Knockdown of vitamin D receptor affects early stages of pectoral fin development in zebrafish.

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.

A median fin derived from the lateral plate mesoderm and the origin of paired fins.

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 territories1. 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 duplication2. 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.

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.

Mechanical role of actinotrichia in shaping the caudal fin of zebrafish.

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.

Tbx5a and Tbx5b paralogues act in combination to control separate vectors of migration in the fin field of zebrafish.

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.

A show of Hands: Novel and conserved expression patterns of teleost hand paralogs during craniofacial, heart, fin, peripheral nervous system and gut development.

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.

Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins.

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.

A calcineurin-mediated scaling mechanism that controls a K+-leak channel to regulate morphogen and growth factor transcription.

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.

Organs, limbs, fins and tails are made of multiple tissues whose growth is controlled by specific signals and genetic programmes. All these different cell populations must work together during development or regeneration to form a complete structure that is the right size in relation to the rest of the body. Growing evidence suggests that this synchronicity might be down to electric signals, which are created by movements of charged particles in and out of cells. In particular, previous work has identified two factors that control the development of fins in fish: the Kcnk5b potassium-leak channel, which allows positive ions to cross the cell membrane; and an enzyme called calcineurin, which can modify the activity of proteins. Kcnk5b and calcineurin seem to play similar roles in the proportional growth of the fins in relation to the body, but exactly how was unknown. To investigate this question, Yi et al. used genetically modified zebrafish to show how the Kcnk5b channel could control genes responsible for appendage growth. However, their tests on different cell types revealed that potassium movement through the Kcnk5b channel leads to different sets of developmental genes being turned on, depending on the tissue type of the cell. This could explain how one type of signal (in this case, movement of ions) can coordinate the growth of a wide range of tissues that use different combinations of developmental genes to form. Kcnk5b therefore appears to coordinate the regulation of the various combinations of genes needed for different fin tissues to develop, so that every component grows in a proportional, synchronized manner. Yi et al. also showed that calcineurin can modify the Kcnk5b channel to control its activity. In turn, this affects the movement of potassium ions across the membrane, changing electrical activity and, as a consequence, the proportional growth of the fin. Further work should explore how Kcnk5b and calcineurin link to other signals that regulate the size of fins and limbs. Ultimately, a finer understanding of the molecules controlling the growth of body parts will be useful in fields such as regenerative medicine or stem cell biology, which attempt to build organs for clinical therapies.

Hoxd13/Bmp2-mediated mechanism involved in zebrafish finfold design.

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 leot1/lofdt1 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.

Latent developmental potential to form limb-like skeletal structures in zebrafish.

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.

Developmental hourglass and heterochronic shifts in fin and limb development.

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.

Animals come in all shapes and sizes. This diversity arose through genetic mutations during evolution, but it is unclear exactly how these variations led to the formation of new shapes. There is increasing evidence to suggest that not all shapes are possible and that variability between animals is limited by a phenomenon known as "developmental constraint". These limitations direct parts of the body towards a specific shape as they develop in the embryo. Therefore, understanding the mechanisms underlying these developmental constraints could help explain how different body shapes evolved. The limbs of humans and other mammals evolved from the fins of fish, and this transition is often used to study the role developmental constraints play in evolution. This is an ideal model as there is already a detailed fossil record mapping this evolutionary event, and data pinpointing some of the genes involved in the development of limbs and fins. But this data is incomplete, and a full comparison between the genes activated in the fin and the limb during embryonic development had not been achieved. This is because most fish used for research have undergone recent genetic changes, making it hard to spot which genetic differences are linked to the evolution of the limb. To overcome this barrier, Onimaru et al. compared genetic data from the developing limbs of mice to the developing fins of the brown-banded bamboo shark, which evolves much slower than other fish. This revealed that although many genes commonly played a role in the development of the fin and the limb in the embryo, the activity of these shared genes was not the same. For example, genes that switched on in the late stages of limb development, switched off in the late stages of fin development. But in the middle of development, those differences were relatively small and both species activated very similar sets of genes. Many of these genes were pleiotropic, which means they have important roles in other tissues and therefore mutate less often. This suggests that the mid-stage of limb development is under the strongest level of constraint. Darwin's theory of natural selection explains that mutations drive evolution. But the theory cannot predict what kinds of new body shapes new mutations will produce. Understanding how the activity levels of different genes affect development could help to fill this knowledge gap. This has potential medical applications, for example, understanding why some genetic changes cause more serious problems than others. This work suggests that mutations in genes that are active during the mid-stage of limb development may have the most serious impact.

