positively-selected single nucleotide variants (SNVs) within brain-exclusive human accelerated enhancers (BE-HAEs) hs1210 (forebrain), hs563 (hindbrain) and hs304 (midbrain/forebrain). By including data from archaic hominins, these SNVs were shown to be Homo sapiens-specific, residing within transcriptional factors binding sites (TFBSs) for SOX2 (hs1210), RUNX1/3 (hs563), and FOS/JUND (hs304). Although these findings suggest that the predicted modifications in TFBSs may have some role in present-day brain structure, work is required to verify the extent to which these changes translate into functional variation. RESULTS: To start to fill this gap, we investigate the SOX2 SNV, with both forebrain expression and strong signal of positive selection in humans. We demonstrate that the HMG box of SOX2 binds in vitro with Homo sapiens-specific derived A-allele and ancestral T-allele carrying DNA sites in BE-HAE hs1210. Molecular docking and simulation analysis indicated highly favourable binding of HMG box with derived A-allele containing DNA site when compared to site carrying ancestral T-allele. CONCLUSION: These results suggest that adoptive changes in TF affinity within BE-HAE hs1210 and other HAR enhancers in the evolutionary history of Homo sapiens might. have brought about changes in gene expression patterns and have functional consequences on forebrain formation and evolution. METHODS: The present study employ electrophoretic mobility shift assays (EMSA) and molecular docking and molecular dynamics simulations approaches.

Ossification patterns of the carpus and tarsus in salamanders and impacts of preaxial dominance on the fin-to-limb transition.

Early limb skeletogenesis in salamanders is characterized by preaxial elements, digits I and II forming earlier than their postaxial counterparts (digits III to V), a phenomenon known as preaxial dominance, whereas in amniotes and anurans, these developmental sequences are reversed. This pattern characterizes the late skeletogenesis of digits and zeugopodium of anamniote tetrapods but remains unknown in carpals/tarsals. To correct this gap in knowledge, we investigate the ossification patterns of the carpals/tarsals in six salamander families/clades based on micro-computed tomography scans. We found that preaxial dominance is seen in the distal carpals/tarsals of several salamander clades and diverse early tetrapods, such as temnospondyls and amniotes. This distribution suggests that preaxial dominance is a primitive developmental pattern in tetrapods. Our results demonstrate that the distal carpals/tarsals are developmentally and evolutionarily independent in the autopodium, and preaxial dominance facilitates stabilization of the number of distal carpals/tarsals during fin-to-limb transition and digit reduction in early tetrapods.

A new elpistostegalian from the Late Devonian of the Canadian Arctic.

A fundamental gap in the study of the origin of limbed vertebrates lies in understanding the morphological and functional diversity of their closest relatives. Whereas analyses of the elpistostegalians Panderichthys rhombolepis, Tiktaalik roseae and Elpistostege watsoni have revealed a sequence of changes in locomotor, feeding and respiratory structures during the transition1-9, an isolated bone, a putative humerus, has controversially hinted at a wider range in form and function than now recognized10-14. Here we report the discovery of a new elpistostegalian from the Late Devonian period of the Canadian Arctic that shows surprising disparity in the group. The specimen includes partial upper and lower jaws, pharyngeal elements, a pectoral fin and scalation. This new genus is phylogenetically proximate to T. roseae and E. watsoni but evinces notable differences from both taxa and, indeed, other described tetrapodomorphs. Lacking processes, joint orientations and muscle scars indicative of appendage-based support on a hard substrate13, its pectoral fin shows specializations for swimming that are unlike those known from other sarcopterygians. This unexpected morphological and functional diversity represents a previously hidden ecological expansion, a secondary return to open water, near the origin of limbed vertebrates.

An Fgf-Shh positive feedback loop drives growth in developing unpaired fins.

The origin and diversification of appendage types is a central question in vertebrate evolution. Understanding the genetic mechanisms that underlie fin and limb development can reveal relationships between different appendages. Here we demonstrate, using chemical genetics, a mutually agonistic interaction between Fgf and Shh genes in the developing dorsal fin of the channel catfish, Ictalurus punctatus. We also find that Fgf8 and Shh orthologs are expressed in the apical ectodermal ridge and zone of polarizing activity, respectively, in the median fins of representatives from other major vertebrate lineages. These findings demonstrate the importance of this feedback loop in median fins and offer developmental evidence for a median fin-first scenario for vertebrate paired appendage origins.

