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Pectoral fins, the anterior paired fins in fish, have enhanced maneuvering abilities due to morphological changes. Teleosts have fewer radial bones in their pectoral fins than basal species, resulting in more-elaborate fins. The mechanism behind this radial constraint change in teleosts is unclear. Here, we found that mutations in hhip, which encodes an antagonist of Hedgehog signaling, led to an increase in radial bones in a localized region. Expression of the Shh genes, encoding ligands of Hedgehog signaling, coincided with notable hhip expression specifically during early development. We suggest that a negative feedback effect of Hedgehog signaling by hhip regulates the constraint of the pectoral fin in zebrafish. Additionally, re-analysis of hhip-related gene expression data in zebrafish and basal species revealed that the notable hhip expression during early development is a characteristic of zebrafish that is not observed in basal species. Region-specific expression of Hox13 genes in the zebrafish pectoral fin indicated that the median region, analogous to the region with abundant radials in basal species, is expanded in hhip-/- zebrafish. These data underscore potential morphological evolution through constrained diversity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The morphological transformation of the pectoral/shoulder girdle is fundamental to the water-to-land transition in vertebrate evolution. Although previous studies have resolved the embryonic origins of tetrapod shoulder girdles, those of fish pectoral girdles remain uncharacterized, creating a gap in the understanding of girdle transformation mechanisms from fish to tetrapods. Here, we identify the embryonic origins of the zebrafish pectoral girdle, including the cleithrum as an ancestral girdle element lost in extant tetrapods. Our combinatorial approach of photoconversion and genetic lineage tracing demonstrates that cleithrum development combines four adjoining embryonic populations. A comparison of these pectoral girdle progenitors with extinct and extant vertebrates highlights that cleithrum loss, indispensable for neck evolution, is associated with the disappearance of its unique developmental environment at the head/trunk interface. Overall, our study establishes an embryological framework for pectoral/shoulder girdle formation and provides evolutionary trajectories from their origin in water to diversification on land., (© 2024. The Author(s).)

Background: Mutations of human WNT10A are associated with odonto-ectodermal dysplasia syndromes. Here, we present analyses of wnt10a loss-of-function mutants in the zebrafish., Results: wnt10a mutant zebrafish embryos display impaired tooth development and a collapsing median fin fold (MFF). Rescue experiments show that wnt10a is essential for MFF maintenance both during embryogenesis and later metamorphosis. The MFF collapse could not be attributed to increased cell death or altered proliferation rates of MFF cell types. Rather, wnt10a mutants show reduced expression levels of dlx2a in distal-most MFF cells, followed by compromised expression of col1a1a and other extracellular matrix proteins encoding genes. Transmission electron microscopy analysis shows that although dermal MFF compartments of wnt10a mutants initially are of normal morphology, with regular collagenous actinotrichia, positioning of actinotrichia within the cleft of distal MFF cells becomes compromised, coinciding with actinotrichia shrinkage and MFF collapse., Conclusions: MFF collapse of wnt10a mutant zebrafish is likely caused by the loss of distal properties in the developing MFF, strikingly similar to the proposed molecular pathomechanisms underlying the teeth defects caused by the loss of Wnt10 in fish and mammals. In addition, it points to thus fur unknown mechanisms controlling the linear growth and stability of actinotrichia and their collagen fibrils., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. In situ hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

The development of paired appendages was a key innovation during evolution and facilitated the aquatic to terrestrial transition of vertebrates. Largely derived from the lateral plate mesoderm (LPM), one hypothesis for the evolution of paired fins invokes derivation from unpaired median fins via a pair of lateral fin folds located between pectoral and pelvic fin territories 1 . Whilst unpaired and paired fins exhibit similar structural and molecular characteristics, no definitive evidence exists for paired lateral fin folds in larvae or adults of any extant or extinct species. As unpaired fin core components are regarded as exclusively derived from paraxial mesoderm, any transition presumes both co-option of a fin developmental programme to the LPM and bilateral duplication 2 . Here, we identify that the larval zebrafish unpaired pre-anal fin fold (PAFF) is derived from the LPM and thus may represent a developmental intermediate between median and paired fins. We trace the contribution of LPM to the PAFF in both cyclostomes and gnathostomes, supporting the notion that this is an ancient trait of vertebrates. Finally, we observe that the PAFF can be bifurcated by increasing bone morphogenetic protein signalling, generating LPM-derived paired fin folds. Our work provides evidence that lateral fin folds may have existed as embryonic anlage for elaboration to paired fins., (© 2023. The Author(s).)

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.

Spear-like collagen complexes, known as actinotrichia, underlie the epidermal cell layer in the tip of teleost fins and are known to contribute toward fin formation; however, their specific role remains largely unclear. In this study, we investigated of actinotrichia in the role of caudal fin formation by generating collagen9a1c (col9a1c)-knockout zebrafish. Although actinotrichia were initially produced normally and aligned correctly in the knockout fish, the number of actinotrichia decreased as the fish grew and their alignment became disordered. Simultaneously, the fin tip gradually shortened in the dorsal-ventral direction and the entire fin became oval-shaped, while the fin-rays rarely bifurcated and instead underwent fusion, suggesting that actinotrichia are essential for spreading fins dorsoventrally. Furthermore, the epithelial cells that are usually thinly spread in normal fish became spherical in the knockout fish, reducing the area covered by each cell and thus the area of the fin tip. Together, these findings suggest that the tight alignment of actinotrichia provides physical support in the dorsal-ventral direction that allows caudal fins to expand in a triangular-shape., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The T-box containing family member, TBX5, has been shown to play important functional roles in the pectoral appendages of a variety of vertebrate species. While a single TBX5 gene exists in all tetrapods studied to date, the zebrafish genome retains two paralogues, designated as tbx5a and tbx5b, resulting from a whole genome duplication in the teleost lineage. Zebrafish deficient in tbx5a lack pectoral fin buds, whereas zebrafish deficient in tbx5b exhibit misshapen pectoral fins, showing that both paralogues function in fin development. The mesenchymal cells of the limb/fin bud are derived from the Lateral Plate Mesoderm (LPM). Previous fate mapping work in zebrafish has shown that wildtype (wt) fin field cells are initially located adjacent to somites (s)1-4. The wt fin field cells migrate in opposing diagonal directions placing the limb bud between s2-3 and lateral to the main body. To better characterize tbx5 paralogue functions in zebrafish, time-lapse analyses of the migrations of fin bud precursors under conditions of tbx5a knock-down, tbx5b knock-down and double-knock-down were performed. Our data suggest that zebrafish tbx5a and tbx5b have functionally separated migration direction vectors, that when combined recapitulate the migration of the wt fin field. We and others have shown that loss of Tbx5a function abolishes an fgf24 signaling cue resulting in fin field cells failing to converge in an Antero-Posterior (AP) direction and migrating only in a mediolateral (ML) direction. We show here that loss of Tbx5b function affects initial ML directed movements so that fin field cells fail to migrate laterally but continue to converge along the AP axis. Furthermore, fin field cells in the double Tbx5a/Tbx5b knock-down zebrafish do not engage in directed migrations along either the ML or AP axis. Therefore, these two paralogues may be acting to instruct separate vectors of fin field migration in order to direct proper fin bud formation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus., Results: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap., Conclusions: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes., (© 2021 American Association for Anatomy.)

