Laminin alpha5 is essential for the formation of the zebrafish fins

The vertebrate fin fold, the presumptive evolutionary antecedent of the paired fins, consists of two layers of epidermal cells extending dorsally and ventrally over the trunk and tail of the embryo, facilitating swimming during the embryonic and larval stages. Development of the fin fold requires dramatic changes in cell shape and adhesion during early development, but the proteins involved in this process are completely unknown. In a screen of mutants defective in fin fold morphogenesis, we identified a mutant with a severe fin fold defect, which also displays malformed pectoral fins. We find that the cause of the defect is a non-sense mutation in the zebrafish lama5 gene that truncates laminin α5 before the C-terminal laminin LG domains, thereby preventing laminin α5 from interacting with its cell surface receptors. Laminin is mislocalized in this mutant, as are the membrane-associated proteins, actin and β-catenin, that normally form foci within the fin fold. Ultrastructural analysis revealed severe morphological abnormalities and defects in cell-cell adhesion within the epidermis of the developing fin fold at 36 hpf, resulting in an epidermal sheet that can not extend away from the body. Examining the pectoral fins, we find that the lama5 mutant is the first zebrafish mutant identified in which the pectoral fins fail to make the transition from an apical epidermal ridge to an apical fold, a transformation that is essential for pectoral fin morphogenesis. We propose that laminin α5, which is concentrated at the distal ends of the fins, organizes the distal cells of the fin fold and pectoral fins in order to promote the morphogenesis of the epidermis. The lama5 mutant provides novel insight into the role of laminins in the zebrafish epidermis, and the molecular mechanisms driving fin formation in vertebrates.     

Morphological and histochemical characterization of the pectoral fin muscle of batoids

Batoids differ from other elasmobranch fishes in that they possess dorsoventrally flattened bodies with enlarged muscled pectoral fins. Most batoids also swim using either of two modes of locomotion: undulation or oscillation of the pectoral fins. In other elasmobranchs (e.g., sharks), the main locomotory muscle is located in the axial myotome; in contrast, the main locomotory muscle in batoids is found in the enlarged pectoral fins. The pectoral fin muscles of sharks have a simple structure, confined to the base of the fin; however, little to no data are available on the more complex musculature within the pectoral fins of batoids. Understanding the types of fibers and their arrangement within the pectoral fins may elucidate how batoid fishes are able to utilize such unique swimming modes. In the present study, histochemical methods including succinate dehydrogenase (SDH) and immunofluoresence were used to determine the different fiber types comprising these muscles in three batoid species: Atlantic stingray (Dasyatis sabina), ocellate river stingray (Potamotrygon motoro) and cownose ray (Rhinoptera bonasus). All three species had muscles comprised of two muscle fiber types (slow-red and fast-white). The undulatory species, D. sabina and P. motoro, had a larger proportion of fast-white muscle fibers compared to the oscillatory species, R. bonasus. The muscle fiber sizes were similar between each species, though generally smaller compared to the axial musculature in other elasmobranch fishes. These results suggest that batoid locomotion can be distinguished using muscle fiber type proportions. Undulatory species are more benthic with fast-white fibers allowing them to contract their muscles quickly, as a possible means of escape from potential predators. Oscillatory species are pelagic and are known to migrate long distances with muscles using slow-red fibers to aid in sustained swimming.     

The morphology of the cephalic lobes and anterior pectoral fins in six species of batoids