The Shh/Gli3 gene regulatory network precedes the origin of paired fins and reveals the deep homology between distal fins and digits.

One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among dorsal and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3-null mutant mice. In limbs, Gli3 controls both anterior-posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and dorsal fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 down-regulation in shh mutant fins rescues fin loss in a manner similar to how Gli3 deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh gene pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of dorsal fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.

Bacterial community dynamics during embryonic development of the little skate (Leucoraja erinacea).

BACKGROUND: Microbial transmission from parent to offspring is hypothesized to be widespread in vertebrates. However, evidence for this is limited as many evolutionarily important clades remain unexamined. There is currently no data on the microbiota associated with any Chondrichthyan species during embryonic development, despite the global distribution, ecological importance, and phylogenetic position of this clade. In this study, we take the first steps towards filling this gap by investigating the microbiota associated with embryonic development in the little skate, Leucoraja erinacea, a common North Atlantic species and popular system for chondrichthyan biology. METHODS: To assess the potential for bacterial transmission in an oviparous chondrichthyan, we used 16S rRNA amplicon sequencing to characterize the microbial communities associated with the skin, gill, and egg capsule of the little skate, at six points during ontogeny. Community composition was analyzed using the QIIME2 pipeline and microbial continuity between stages was tracked using FEAST. RESULTS: We identify site-specific and stage-specific microbiota dominated by the bacterial phyla Proteobacteria and Bacteroidetes. This composition is similar to, but distinct from, that of previously published data on the adult microbiota of other chondrichthyan species. Our data reveal that the skate egg capsule harbors a highly diverse bacterial community-particularly on the internal surface of the capsule-and facilitates intergenerational microbial transfer to the offspring. Embryonic skin and external gill tissues host similar bacterial communities; the skin and gill communities later diverge as the internal gills and skin denticles develop. CONCLUSIONS: Our study is the first exploration of the chondrichthyan microbiota throughout ontogeny and provides the first evidence of vertical transmission in this group.

Evolution: The deep genetic roots of tetrapod-specific traits.

Based on embryology and comparative genomics, recent studies reveal that genetic pathways and gene regulatory elements responsible for the invasion of land by tetrapod ancestors are deeply conserved in fish.

The feeding system of Tiktaalik roseae: an intermediate between suction feeding and biting.

Changes to feeding structures are a fundamental component of the vertebrate transition from water to land. Classically, this event has been characterized as a shift from an aquatic, suction-based mode of prey capture involving cranial kinesis to a biting-based feeding system utilizing a rigid skull capable of capturing prey on land. Here we show that a key intermediate, Tiktaalik roseae, was capable of cranial kinesis despite significant restructuring of the skull to facilitate biting and snapping. Lateral sliding joints between the cheek and dermal skull roof, as well as independent mobility between the hyomandibula and palatoquadrate, enable the suspensorium of T. roseae to expand laterally in a manner similar to modern alligator gars and polypterids. This movement can expand the spiracular and opercular cavities during feeding and respiration, which would direct fluid through the feeding apparatus. Detailed analysis of the sutural morphology of T. roseae suggests that the ability to laterally expand the cheek and palate was maintained during the fish-to-tetrapod transition, implying that limited cranial kinesis was plesiomorphic to the earliest limbed vertebrates. Furthermore, recent kinematic studies of feeding in gars demonstrate that prey capture with lateral snapping can synergistically combine both biting and suction, rather than trading off one for the other. A "gar-like" stage in early tetrapod evolution might have been an important intermediate step in the evolution of terrestrial feeding systems by maintaining suction-generation capabilities while simultaneously elaborating a mechanism for biting-based prey capture.

Comparative genomic analysis of human GLI2 locus using slowly evolving fish revealed the ancestral gnathostome set of early developmental enhancers.