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

While it is well known that the notochord of bony fishes changes over developmental time, less is known about how it varies across different body regions. In the development of the Atlantic salmon, Salmo salar L., cranial and caudal ends of the notochord are overlaid by the formation of the bony elements of the neurocranium and caudal fin, respectively. To investigate, we describe how the notochord of the cranium and caudal fin changes from embryo to spawning adult, using light microscopy, SEM, TEM, dissection, and CT scanning. The differences are dramatic. In contrast to the abdominal and caudal regions, at the ends of the notochord vertebrae never develop. While the cranial notochord builds a tapering, unsegmented cone of chordal bone, the urostylic notochordal sheath never ossifies: adjacent, irregular bony elements form from the endoskeleton of the caudal fin. As development progresses, two previously undescribed processes occur. First, the bony cone of the cranial notochord, and its internal chordocytes, are degraded by chordoclasts, an undescribed function of the clastic cell type. Second, the sheath of the urostylic notochord creates transverse septae that partly traverse the lumen in an irregular pattern. By the adult stage, the cranial notochord is gone. In contrast, the urostylic notochord in adults is robust, reinforced with septae, covered by irregularly shaped pieces of cellular bone, and capped with an opistural cartilage that develops from the sheath of the urostylic notochord. A previously undescribed muscle, with its origin on the opistural cartilage, inserts on the lepidotrich ventral to it., (© 2020 The Authors. The Anatomical Record published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

The increase in activity of the two-pore potassium-leak channel Kcnk5b maintains allometric juvenile growth of adult zebrafish appendages. However, it remains unknown how this channel maintains allometric growth and how its bioelectric activity is regulated to scale these anatomical structures. We show the activation of Kcnk5b is sufficient to activate several genes that are part of important development programs. We provide in vivo transplantation evidence that the activation of gene transcription is cell autonomous. We also show that Kcnk5b will induce the expression of different subsets of the tested developmental genes in different cultured mammalian cell lines, which may explain how one electrophysiological stimulus can coordinately regulate the allometric growth of diverse populations of cells in the fin that use different developmental signals. We also provide evidence that the post-translational modification of serine 345 in Kcnk5b by calcineurin regulates channel activity to scale the fin. Thus, we show how an endogenous bioelectric mechanism can be regulated to promote coordinated developmental signaling to generate and scale a vertebrate appendage., Competing Interests: CY, TS, EA, SW, TX, SC, XY, KG, MW, AE, CA No competing interests declared, (© 2021, Yi et al.)

The overexpression of hoxd13a during zebrafish fin development causes distal endochondral expansion and simultaneous reduction of the finfold, mimicking the major events thought to have happened during the fin-to-limb transition in Vertebrates. We investigated the effect of hoxd13a overexpression on putative downstream targets and found it to cause downregulation of proximal fin identity markers (meis1 and emx2) and upregulation of genes involved in skeletogenesis/patterning (fbn1, dacha) and AER/Finfold maintenance (bmps). We then show that bmp2b overexpression leads to finfold reduction, recapitulating the phenotype observed in hoxd13a-overexpressing fins. In addition, we show that during the development of the long finfold in leo t1 /lof dt1 mutants, hoxd13a and bmp2b are downregulated. Our results suggest that modulation of the transcription factor Hoxd13 during evolution may have been involved in finfold reduction through regulation of the Bmp signalling that then activated apoptotic mechanisms impairing finfold elongation.

Sexual selection results in sex-specific characters like the conspicuously pigmented extension of the ventral tip of the caudal fin-the "sword"-in males of several species of Xiphophorus fishes. To uncover the genetic architecture underlying sword formation and to identify genes that are associated with its development, we characterized the sword transcriptional profile and combined it with genetic mapping approaches. Results showed that the male ornament of swordtails develops from a sexually non-dimorphic prepattern of transcription factors in the caudal fin. Among genes that constitute the exclusive sword transcriptome and are located in the genomic region associated with this trait we identify the potassium channel, Kcnh8, as a sword development gene. In addition to its neural function kcnh8 performs a known role in fin growth. These findings indicate that during evolution of swordtails a brain gene has been co-opted for an additional novel function in establishing a male ornament., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hoxc6 gene can be described as having roles in axial patterning in early embryogenesis, and in at least some species, having a contribution to limb positioning. In this study, we cloned and characterised Pampus argenteus Hoxc6. The highly conserved HOXC6 protein sequence contains a homeodomain and a low-complexity region. Expression of Hoxc6 mRNA was measured at different developmental stages and in different tissues by real-time PCR (p < 0.05), and was high during eye capsule and brain differentiation stages, but low in 7 and 13-day-old larvae. Hoxc6 mRNA was more abundant in fin tissue than brain and eye tissues. Western blotting showed that HOXC6 protein levels were high at embryonic stages, but decreased significantly in 7, 13, 16 and 19-day-old larvae, and levels were essentially consistent with those of mRNA measured by real-time PCR in different tissues. In situ hybridisation showed that the Hoxc6 transcript was strongly expressed in the whole brain and anterior part of the body axis in 1-day-old larvae, but in the hindbrain, pectoral fin, mandible and hypothetical pelvic fin region in 7, 13, 16 and 19-day-old organisms. These results clarify the expression and localisation characteristics of Hoxc6 gene in P. argenteus, and provide a theoretical basis for the molecular mechanism of pelvic fin loss in silver pomfret., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Changes in appendage structure underlie key transitions in vertebrate evolution. Addition of skeletal elements along the proximal-distal axis facilitated critical transformations, including the fin-to-limb transition that permitted generation of diverse modes of locomotion. Here, we identify zebrafish mutants that form supernumerary long bones in their pectoral fins. These new bones integrate into musculature, form joints, and articulate with neighboring elements. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage patterning, vav2 and waslb, that function in a common pathway. This pathway is required for appendage development across vertebrates, and loss of Wasl in mice causes defects similar to those seen in murine Hox mutants. Concordantly, formation of supernumerary bones requires Hox11 function, and mutations in the vav2/wasl pathway drive enhanced expression of hoxa11b, indicating developmental homology with the forearm. Our findings reveal a latent, limb-like pattern ability in fins that is activated by simple genetic perturbation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