Many benthic batoids utilize their pectoral fins for both undulatory locomotion and feeding. Certain derived, pelagic species of batoids possess cephalic lobes, which evolved from the anterior pectoral fins. These species utilize the pectoral fins for oscillatory locomotion while the cephalic lobes are used for feeding. The goal of this article was to compare the morphology of the cephalic lobes and anterior pectoral fins in species that possess and lack cephalic lobes. The skeletal elements (radials) of the cephalic lobes more closely resembled the radials in the pectoral fin of undulatory species. Second moment of area (I), calculated from cephalic lobe radial cross sections, and the number of joints revealed greater flexibility and resistance to bending in multiple directions as compared to pectoral fin radials of oscillatory species. The cephalic lobe musculature was more complex than the anterior pectoral fin musculature, with an additional muscle on the dorsal side, with fiber angles running obliquely to the radials. In Rhinoptera bonasus, a muscle presumably used to help elevate the cephalic lobes is described. Electrosensory pores were found on the cephalic lobes (except Mobula japonica) and anterior pectoral fins of undulatory swimmers, but absent from the anterior pectoral fins of oscillatory swimmers. Pore distributions were fairly uniform except in R. bonasus, which had higher pore numbers at the edges of the cephalic lobes. Overall, the cephalic lobes are unique in their anatomy but are more similar to the anterior pectoral fins of undulatory swimmers, having more flexibility and maneuverability compared to pectoral fins of oscillatory swimmers. The maneuverable cephalic lobes taking on the role of feeding may have allowed the switch to oscillatory locomotion and hence, a more pelagic lifestyle. J. Morphol. 274:1070–1083, 2013. © 2013 Wiley Periodicals, Inc.     

Notch Signalling Is Required for the Formation of Structurally Stable Muscle Fibres in Zebrafish

Background

Accurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown.

Methodology/Principal Findings

Here we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis.

Conclusions/Significance

We propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin.     

Functional Morphology of Aquatic Flight in Fishes: Kinematics, Electromyography, and Mechanical Modeling of Labriform Locomotion

Labriform locomotion is the primary swimming mode for many fishesthat use the pectoral fins to generate thrust across a broadrange of speeds. A review of the literature on hydrodynamics,kinematics, and morphology of pectoral fin mechanisms in fishesreveals that we lack several kinds of morphological and kinematicdata that are critical for understanding thrust generation inthis mode, particularly at higher velocities. Several needsinclude detailed three-dimensional kinematic data on speciesthat are pectoral fin swimmers across a broad range of speeds,data on the motor patterns of pectoral fin muscles, and thedevelopment of a mechanical model of pectoral fin functionalmorphology. New data are presented here on pectoral fin locomotionin Gomphosus varius, a labrid fish that uses the pectoral finsat speeds of 1 –6 total body lengths per second. Three-dimensionalkinematic data for the pectoral fins of G. varius show thata typical "drag-based" mechanism is not used in this species.Instead, the thrust mechanics of this fish are dominated bylift forces and acceleration reaction forces. The fin is twistedlike a propeller during the fin stroke, so that angles of attackare variable along the fin length. Electromyographic data onsix fin muscles indicate the sequence of muscle activity thatproduces antagonistic fin abduction and adduction and controlsthe leading edge of the fin. EMG activity in abductors and adductorsis synchronous with the start of abduction and adduction, respectively,so that muscle mechanics actuate the fin with positive work.A mechanical model of the pectoral fin is proposed in whichfin morphometrics and computer simulations allow predictionsof fin kinematics in three dimensions. The transmission of forceand motion to the leading edge of the fin depends on the mechanicaladvantage of fin ray levers. An integrative program of researchis suggested that will synthesize data on morphology, physiology,kinematics, and hydrodynamics to understand the mechanics ofpectoral fin swimming.     

Larval development of Hypsophrys nicaraguensis (Pisces: Cichlidae) under laboratory conditions

The cichlid Hypsophrys nicaraguensis is a popular fish known as butterfly, and despite its widespread use as pets, little is known about its reproductive biology. In order to contribute to this knowledge, the study describes the relevant larval development characteristics, from adult and larval cultures in captivity. Every 12h, samples of larvae were collected and observed under the microscope for larval stage development, and every 24h morphometric measurements were taken. Observations showed that at 120h, some larvae had swimming activity and the pectoral fins development was visible; at 144h, the dorsal fin appear and all larvae started food intake; at 168h, the formation of anal fins begins, small rudiments of pelvic fins emerge, the separation of caudal fin from anal and dorsal fins starts, and the yolk sac is reabsorbed almost completely; at 288h, the pelvic fins starts to form; at 432h, the rays and spines of dorsal and anal fins can be distinguished, both the anal and the dorsal fins have the same number of spines and rays as in adults. After 480h larvae have the first scales, ending the larval stages and starting the transformation to fingerlings. Larvae were successfully fed with commercial diet.     