The Developmental and Genetic Architecture of the Sexually Selected Male Ornament of Swordtails.

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.

Cloning and expression of Hoxc6 gene from Pampus argenteus and its relationship with pelvic fin absence.

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.

Heads and tails: The notochord develops differently in the cranium and caudal fin of Atlantic Salmon (Salmo salar, L.).

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.

Deformación de la aleta caudal en alevín de salmón del atlántico (Salmo salar), en etapa de saco vitelino / Deformation of the caudal fin in atlantic salmon fry (Salmo salar), in the yolk sac stage

RESUMEN: En este estudio se analiza una deformación que afectó la aleta caudal de los alevines de salmón del Atlántico (Salmo salar) y que les proporcionó un aspecto de "cola aguzada". Al momento de la eclosión se observaron completamente normales pero la deformación se detectó con posterioridad. El objetivo de este trabajo es describir los cambios anatómicos e histológicos de la aleta caudal deformada del alevín de salmón del Atlántico (Salmo salar). Para esto se utilizaron 60 alevines para realizar análisis de laboratorio y descartar la presencia de patógenos virales o bacterianos. Otros 60 alevines con un estado de desarrollo entre 600 y 700 Unidades térmicas acumuladas (UTAs) fueron anestesiados con Benzocaína 5 %, fijados en formalina al 10 % pesados y medidos. De estos un grupo de 30 alevines (15 normales y 15 deformes) fueron sometidos a la técnica de Hanken y Wassersug para evaluar Lepidotriquias. Los otros 30 alevines (15 normales y 15 deformes), fueron procesados mediante las técnicas histoquímicas: H&E/azul de Alcián para evaluar las características histológicas generales. Adicionalmente se utilizó técnicas inmunohistoquímicas para reconocer la ubicación y la presencia de los centros de señales Sonic hedgehog (Shh) para la formación de Lepidotriquias. A los valores obtenidos para las variables cuantitativas peso y longitud de cuerpo, largo y ancho de aleta caudal, se les realizó estadística descriptiva y fueron sometidos a prueba de normalidad de Shapiro-Wilk. Las diferencias observadas entre peces normales y deformes, fueron analizadas mediante prueba t de Student o U de Mann Whitney, utilizando el paquete estadístico IBM SPSS 20.0. La deformación se observó desde las 600 UTA. El peso de los alevines deformes fue similar al de los peces normales (p>0,05), lo mismo sucedió con la longitud de la aleta (p>0,05). Por el contrario, el ancho de la aleta de los deformes fue muy reducida (p<0,05). El fenotipo de aleta aguzada presentó un cambio en la integridad de los bordes, fracturas de Lepidotriquias. La epidermis de la aleta caudal de los alevines deformados presentó sus centros de señalización Shh activos, pero el blastema interrayos presentó vasodilatación, congestión y hemorragias. La presentación de este caso se relacionó con incrementos bruscos de temperatura peri eclosional.

SUMMARY: This study analyzes the deformity of Atlantic salmon fry (Salmo salar) caudal fin, which gives it a "pointed tail" appearance. Although at hatching specimens were normal, the deformities were detected later. The objective of this work is to describe the anatomical and histological changes of the deformed caudal fin of the Atlantic salmon fry (Salmo salar). In this analysis we used 60 specimens for laboratory analyses, to rule out the presence of viral or bacterial pathogens. Another 60 fry, developmental stage between 600 and 700 Accumulated Thermal Units (UTAs) were anesthetized with 5 % Benzocaine, fixed in 10 % formalin, weighed and measured. Of these, a group of 30 fry (15 normal and 15 deformed) underwent the Hanken and Wassersug technique to evaluate lepidotrychia or dermal rays. The remaining 30 fry (15 normal and 15 deformed) were processed using H & E / Alcián blue histochemical techniques to evaluate general histological characteristics. Additionally, immunohistochemical techniques were used to determine the location and presence of Sonic hedgehog (Shh) signal centers for lepidotrychia development. The values obtained for the quantitative variables body weight, length and width of the tail fin were described using the arithmetic mean and standard deviation. The deformity was observed from 600 UTA. Weight of deformed fry was less than normal fish, length of the fin was similar in normal and deformed fish. In contrast, width of the deformed fin was significantly reduced. The sharp fin phenotype presented a change in the conformity of the edges, lepidotrychia fractures. The epidermis presented active Shh signaling centers, but the interray blastema showed vasodilation, congestion and hemorrhages. The presentation of this case was related to sudden increases in perieclosional temperature.