How genetic changes are linked to morphological novelties and developmental constraints remains elusive. Here, we investigate genetic apparatuses that distinguish fish fins from tetrapod limbs by analyzing transcriptomes and open-chromatin regions (OCRs). Specifically, we compared mouse forelimb buds with the pectoral fin buds of an elasmobranch, the brown-banded bamboo shark ( Chiloscyllium punctatum ). A transcriptomic comparison with an accurate orthology map revealed both a mass heterochrony and hourglass-shaped conservation of gene expression between fins and limbs. Furthermore, open-chromatin analysis suggested that access to conserved regulatory sequences is transiently increased during mid-stage limb development. During this stage, stage-specific and tissue-specific OCRs were also enriched. Together, early and late stages of fin/limb development are more permissive to mutations than middle stages, which may have contributed to major morphological changes during the fin-to-limb evolution. We hypothesize that the middle stages are constrained by regulatory complexity that results from dynamic and tissue-specific transcriptional controls., Competing Interests: KO, KT, CT, MK, ON, SK No competing interests declared, (© 2021, Onimaru et al.)

Paired fins are a defining feature of the jawed vertebrate body plan, but their evolutionary origin remains unresolved. Gegenbaur proposed that paired fins evolved as gill arch serial homologues, but this hypothesis is now widely discounted, owing largely to the presumed distinct embryonic origins of these structures from mesoderm and neural crest, respectively. Here, we use cell lineage tracing to test the embryonic origin of the pharyngeal and paired fin skeleton in the skate ( Leucoraja erinacea ). We find that while the jaw and hyoid arch skeleton derive from neural crest, and the pectoral fin skeleton from mesoderm, the gill arches are of dual origin, receiving contributions from both germ layers. We propose that gill arches and paired fins are serially homologous as derivatives of a continuous, dual-origin mesenchyme with common skeletogenic competence, and that this serial homology accounts for their parallel anatomical organization and shared responses to axial patterning signals., Competing Interests: VS, JG No competing interests declared, (© 2020, Sleight and Gillis.)

Mammalian articular cartilage is an avascular tissue with poor capacity for spontaneous repair. Here, we show that embryonic development of cartilage in the skate ( Leucoraja erinacea ) mirrors that of mammals, with developing chondrocytes co-expressing genes encoding the transcription factors Sox5, Sox6 and Sox9. However, in skate, transcriptional features of developing cartilage persist into adulthood, both in peripheral chondrocytes and in cells of the fibrous perichondrium that ensheaths the skeleton. Using pulse-chase label retention experiments and multiplexed in situ hybridization, we identify a population of cycling Sox5/6/9 + perichondral progenitor cells that generate new cartilage during adult growth, and we show that persistence of chondrogenesis in adult skates correlates with ability to spontaneously repair cartilage injuries. Skates therefore offer a unique model for adult chondrogenesis and cartilage repair and may serve as inspiration for novel cell-based therapies for skeletal pathologies, such as osteoarthritis., Competing Interests: AM, AH, JG No competing interests declared, (© 2020, Marconi et al.)

Nucleosome assembly protein 1-like (Nap1l) family plays numerous biological roles including nucleosome assembly, transcriptional regulation, and cell cycle progression. However, the tissue specific in vivo functions of the Nap1l family members remain largely unknown. In this study, we finished the complete expression patterns of nap1l1 and nap1l4a in zebrafish embryos by whole-mount in situ hybridization. We observed maternal existence of nap1l1 transcript and that its zygotic expression is abundant and not spatially restricted at 6 somite stage, while nap1l4a mRNA is not detectable until 6 somite stage when it is weakly transcribed throughout the embryo. At 24 h post-fertilization (hpf), nap1l1 is predominantly expressed in the central nervous system, neural tube, ventral mesoderm, branchial arches, and pectoral fins, while nap1l4a mRNA is throughout the embryo, enriched in the eyes, tectum, and myotomes. As the embryo develops, nap1l1 expression maintains throughout the head, with gradually enriched in the tectum, olfactory vesicle, lens, optic cups, heart, branchial arches, pectoral fins, axial vasculature, pronephros, and lateral line neuromasts, whereas nap1l4a expression is weak in the tectum, branchial arches, and pectoral fins. Overall, these expression analyses provide a valuable basis for the functional study of nap1l family in zebrafish development., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In zebrafish, pelvic fin buds appear at 3 weeks post fertilization (wpf) during the larval to juvenile transition (metamorphosis), but their fate is already determined during embryogenesis. Thus, presumptive pelvic fin cells appear to memorize their positional information for three weeks, but no factors expressed in the pelvic fin field from the embryonic to the metamorphic stages have been identified. In mice, Islet1 is proposed to promote nuclear accumulation of β-catenin in the hindlimb field, which leads to the initiation of hindlimb bud outgrowth through activation of the Wnt/βcatenin pathway. Here, we examined the distribution of β-catenin and islet proteins in the pelvic fin field of zebrafish from the embryonic to the metamorphic stages. We found that transcripts of islet2a , but not islet1 , are detected in the posterior lateral plate mesoderm, including the presumptive pelvic fin field, at the embryonic stage as well as in the pelvic fin bud at the metamorphic stage. Immunolocalization revealed that β-catenin and islet proteins, which are synthesized during the embryonic stage, remain in the cytoplasm of the presumptive pelvic fin cells during the larval stage, and are then translocated into the nuclei of the pelvic fin bud at the metamorphic stage. We propose that cytoplasmic localization of these proteins in the presumptive pelvic fin cells that remained during the larval stage may underlie the mechanism by which pelvic fin cells memorize their positional information from the embryonic stage to the metamorphic stage.