The Embryonic and Larval Development of Polypterus senegalusCuvier, 1829: its Staging with Reference to External and Skeletal Features, Behaviour and Locomotory Habits

The embryonic and larval development of the Polypteriformes, the presumed sister group of all other living actinopterygians, is poorly known. The main reason is the scarcity of successful breedings in captivity and therefore the lack of developmental series of any one polypterid species. A series of five successful breedings of P. senegalusnow makes it possible to define developmental stages of this species based on numerous closely timed specimens. The staging given here focuses on external embryonic and larval features: epidermal surface structures documented by SEM, colour pattern, development of fins and squamation, larval feeding and locomotory behaviour. The development of P. senegalusis characterized by a long free embryonic phase. Suction feeding is performed from the beginning of larval life (apterolarval phase). The pectoral fins start to become employed for slow locomotion and as supportive structures at around the same time. Olfactorily guided prey capture, however, is observed later in the pterolarval phase. Quantitative kinematic data also demonstrate a change in the mode of undulatory locomotion during this phase. Sustained axial undulation becomes confined to the posterior abdominal and caudal region of the body. At about the same time the paraxial high frequency undulation of the pectoral fin fold is replaced by the characteristic propeller-like movement of much greater amplitude and wavelength. Surfacing for aerial breathing is not seen before a marked change in colouration has taken place at the beginning of the juvenile period. The external gills slowly become reduced during this period. The definitions of larval and juvenile stages given here may advance understanding of developmental processes in the ontogeny of these primitive actinopterygians, and may serve as a tool for comparison with the ontogeny of Tetrapoda and Dipnoi, as well as to that of some “primitive” groups of Actinopterygii. Judging from its distribution among extant taxa, embryonic and larval ciliation is a character that most probably belongs to the grundplan? of Osteognathostomata. Phylogenetic evaluation is not so clear for the two other prominent embryonic and larval specializations found in Polypterus: upper labial attachment glands and opercular external gills. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

Expression analysis of zebrafish membrane type-2 matrix metalloproteinases during embryonic development

Membrane tethered matrix metalloproteinases (MMPs) cleave a variety of extracellular matrix (ECM) and non-ECM targets and play important roles during embryonic development and tumor progression. Membrane tethered MMPs in particular are important regulators of both tissue invasion and morphogenesis. Much attention has been given to understanding the function of human and mouse MMP14 (also called membrane type-1 MMP, MT1-MMP) and our own data have linked zebrafish Mmp14 to the regulation of gastrulation cell movements. However, less is known regarding the expression and function of other membrane tethered MMPs. We report the cloning and gene expression analysis of zebrafish mmp15a and mmp15b (MT2-MMP) during early embryonic and larval development. Our data show that mmp15a exhibits limited expression prior to segmentation stages and is first detected in the tectum and posterior tailbud. At 24hours post-fertilization (hpf) mmp15a localizes to the caudal hematopoietic tissue, pectoral fin buds, and mandibular arch. By contrast, mmp15b is strongly expressed during gastrula stages before becoming restricted to the polster and anterior neural plate. From 24 to 48hpf, mmp15b expression is detected in the pharyngeal arches, fin buds, otic vesicle, pronephric ducts, proctodeum, tail epidermis, posterior lateral line primordia, and caudal notochord. During the larval period beginning at 72hpf, mmp15b expression becomes restricted to the brain ventricular zone, pharyngeal arches, pectoral fins, and the proctodeum. Many of the mmp15-expressing tissues have been shown to express genes encoding components of the ECM including collagens, fibronectin, and laminins. Our data thus provide a foundation for uncovering the role of Mmp15-dependent pericellular proteolysis during zebrafish embryonic development.     