Embryonic origin and serial homology of gill arches and paired fins in the skate, Leucoraja erinacea.

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.

A common way to evolve new body parts is to copy existing ones and to remodel them. In insects for example, the antennae, mouth parts and legs all follow the same basic body plan, with modifications that adapt them for different uses. In the late 19th century, anatomist Karl Gegenbaur noticed a similar pattern in fish. He saw similarities between pairs of fins and pairs of gills, suggesting that one evolved from the other. But there is currently no fossil evidence documenting such a transformation. Modern research has shown that the development of both gill and fin skeletons shares common genetic pathways. But the cells that form the two structures do not come from the same place. Gill skeletons develop from a part of the embryo called the neural crest, while fin skeletons come from a region called the mesoderm. One way to test Gegenbaur's idea is to look more closely at the cells that form gill and fin skeletons as fish embryos develop. Here, Sleight and Gillis examined the gills and fins of a cartilaginous fish called Leucoraja erinacea, also known as the little skate. Sleight and Gillis labelled the cells from the neural crest and mesoderm of little skate embryos with a fluorescent dye and then tracked the cells over several weeks. While the fins did form from mesoderm cells, the gills did not develop as expected. The first gill contained only neural crest cells, but the rest were a mixture of both cell types. This suggests that fins and gills develop from a common pool of cells that consists of both neural crest and mesoderm cells, which have the potential to develop into either body part. This previously unrecognised embryonic continuity between gills and fins explains why these structures respond in the same way to the same genetic cues, regardless of what cell type they develop from. Based on this new evidence, Sleight and Gillis believe that Gegenbaur was right, and that fins and gills do indeed share an evolutionary history. While firm evidence for the transformation of gills into fins remains elusive, this work suggests it is possible. A deeper understanding of the process could shed light on the development of other repeated structures in nature. Research shows that animals use a relatively small number of genetic cues to set out their body plans. This can make it hard to use genetics alone to study their evolutionary history. But, looking at how different cell types respond to those cues to build anatomical features, like fins and gills, could help to fill in the gaps.

Adult chondrogenesis and spontaneous cartilage repair in the skate, Leucoraja erinacea.

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.

For our joints to move around freely, they are lubricated with cartilage. In growing mammals, this tissue is continuously made by the body. But, by adulthood, this cartilage will have been almost entirely replaced by bone. It is also difficult for adult bodies to replenish what cartilage does remain ­ such as that in the joints. When growing new cartilage, the body uses so-called progenitor cells, which have the ability to turn into different cell types. Progenitor cells are recruited to the joints, where they transform into cartilage cells called chondrocytes, which generate new cartilage. But adults lack these progenitor cells, leaving them unfit to heal damaged cartilage after injury or diseases like osteoarthritis. In contrast, certain groups of fishes, such as skates, sharks and rays, produce cartilage throughout their life ­ indeed their whole skeleton is made of cartilage. So, what is the difference between these cartilaginous fishes and mammals? Why can they generate cartilage throughout their lives, while humans are unable to? And does this mean that these adult fish are better at healing injured cartilage? Marconi et al. used skates (Leucoraja erinacea) to study how cartilage develops, grows and heals in a cartilaginous fish. Progenitor cells were found in a layer that wraps around the cartilage skeleton (called the perichondrium). These cells were also shown to activate genes that control cartilage development. By labelling these progenitor cells, their presence and movements could be tracked around the fish. Marconi et al. found progenitor cells in adult skates that were able to generate chondrocytes. Skates were also shown to spontaneously repair damaged cartilage in experiments where cartilage was injured. Marconi et al. have identified the skate as a new animal model for studying cartilage growth and repair. Studying the mechanisms that skate progenitor cells use for generating cartilage could lead to improvements in current therapies used for repairing cartilage in the joints.

Expression of nucleosome assembly protein 1 like genes in zebrafish embryos.

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.

3D + Time Imaging and Image Reconstruction of Pectoral Fin During Zebrafish Embryogenesis.

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.

Ontogeny of the catfish pectoral-fin spine (Teleostei: Siluriformes).

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).