The characteristic and morphologically variable pectoral-fin spine of catfishes (order Siluriformes) has been well-investigated based on later developmental stages (juveniles and adults) but information on the earliest life stages are lacking. Here, we document the ontogeny of pectoral-fin spines in four siluroid (Ictalurus punctatus, Noturus gyrinus, Silurus glanis and Akysis vespa) and two loricarioid catfishes (Corydoras panda and Ancistrus sp.). To further our understanding of pectoral-fin spine development, we also examined adult and juvenile specimens representing 41 of the currently 43 recognized families of catfishes. Development of the pectoral-fin spine is similar in all catfishes and resembles the development of a typical soft fin ray. Fusion between hemitrichia of the anteriormost lepidotrichium occurs proximally first, forming the spine proper, with growth of the spine occurring through the subsequent fusion of developing distal hemitrichial segments that comprise the spurious ray. The variation of pectoral-fin spine morphology observed is largely attributed to the presence/absence of five traits, which either develop as part of the hemitrichial segments that are added to the distal tip of the spine during growth (distal rami, anterior/posterior serrae) or develop independent of these segments (denticuli and odontodes)., (© 2019 Wiley Periodicals, Inc.)

Morphogenesis is the fundamental developmental process during which the embryo body is formed. Proper shaping of different body parts depends on cellular divisions and rearrangements in the growing embryo. Understanding three-dimensional shaping of organs is one of the basic questions in developmental biology. Here, we consider the early stages of pectoral fin development in zebrafish, which serves as a model for limb development in vertebrates, to study emerging shapes during embryogenesis. Most studies on pectoral fin are concerned with late stages of fin development when the structure is morphologically distinct. However, little is known about the early stages of pectoral fin formation because of the experimental difficulties in establishing proper imaging conditions during these stages to allow long-term live observation. In this protocol, we address the challenges of pectoral fin imaging during the early stages of zebrafish embryogenesis and provide a strategy for three-dimensional shape analysis of the fin. The procedure outlined here is aimed at studying pectoral fin during the first 24 h of its formation corresponding to the time period between 24 and 48 h of zebrafish development. The same principles could also be applied when studying three-dimensional shape establishment of other embryonic structures. We first discuss the imaging procedure and then propose strategies of extracting quantitative information regarding fin shape and dimensions.

Low-density lipoprotein receptor-related protein 4 (LRP4) is a multi-functional protein implicated in bone, kidney and neurological diseases including Cenani-Lenz syndactyly (CLS), sclerosteosis, osteoporosis, congenital myasthenic syndrome and myasthenia gravis. Why different LRP4 mutation alleles cause distinct and even contrasting disease phenotypes remain unclear. Herein, we utilized the zebrafish model to search for pathways affected by a deficiency of LRP4. The lrp4 knockdown in zebrafish embryos exhibits cyst formations at fin structures and the caudal vein plexus, malformed pectoral fins, defective bone formation and compromised kidney morphogenesis; which partially phenocopied the human LRP4 mutations and were reminiscent of phenotypes resulting form a perturbed Notch signaling pathway. We discovered that the Lrp4-deficient zebrafish manifested increased Notch outputs in addition to enhanced Wnt signaling, with the expression of Notch ligand jagged1b being significantly elevated at the fin structures. To examine conservatism of signaling mechanisms, the effect of LRP4 missense mutations and siRNA knockdowns, including a novel missense mutation c.1117C > T (p.R373W) of LRP4, were tested in mammalian kidney and osteoblast cells. The results showed that LRP4 suppressed both Wnt/β-Catenin and Notch signaling pathways, and these activities were perturbed either by LRP4 missense mutations or by a knockdown of LRP4. Our finding underscore that LRP4 is required for limiting Jagged-Notch signaling throughout the fin/limb and kidney development, whose perturbation representing a novel mechanism for LRP4-related diseases. Moreover, our study reveals an evolutionarily conserved relationship between LRP4 and Jagged-Notch signaling, which may shed light on how the Notch signaling is fine-tuned during fin/limb development.

All living tetrapods have a one-to-two branching pattern in the embryonic proximal limb skeleton, with a single element at the base of the limb (the humerus or femur) that articulates distally with two parallel radials (the ulna and radius or the tibia and fibula). This pattern is also seen in the fossilized remains of stem-tetrapods, including the fishlike members of the group, in which despite the absence of digits, the proximal parts of the fin skeleton clearly resemble those of later tetrapods. However, little is known about the developmental mechanisms that establish and canalize this highly conserved pattern. We describe the well-preserved pelvic fin skeleton of Rhizodus hibberti , a Carboniferous sarcopterygian (lobe-finned) fish, and member of the tetrapod stem group. In this specimen, three parallel radials, each robust with a distinct morphology, articulate with the femur. We review this unexpected morphology in a phylogenetic and developmental context. It implies that the developmental patterning mechanisms seen in living tetrapods, now highly constrained, evolved from mechanisms flexible enough to accommodate variation in the zeugopod (even between pectoral and pelvic fins), while also allowing each element to have a unique morphology., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Using multiple taxa to research development is necessary for making general conclusions about developmental patterns and mechanisms. We present a staging series for Cyprinodon variegatus as a basis for further study of the developmental biology of fishes in the genus Cyprinodon and for comparative work on teleost fishes beyond the standard models. Cyprinodon are small, euryhaline fishes, widely distributed in fresh, brackish, and hypersaline waters of southern and eastern North America. Cyprinodontids are closely related to fundulids, providing a comparative reference point to the embryological model, Fundulus heteroclitus. Ecologists and evolutionary biologists commonly study Cyprinodon, and we have been using Cyprinodon to study skull variation and its genetic basis among closely related species. We divided embryonic development of C. variegatus into 34 morphologically identifiable stages. We reference our staging series to that already defined for a related model species, Oryzias latipes (medaka) that is studied by a large community of researchers. We provide a description of the early chondrogenesis and ossification of skull and caudal fin bones during the latter stages of embryonic development. We show that Cyprinodon are tractable for studying development. Eggs can be obtained easily from breeding pairs and our study provides a staging system to facilitate future developmental studies., (© 2018 Wiley Periodicals, Inc.)