The fine structure of the fin musculature in two teleost species with different swimming modes,the puffer,Tetraodon steindachneri,and the goldfish,Carassius auratus

Summary Puffer fish (Tetraodon steindachneri) can execute precise maneuvers due to their highly specialized mode of propulsion. In the conventional locomotion exemplified by the goldfish (Carassius auratus), the fish thrusts are generated by lateral beating of the caudal fin. In contrast, the puffer generates its propulsive force by very rapid undulating movements of the pectoral, dorsal and anal fins. The fine structure of the fin muscles is identical in the two species of fishes, despite the differences in fin movement; cytologically, the fibers are intermediate between those of red and of white muscle. On the other hand, both the fusion frequency and the number of motor endplates are considerably higher in the fin muscles of the puffer than in those of the goldfish.     

The mechanism of cartilage subdivision in the reorganization of the zebrafish pectoral fin endoskeleton

A cartilaginous pectoral fin endoskeleton in zebrafish (Danio rerio) develops early, after which the cartilage of the larval fin endoskeleton undergoes a complete transformation into the adult morphology. This transformation includes multiple subdivisions of a single cartilaginous disk. The type of cartilage subdivision is unique to teleost fish. In this study, we present the timing and the developmental features of these subdivisions and we discuss variation in this process, caused by differences in growth rate. We establish that the cartilage subdivisions are developmentally linked to the formation of lepidotrichia in the fin fold. At the cellular level, we show that neither apoptosis nor resorption by chondroclasts and/or macrophages contributes to the cartilage subdivision. Ultrastructural observations show dedifferentiation of chondrocytes in subdivision zones. Different from forelimb development in other vertebrates, dedifferentiation is an important mechanism in the development of the adult pectoral fin skeleton. We here provide further support for the idea that the phenotype of skeletal tissues is not terminal and that plasticity of differentiated connective tissues can play an important role in various developmental and homeostatic processes.     

Growth of inhibitory innervation in a lobster muscle

The fine structure of inhibitory innervation to a limb muscle was examined in larval, juvenile, and adult lobsters. The innervation is essentially similar in qualitative features among these different stages, although there are some marked quantitative changes associated with growth. From being localized to discrete regions in the larval muscle, the inhibitory innervation spreads to groups of muscle fibers in the early juvenile muscle and to single fibers in the late juvenile and adult muscles. Concurrently, its neuromuscular synapses enlarge in area, become perforated, and acquire more active sites of transmitter release. Inhibitory nerve terminals occur in close proximity to their excitatory counterparts in the muscles of larval and early juvenile stages, although in later stages this juxtaposition occurs preferentially in some muscle fibers but not others. The inhibitory innervation is, nevertheless, much more restricted in occurrence than is the excitatory innervation.     

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages

During development of the limbs, Hox genes belonging to the paralogous groups 9-13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9-13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.     

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.     

Synchronized swimming: coordination of pelvic and pectoral fins during augmented punting by the freshwater stingray Potamotrygon orbignyi

Benthic animals live at the juncture of fluid and solid environments, an interface that shapes many aspects of their behavior, including their means of locomotion. Aquatic walking and similar substrate-dependent forms of underwater propulsion have evolved multiple times in benthic invertebrate and vertebrate taxa, including batoid elasmobranchs. Skates (Rajidae) use the pelvic fins to punt across the substrate, keeping the pectoral fin disc still. Other batoids combine pelvic fin motions with pectoral fin undulation in augmented punting, but the coordination of these two modes has not been described. In this study of an augmented punter, the freshwater stingray Potamotrygon orbignyi, we demonstrate the synchrony of pelvic and pectoral fin cycles. The punt begins as the pelvic fins, held in an anterior position, are planted into the substrate and used to push the body forward. Meanwhile, a wave of pectoral fin undulation begins, increasing to maximum height just before the cycle's halfway point, when the pelvic fins reach their furthest posterior extension. The pectoral fin wave subsides as the pelvic fins return to their starting position for subsequent punts. Despite definitive links between pectoral and pelvic fin activity, we find no significant relationship between pectoral fin kinematics (frequency, wave height, and wave speed) and punt performance. However, slip calculations indicate that pectoral undulation can produce thrust and augment punting. Pelvic fin kinematics (frequency and duty factor) have significant effects, suggesting that while both sets of fins contribute to thrust generation, the pelvic fins likely determine punt performance.     