BACKGROUND: The zinc finger-containing transcription factor Gli2, is a key mediator of Hedgehog (Hh) signaling and participates in embryonic patterning of various organs including the central nervous system (CNS) and limbs. Abnormal expression of Gli2 can impede the transcription of Hh target genes through disruption of proper balance between Gli2 and Gli3 functions. Therefore, delineation of enhancers that are required for complementary roles of Glis would allow the interrogation of those pathogenic variants that cause gene dysregulation, and a corresponding abnormal phenotype. Previously, we reported tissue-specific enhancers for Gli family including Gli2 through direct tetrapod-teleost comparisons. RESULTS: Here, we employed the sequence alignments of slowly evolving spotted gar and elephant shark and have identified six novel conserved noncoding elements in human GLI2 containing locus. Zebrafish-based transgenic assays revealed that combined action of these autonomous CNEs reflects many aspects of Gli2 specific endogenous transcriptional activity, including CNS and pectoral fins. CONCLUSION: Taken together with our previous findings, this study suggests that Hh-signaling controlled deployment of Gli2 activity in embryonic patterning arose in the common ancestor of gnathostomes. These GLI2 specific cis-regulatory modules will help to identify DNA variants that probably reside outside of coding intervals and are associated with congenital anomalies.

Salamander-like tail regeneration in the West African lungfish.

Salamanders, frog tadpoles and diverse lizards have the remarkable ability to regenerate tails. Palaeontological data suggest that this capacity is plesiomorphic, yet when the developmental and genetic architecture of tail regeneration arose is poorly understood. Here, we show morphological and molecular hallmarks of tetrapod tail regeneration in the West African lungfish Protopterus annectens, a living representative of the sister group of tetrapods. As in salamanders, lungfish tail regeneration occurs via the formation of a proliferative blastema and restores original structures, including muscle, skeleton and spinal cord. In contrast with lizards and similar to salamanders and frogs, lungfish regenerate spinal cord neurons and reconstitute dorsoventral patterning of the tail. Similar to salamander and frog tadpoles, Shh is required for lungfish tail regeneration. Through RNA-seq analysis of uninjured and regenerating tail blastema, we show that the genetic programme deployed during lungfish tail regeneration maintains extensive overlap with that of tetrapods, with the upregulation of genes and signalling pathways previously implicated in amphibian and lizard tail regeneration. Furthermore, the lungfish tail blastema showed marked upregulation of genes encoding post-transcriptional RNA processing components and transposon-derived genes. Our results show that the developmental processes and genetic programme of tetrapod tail regeneration were present at least near the base of the sarcopterygian clade and establish the lungfish as a valuable research system for regenerative biology.

Ontogeny of the anuran urostyle and the developmental context of evolutionary novelty.

Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan.

Fin ray patterns at the fin-to-limb transition.

The fin-to-limb transition was marked by the origin of digits and the loss of dermal fin rays. Paleontological research into this transformation has focused on the evolution of the endoskeleton, with little attention paid to fin ray structure and function. To address this knowledge gap, we study the dermal rays of the pectoral fins of 3 key tetrapodomorph taxa-Sauripterus taylori (Rhizodontida), Eusthenopteron foordi (Tristichopteridae), and Tiktaalik roseae (Elpistostegalia)-using computed tomography. These data show several trends in the lineage leading to digited forms, including the consolidation of fin rays (e.g., reduced segmentation and branching), reduction of the fin web, and unexpectedly, the evolution of asymmetry between dorsal and ventral hemitrichia. In Eusthenopteron, dorsal rays cover the preaxial endoskeleton slightly more than ventral rays. In Tiktaalik, dorsal rays fully cover the third and fourth mesomeres, while ventral rays are restricted distal to these elements, suggesting the presence of ventralized musculature at the fin tip analogous to a fleshy "palm." Asymmetry is also observed in cross-sectional areas of dorsal and ventral rays. Eusthenopteron dorsal rays are slightly larger than ventral rays; by contrast, Tiktaalik dorsal rays can be several times larger than ventral rays, and degree of asymmetry appears to be greater at larger sizes. Analysis of extant osteichthyans suggests that cross-sectional asymmetry in the dermal rays of paired fins is plesiomorphic to crown group osteichthyans. The evolution of dermal rays in crownward stem tetrapods reflects adaptation for a fin-supported elevated posture and resistance to substrate-based loading prior to the origin of digits.

The evolutionary origins and diversity of the neuromuscular system of paired appendages in batoids.