The embryonic fins and the tip of adult fins of teleost fish are supported by rows of straight, unmineralized fibrils called actinotrichia. The proximal ends of the actinotrichia are bundled and the mineralized bones called lepidotrichia are made along them. Since malformation in actinotrichia causes wavy fin bones, the correct configuration of actinotrichia is essential for the correct construction of the fin shape. Past studies suggested that two types of cells, basal keratinocytes, and mesenchymal cells involve in the formation of actinotrichia. However, the mechanism how these cells contribute is unknown. In this study, we elucidated the role of basal keratinocytes in actinotrichia formation. First, we developed the imaging tool that specifically visualizes the basal keratinocytes and actinotrichia. Then, we established the in vitro culture method of the basal keratinocytes and found that the keratinocytes developed fine needle-like structures in it. The TEM image of them showed the specific shadow pattern of actinotrichia, indicating that the fine needle-like structures are the newly made actinotrichia. Finally, we cultured the basal keratinocytes with mature actinotrichia and observed that the basal keratinocytes actively holded actinotrichia with their membrane, and often generated a linear array of cells holding a single actinotrichium. This behavior suggested a mechanism with which long actinotrichia are made by relatively small cells. Our results clarified the role of basal keratinocyte and provided a novel insight into understanding the mechanism of actinotrichia formation., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Caudal-fin lepidotrichia is composed of numerous segments, which are linked to each other by intersegmental joints. During fish growth, lepidotrichia elongate by the addition of new segments at their distal margin, whereas the length of each segment remains constant after it is formed. In the present paper, we examined whether the water temperature affects the segmentation pattern of the juvenile and adult caudal fin. For this purpose, zebrafish (Danio rerio) embryos and larvae were exposed to three different temperature conditions (24°C, 28°C, and 32°C) from the pharyngula stage (1 day postfertilization [dpf]) to metamorphosis, whereas the control temperature (28°C) was applied to all the groups before and after this period. Results demonstrated that water temperature had a significant effect on the length of the segments of each lepidotrichium, at both the juvenile and adult stages. Moreover, at higher temperatures, there was a significant proximal shift of the position of the first bifurcation of the second lepidotrichium of the dorsal lobe. At all the experimental conditions, the length of proximal segment was not constant during fish growth, but it followed a discontinuous saltatory growth. Histological analysis of the proximal lepidotrichia segments revealed that the observed apparent growth of segments is the result of fusions between segments. Fusion occurs not by mineralization of the intersegmental joints, but by bone deposition around the joints., (© 2018 Wiley Periodicals, Inc.)

Limb muscles are formed from migratory muscle precursor cells (MMPs) that delaminate from the ventral region of dermomyotomes and migrate into the limb bud. MMPs remain undifferentiated during migration, commencing differentiation into skeletal muscle after arrival in the limb. However, it is still unclear whether the developmental mechanisms of MMPs are conserved in teleost fishes. Here, we investigate the development of pectoral fin muscles in the teleost medaka Oryzias latipes. Expression of the MMP marker lbx1 is first observed in several somites prior to the appearance of fin buds. lbx1-positive cells subsequently move anteriorly and localize in the prospective fin bud region to differentiate into skeletal muscle cells. To address the developmental mechanisms underlying fin muscle formation, we knocked down tbx5, a gene that is required for fin bud formation. tbx5 morphants showed loss of fin buds, whereas lbx1 expression initiated normally in anterior somites. Unlike in normal embryos, expression of lbx1 was not maintained in migrating fin MMPs or within the fin buds. We suggest that fin MMPs appear to undergo two phases in their development, with an initial specification of MMPs occurring independent of fin buds and a second fin bud-dependent phase of MMP migration and proliferation. Our results showed that medaka fin muscle is composed of MMPs. It is suggested that the developmental mechanism of fin muscle formation is conserved in teleost fishes including medaka. Through this study, we also propose new insights into the developmental mechanisms of MMPs in fin bud formation.

Early development of the Adriatic sturgeon Acipenser naccarii from its free embryo after hatching (stage 36), until late embryo stage, when the transition to exogenous feeding starts (stage 45) is described. Special emphasis is given to morphological development and description of the different structures that are formed at each life stage. After hatching, free embryos still present embryonic characteristics, little pigmentation and an ovoid yolk sac. The mouth begins to open on the second day post hatch (dph) and is fully open at 3 dph. The head begins to separate from the body at 4 dph and straightens at 6 dph. The first fins to appear are the pectoral fins on the yolk sac and an embryological fin fold that extends from behind the head to the posterior part of the yolk sac. All other fins will develop from this fold. At 7 dph the caudal fin begins to take a heterocercal form and dorsal scutes are observed. This study provides information that will assist aquaculturists by establishing a reference for the normal development of A. naccarii, which may be useful for evaluating the suitability and quality of fish produced for restocking., (© 2018 The Fisheries Society of the British Isles.)

The lunate-shaped caudal fin in lamnid sharks is a morphological specialization for their thunniform mode of locomotion, but its developmental process during gestation has been poorly investigated. Observations of 21 embryonic specimens of the white shark (Carcharodon carcharias) revealed that their caudal fin morphology drastically changes from strongly heterocercal to lunate-shaped through ontogeny. This morphological change involves (1) rapid elongation of the ventral lobe, (2) increased upward curvature of the vertebra within the caudal fin, and (3) formation of keels at both lateral sides of the caudal fin base. These morphological changes are probably shared among the members of the family Lamnidae and are in contrast with the developmental process of the heterocercal tail in the lamniform Carcharias taurus, in which the caudal fin morphology is almost unchanged through the late gestation period. Anat Rec, 301:1068-1073, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: The caudal fin of actinopterygians experienced substantial morphological changes during evolution. In basal actinopterygians, the caudal fin skeleton supports an asymmetrical heterocercal caudal fin, while most teleosts have a symmetrical homocercal caudal fin. The transition from the ancestral heterocercal form to the derived homocercal caudal fin remains poorly understood. Few developmental studies provide an understanding of derived and ancestral characters among basal actinopterygians. To fill this gap, we examined the development of the caudal fin of spotted gar Lepisosteus oculatus, one of only eight living species of Holostei, the sister group to the teleosts., Results: Our observations of animals from fertilization to more than a year old provide the most detailed description of the development of caudal fin skeletal elements in any Holostean species. We observed two different types of distal caudal radials replacing two transient plates of connective tissue, identifying two hypaxial ensembles separated by a space between hypurals 2 and 3. These features have not been described in any gar species, but can be observed in other gar species, and thus represent anatomical structures common to lepisosteiformes., Conclusions: The present work highlights the power and importance of ontogenic studies and provides bases for future evolutionary and morphological investigations on actinopterygians fins. Developmental Dynamics 247:724-740, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: The experimental approach to the evolution and development of the vertebrate skeleton has to a large extent relied on "direct-developing" amniote model organisms, such as the mouse and the chicken. These organisms can however only be partially informative where it concerns secondarily lost features or anatomical novelties not present in their lineages. The widely used anamniotes Xenopus and zebrafish are "indirect-developing" organisms that proceed through an extended time as free-living larvae, before adopting many aspects of their adult morphology, complicating experiments at these stages, and increasing the risk for lethal pleiotropic effects using genetic strategies., Results: Here, we provide a detailed description of the development of the osteology of the African mouthbrooding cichlid Astatotilapia burtoni, primarily focusing on the trunk (spinal column, ribs and epicentrals) and the appendicular skeleton (pectoral, pelvic, dorsal, anal, caudal fins and scales), and to a lesser extent on the cranium. We show that this species has an extremely "direct" mode of development, attains an adult body plan within 2 weeks after fertilization while living off its yolk supply only, and does not pass through a prolonged larval period., Conclusions: As husbandry of this species is easy, generation time is short, and the species is amenable to genetic targeting strategies through microinjection, we suggest that the use of this direct-developing cichlid will provide a valuable model system for the study of the vertebrate body plan, particularly where it concerns the evolution and development of fish or teleost specific traits. Based on our results we comment on the development of the homocercal caudal fin, on shared ontogenetic patterns between pectoral and pelvic girdles, and on the evolution of fin spines as novelty in acanthomorph fishes. We discuss the differences between "direct" and "indirect" developing actinopterygians using a comparison between zebrafish and A. burtoni development.

Regeneration of the zebrafish caudal fin following amputation occurs through wound healing, followed by formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. We show that ptpn11a -/- ptpn11b -/- zebrafish embryos, lacking functional Shp2, fail to regenerate their caudal fin folds. Rescue experiments indicated that Shp2a has a functional signaling role, requiring its catalytic activity and SH2 domains but not the two C-terminal tyrosine phosphorylation sites. Surprisingly, expression of Shp2a variants with increased and reduced catalytic activity, respectively, rescued caudal fin fold regeneration to similar extents. Expression of mmp9 and junbb , indicative of formation of the wound epidermis and distal blastema, respectively, suggested that these processes occurred in ptpn11a -/- ptpn11b -/- zebrafish embryos. However, cell proliferation and MAPK phosphorylation were reduced. Pharmacological inhibition of MEK1 in wild-type zebrafish embryos phenocopied loss of Shp2. Our results suggest an essential role for Shp2a-mitogen-activated protein kinase (MAPK) signaling in promoting cell proliferation during zebrafish embryo caudal fin fold regeneration., (Copyright © 2018 Hale and den Hertog.)

TBX5 plays a critical role in heart and forelimb development. Mutations in TBX5 cause Holt-Oram syndrome, an autosomal dominant condition that affects the formation of the heart and upper-limb. Several studies have provided significant insight into the role of TBX5 in cardiogenesis; however, how TBX5 activity is regulated by other factors is still unknown. Here we report that histone acetyltransferases KAT2A and KAT2B associate with TBX5 and acetylate it at Lys339. Acetylation potentiates its transcriptional activity and is required for nuclear retention. Morpholino-mediated knockdown of kat2a and kat2b transcripts in zebrafish severely perturb heart and limb development, mirroring the tbx5a knockdown phenotype. The phenotypes found in MO-injected embryos were also observed when we introduced mutations in the kat2a or kat2b genes using the CRISPR-Cas system. These studies highlight the importance of KAT2A and KAT2B modulation of TBX5 and their impact on heart and limb development., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In amniote embryos, skeletal muscles in the trunk are derived from epithelial dermomyotomes, the ventral margin of which extends ventrally to form body wall muscles. At limb levels, ventral dermomyotomes also generate limb-muscle precursors, an Lbx1-positive cell population that originates from the dermomyotome and migrates distally into the limb bud. In elasmobranchs, however, muscles in the paired fins were believed to be formed by direct somitic extension, a developmental pattern used by the amniote body wall muscles. Here we re-examined the development of pectoral fin muscles in catsharks, Scyliorhinus, and found that chondrichthyan fin muscles are indeed formed from Lbx-positive muscle precursors. Furthermore, these precursors originate from the ventral edge of the dermomyotome, the rest of which extends towards the ventral midline to form body wall muscles. Therefore, the Lbx1-positive, de-epithelialized appendicular muscle precursors appear to have been established in the body plan before the divergence of Chondrichthyes and Osteichthyes.

In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO 3 - to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.