Appendage patterning and evolution have been active areas of inquiry for the past two centuries. While most work has centred on the skeleton, particularly that of amniotes, the evolutionary origins and molecular underpinnings of the neuromuscular diversity of fish appendages have remained enigmatic. The fundamental pattern of segmentation in amniotes, for example, is that all muscle precursors and spinal nerves enter either the paired appendages or body wall at the same spinal level. The condition in finned vertebrates is not understood. To address this gap in knowledge, we investigated the development of muscles and nerves in unpaired and paired fins of skates and compared them to those of chain catsharks. During skate and shark embryogenesis, cell populations of muscle precursors and associated spinal nerves at the same axial level contribute to both appendages and body wall, perhaps representing an ancestral condition of gnathostome appendicular neuromuscular systems. Remarkably in skates, this neuromuscular bifurcation as well as colinear Hox expression extend posteriorly to pattern a broad paired fin domain. In addition, we identified migratory muscle precursors (MMPs), which are known to develop into paired appendage muscles with Pax3 and Lbx1 gene expression, in the dorsal fins of skates. Our results suggest that muscles of paired fins have evolved via redeployment of the genetic programme of MMPs that were already involved in dorsal fin development. Appendicular neuromuscular systems most likely have emerged as side branches of body wall neuromusculature and have been modified to adapt to distinct aquatic and terrestrial habitats.

Feeding kinematics and morphology of the alligator gar (Atractosteus spatula, Lacépède, 1803).

Living gars are a small clade of seven species that occupy an important position on the actinopterygian phylogenetic tree as members of Holostei, sister-group to teleosts, and exhibit many plesiomorphic traits used to interpret and reconstruct early osteichthyan feeding mechanisms. Previous studies of gar feeding kinematics have focused on the ram-based, lateral-snapping mode of prey capture found in the narrow-snouted Lepisosteus genus, whereas this study focuses on a member of the broad-snouted Atractosteus sister-genus, the alligator gar (Atractosteus spatula, Lacépède, 1803). High-speed videography reveals that the feeding system of alligator gars is capable of rapid expansion from anterior to posterior, timed in a way to generate suction, counteract the effects of a bow-wave during ram-feeding, and direct a unidirectional flow of water through the feeding system. Reconstructed contrast-enhanced µCT-based cranial anatomy and three-dimensional modeling of linkage mechanics show that a lateral-sliding palatoquadrate, flexible intrasuspensorial joint, pivoting interhyal, and retractable pectoral girdle increase the range of motion and expansive capabilities of the alligator gar feeding mechanism. Reconstructions of muscular anatomy, inferences from in vivo kinematics, and in situ manipulations show that input from the hyoid constrictors and hypaxials play an important role in decoupling and modulating the dual roles of the sternohyoideus during feeding: hyoid retraction (jaw opening) and hyoid rotation (pharyngeal expansion). The alligator gar possesses an intricate feeding mechanism, capable of precise control with plesiomorphic muscles that represent one of the many ways the ancestral osteichthyan feeding mechanism has been modified for prey capture.

Chemokine C-C motif ligand 33 is a key regulator of teleost fish barbel development.

Barbels are important sensory organs in teleosts, reptiles, and amphibians. The majority of ∼4,000 catfish species, such as the channel catfish (Ictalurus punctatus), possess abundant whisker-like barbels. However, barbel-less catfish, such as the bottlenose catfish (Ageneiosus marmoratus), do exist. Barbeled catfish and barbel-less catfish are ideal natural models for determination of the genomic basis for barbel development. In this work, we generated and annotated the genome sequences of the bottlenose catfish, conducted comparative and subtractive analyses using genome and transcriptome datasets, and identified differentially expressed genes during barbel regeneration. Here, we report that chemokine C-C motif ligand 33 (ccl33), as a key regulator of barbel development and regeneration. It is present in barbeled fish but absent in barbel-less fish. The ccl33 genes are differentially expressed during barbel regeneration in a timing concordant with the timing of barbel regeneration. Knockout of ccl33 genes in the zebrafish (Danio rerio) resulted in various phenotypes, including complete loss of barbels, reduced barbel sizes, and curly barbels, suggesting that ccl33 is a key regulator of barbel development. Expression analysis indicated that paralogs of the ccl33 gene have both shared and specific expression patterns, most notably expressed highly in various parts of the head, such as the eye, brain, and mouth areas, supporting its role for barbel development.