Background: How joints are correctly positioned in the vertebrate skeleton remains poorly understood. From our studies on the regenerating fin, we have evidence that the gap junction protein Cx43 suppresses joint formation by suppressing the expression of the evx1 transcription factor. Joint morphogenesis proceeds through at least two discrete stages. First, cells that will produce the joint condense in a single row on the bone matrix ("initiation"). Second, these cells separate coincident with articulation of the bone matrix. We propose that Cx43 activity is transiently reduced prior to joint initiation., Results: We first define the timing of joint initiation with respect to regeneration. We next correlate reduced cx43 expression and increased evx1 expression with initiation. Through manipulation of cx43 expression, we demonstrate that Cx43 negatively influences evx1 expression and joint formation. We further demonstrate that Cx43 activity in the dermal fibroblasts is required to rescue joint formation in the cx43 mutant, short fin b123 ., Conclusions: We conclude that Cx43 activity in the dermal fibroblasts influences the expression of evx1, and therefore the differentiation of the precursor cells that give rise to the joint-forming osteoblasts. Developmental Dynamics 246:691-699, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Cranial neural crest cells are a multipotent cell population that generate all the elements of the pharyngeal cartilage with differentiation into chondrocytes tightly regulated by temporal intracellular and extracellular cues. Here, we demonstrate a novel role for miR-27, a highly enriched microRNA in the pharyngeal arches, as a positive regulator of chondrogenesis. Knock down of miR-27 led to nearly complete loss of pharyngeal cartilage by attenuating proliferation and blocking differentiation of pre-chondrogenic cells. Focal adhesion kinase (FAK) is a key regulator in integrin-mediated extracellular matrix (ECM) adhesion and has been proposed to function as a negative regulator of chondrogenesis. We show that FAK is downregulated in the pharyngeal arches during chondrogenesis and is a direct target of miR-27. Suppressing the accumulation of FAK in miR-27 morphants partially rescued the severe pharyngeal cartilage defects observed upon knock down of miR-27. These data support a crucial role for miR-27 in promoting chondrogenic differentiation in the pharyngeal arches through regulation of FAK., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Jawed vertebrates, or gnathostomes, have two sets of paired appendages, pectoral and pelvic fins in fishes and fore- and hindlimbs in tetrapods. As for paired limbs, paired fins are purported serial homologues, and the advent of pelvic fins has been hypothesized to have resulted from a duplication of the developmental mechanisms present in the pectoral fins, but re-iterated at a posterior location. Developmental similarity of gene expression between pectoral and pelvic fins has been documented in chondrichthyans, but a detailed morphological description of the progression of paired fin development for this group is still lacking. We studied paired fin development in an ontogenetic series of a phylogenetically basal chondrichthyan, the elephant shark Callorhinchus milii. A strong similarity in the morphology and progression of chondrification between the pectoral and pelvic fins was found, which could be interpretated as further evidence of serial homology in paired fins, that could have arisen by duplication. Furthermore, this high degree of morphological and developmental similarity suggests the presence of morphological and developmental modules within paired fins, as observed in paired limbs. This is the first time morphological and developmental modules are described for the paired fins of chimaeras.

Crude oils from distinct geographical regions have distinct chemical compositions, and, as a result, their toxicity may be different. However, developmental toxicity of crude oils derived from different geographical regions has not been extensively characterized. In this study, flounder embryos were separately exposed to effluents contaminated by three crude oils including: Basrah Light (BLO), Pyrenees (PCO), and Sakhalin Vityaz (SVO), in addition to a processed fuel oil (MFO-380), to measure developmental toxicity and for gene expressions. Each oil possessed a distinct chemical composition. Edema defect was highest in embryos exposed to PCO and MFO-380 that both have a greater fraction of three-ring PAHs (33% and 22%, respectively) compared to BLO and SVO. Observed caudal fin defects were higher in embryos exposed to SVO and MFO-380, which are both dominated by naphthalenes (81% and 52%, respectively). CYP1A gene expressions were also highest in embryos exposed to SVO and MFO-380. Higher incidence of cardiotoxicity and lower nkx 2.5 expression were detected in embryos exposed to PCO. Unique gene expression profiles were observed in embryos exposed to crude oils with distinct compositions. This study demonstrates that crude oils of different geographical origins with different compositional characteristics induce developmental toxicity to different degrees., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Understanding the evolutionary transformation of fish fins into tetrapod limbs is a fundamental problem in biology. The search for antecedents of tetrapod digits in fish has remained controversial because the distal skeletons of limbs and fins differ structurally, developmentally, and histologically. Moreover, comparisons of fins with limbs have been limited by a relative paucity of data on the cellular and molecular processes underlying the development of the fin skeleton. Here, we provide a functional analysis, using CRISPR/Cas9 and fate mapping, of 5' hox genes and enhancers in zebrafish that are indispensable for the development of the wrists and digits of tetrapods. We show that cells marked by the activity of an autopodial hoxa13 enhancer exclusively form elements of the fin fold, including the osteoblasts of the dermal rays. In hox13 knockout fish, we find that a marked reduction and loss of fin rays is associated with an increased number of endochondral distal radials. These discoveries reveal a cellular and genetic connection between the fin rays of fish and the digits of tetrapods and suggest that digits originated via the transition of distal cellular fates.

The fin-to-limb transition is an important evolutionary step in the colonization of land and diversification of all terrestrial vertebrates. We previously identified a gene family in zebrafish, termed actinodin, which codes for structural proteins crucial for the formation of actinotrichia, rigid fibrils of the teleost fin. Interestingly, this gene family is absent from all tetrapod genomes examined to date, suggesting that it was lost during limb evolution. To shed light on the disappearance of this gene family, and the consequences on fin-to-limb transition, we characterized actinodin regulatory elements. Using fluorescent reporters in transgenic zebrafish, we identified tissue-specific cis-acting regulatory elements responsible for actinodin1 (and1) expression in the ectodermal and mesenchymal cell populations of the fins, respectively. Mutagenesis of potential transcription factor binding sites led to the identification of one binding site crucial for and1 expression in ectodermal cells. We show that these regulatory elements are partially functional in mouse limb buds in a tissue-specific manner. Indeed, the zebrafish regulatory elements target expression to the dorsal and ventral ectoderm of mouse limb buds. Absence of expression in the apical ectodermal ridge is observed in both mouse and zebrafish. However, cells of the mouse limb bud mesoderm do not express the transgene, in contrast to zebrafish. Altogether these results hint for a change in regulation of and1 during evolution that led to the downregulation and eventual loss of this gene from tetrapod genomes., (Copyright © 2016. Published by Elsevier Inc.)

Gambogic acid (GA), the major active ingredient of gamboge, has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients due to its strong anticancer activity. However, our previous research showed that GA was teratogenic against zebrafish fin development. To explore the teratogenicity and the underlying mechanisms, zebrafish (Danio rerio) embryos were used. The morphological observations revealed that GA caused fin defects in zebrafish embryos in a concentration-dependent manner. The critical exposure time of GA to reveal teratogenicity was before 8 hpf (hours post fertilization). LC/MS/MS analysis revealed that a maximum bioconcentration of GA was occurred at 4 hpf. Q-PCR data showed that GA treatment resulted in significant inactivation of RA signaling which could be partially rescued by the exogenous supply of RA. These results indicate the potential teratogenicity of GA and provide evidence for a caution in its future clinic use., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.

Tamoxifen is a widely used anticancer drug with both an estrogen agonist and antagonist effect. This study focused on its endocrine disrupting effect, and overall environmental significance. Zebrafish embryos were exposed to different concentrations (0.5, 5, 50 and 500 µg l(-1) ) of tamoxifen for 96 h. The results showed a complex effect of tamoxifen on zebrafish embryo development. For the 500 µg l(-1) exposure group, the heart rate was decreased by 20% and mild defects in caudal fin and skin were observed. Expressions of a series of genes related to endocrine and morphological changes were subsequently tested through quantitative real-time polymerase chain reaction. Bisphenol A as a known estrogen was also tested as an endocrine-related comparison. Among the expression of endocrine-related genes, esr1, ar, cyp19a1b, hsd3b1 and ugt1a1 were all increased by tamoxifen exposure, similar to bisphenol A. The cyp19a1b is a key gene that controls estrogen synthesis. Exposure to 0.5, 5, 50 and 500 µg l(-1) of tamoxifen caused upregulation of cyp19a1b expression to 152%, 568%, 953% and 2024% compared to controls, higher than the effects from the same concentrations of bisphenol A treatment, yet vtg1 was suppressed by 24% from exposure to 500 µg l(-1) tamoxifen. The expression of metabolic-related genes such as cyp1a, cyp1c2, cyp3a65, gpx1a, gstp1, gsr and genes related to observed morphological changes such as krt17 were also found to be upregulated by high concentrations of tamoxifen. These findings indicated the potential environmental effect of tamoxifen on teleost early development. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

A Turing mechanism implemented by BMP, SOX9 and WNT has been proposed to control mouse digit patterning. However, its generality and contribution to the morphological diversity of fins and limbs has not been explored. Here we provide evidence that the skeletal patterning of the catshark Scyliorhinus canicula pectoral fin is likely driven by a deeply conserved Bmp-Sox9-Wnt Turing network. In catshark fins, the distal nodular elements arise from a periodic spot pattern of Sox9 expression, in contrast to the stripe pattern in mouse digit patterning. However, our computer model shows that the Bmp-Sox9-Wnt network with altered spatial modulation can explain the Sox9 expression in catshark fins. Finally, experimental perturbation of Bmp or Wnt signalling in catshark embryos produces skeletal alterations which match in silico predictions. Together, our results suggest that the broad morphological diversity of the distal fin and limb elements arose from the spatial re-organization of a deeply conserved Turing mechanism.

The role of Homeobox transcription factors during fin and limb development have been the focus of recent work investigating the evolutionary origin of limb-specific morphologies. Here we characterize the expression of HoxD genes, as well as the cluster-associated genes Evx2 and LNP, in the paddlefish Polyodon spathula, a basal ray-finned fish. Our results demonstrate a collinear pattern of nesting in early fin buds that includes HoxD14, a gene previously thought to be isolated from global Hox regulation. We also show that in both Polyodon and the catshark Scyliorhinus canicula (a representative chondrichthyan) late phase HoxD transcripts are present in cells of the fin-fold and co-localize with And1, a component of the dermal skeleton. These new data support an ancestral role for HoxD genes in patterning the fin-folds of jawed vertebrates, and fuel new hypotheses about the evolution of cluster regulation and the potential downstream differentiation outcomes of distinct HoxD-regulated compartments.

In humans, genetic diseases affecting skin integrity (genodermatoses) are generally caused by mutations in a small number of genes that encode structural components of the dermal-epidermal junctions. In this article, we first show that inactivation of both exosc9, which encodes a component of the RNA exosome, and ptbp1, which encodes an RNA-binding protein abundant in Xenopus embryonic skin, impairs embryonic Xenopus skin development, with the appearance of dorsal blisters along the anterior part of the fin. However, histological and electron microscopy analyses revealed that the two phenotypes are distinct. Exosc9 morphants are characterized by an increase in the apical surface of the goblet cells, loss of adhesion between the sensorial and peridermal layers, and a decrease in the number of ciliated cells within the blisters. Ptbp1 morphants are characterized by an altered goblet cell morphology. Gene expression profiling by deep RNA sequencing showed that the expression of epidermal and genodermatosis-related genes is also differentially affected in the two morphants, indicating that alterations in post-transcriptional regulations can lead to skin developmental defects through different routes. Therefore, the developing larval epidermis of Xenopus will prove to be a useful model for dissecting the post-transcriptional regulatory network involved in skin development and stability with significant implications for human diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Extreme novelties in the shape and size of paired fins are exemplified by extinct and extant cartilaginous and bony fishes. Pectoral fins of skates and rays, such as the little skate (Batoid, Leucoraja erinacea), show a strikingly unique morphology where the pectoral fin extends anteriorly to ultimately fuse with the head. This results in a morphology that essentially surrounds the body and is associated with the evolution of novel swimming mechanisms in the group. In an approach that extends from RNA sequencing to in situ hybridization to functional assays, we show that anterior and posterior portions of the pectoral fin have different genetic underpinnings: canonical genes of appendage development control posterior fin development via an apical ectodermal ridge (AER), whereas an alternative Homeobox (Hox)-Fibroblast growth factor (Fgf)-Wingless type MMTV integration site family (Wnt) genetic module in the anterior region creates an AER-like structure that drives anterior fin expansion. Finally, we show that GLI family zinc finger 3 (Gli3), which is an anterior repressor of tetrapod digits, is expressed in the posterior half of the pectoral fin of skate, shark, and zebrafish but in the anterior side of the pelvic fin. Taken together, these data point to both highly derived and deeply ancestral patterns of gene expression in skate pectoral fins, shedding light on the molecular mechanisms behind the evolution of novel fin morphologies.