Morphogenesis in hatchery-reared larvae of the black rockfish,Sebastes schlegeli,and its relationship to the development of swimming and feeding functions

The developmental sequence of morphological characteristics related to swimming and feeding functions was investigated in hatchery-reared larvae and juveniles ofSebastes schlegeli, a viviparous scorpaenid. The fish were extruded at an early larval stage, when the mean body size was 6.23 mm TL. Fin-ray rudiments became visible at 9.0 mm TL in the dorsal and anal fins, at 8.0 mm TL in the pectoral and pelvic fins and 6.0 mm TL (size at extrusion) in the caudal fin. Completion of segmentation of soft rays in the dorsal and anal fins was attained by 14 mm TL and in all fins by 17 mm TL. Branching of soft rays in the respective fins started and was completed considerably later than the completion of segmentation, as well as ossification of the fin-supports. Morphological transformation from larva to juvenile was apparently completed by about 17 mm TL. Although the completion of basic juvenile structures was attained by transformation at that body size, succeeding morphological changes occurred between 17 mm and 32 mm TL. Newly-extruded larvae possessed one or two teeth on the lower pharyngeal and pharyngobranchials 3 and 4, but lacked premaxillary, dentary, palatine and prevomer teeth. The fish attained full development of gill rakers and gill teeth by 15 mm TL, the upper and lower pharyngeal teeth subsequently developing into a toothplate. Development of the premaxillary, dentary and palatine teeth was completed at about 30 mm TL, by which time loop formation of the digestive canal and the number of pyloric caeca had attained the adult condition. The developmental sequence of swimming and feeding functions during larval and early juvenile periods appeared to proceed from primitive functions to advanced or complex ones, from the ability to produce propulsive force to that of swimming with high maneuverability and from development of the irreducible minimum function of passing food into the stomach to the ability to actively capture prey via passive food acquisition with the gill rakers and gill teeth. The relationship of morphological development to the behavior and feeding activity of artificially-produced hatchlings is also discussed.     

Diversity of pectoral fin structure and function in fishes with labriform propulsion

Aquatic propulsion generated by the pectoral fins occurs in many groups of perciform fishes, including numerous coral reef families. This study presents a detailed survey of pectoral fin musculoskeletal structure in fishes that use labriform propulsion as the primary mode of swimming over a wide range of speeds. Pectoral fin morphological diversity was surveyed in 12 species that are primarily pectoral swimmers, including members of all labroid families (Labridae, Scaridae, Cichlidae, Pomacentridae, and Embiotocidae) and five additional coral reef fish families. The anatomy of the pectoral fin musculature is described, including muscle origins, insertions, tendons, and muscle masses. Skeletal structures are also described, including fin shape, fin ray morphology, and the structure of the radials and pectoral girdle. Three novel muscle subdivisions, including subdivisions of the abductor superficialis, abductor profundus, and adductor medialis were discovered and are described here. Specific functional roles in fin control are proposed for each of the novel muscle subdivisions. Pectoral muscle masses show broad variation among species, particularly in the adductor profundus, abductor profundus, arrector dorsalis, and abductor superficialis. A previously undescribed system of intraradial ligaments was also discovered in all taxa studied. The morphology of these ligaments and functional ramifications of variation in this connective tissue system are described. Musculoskeletal patterns are interpreted in light of recent analyses of fin behavior and motor control during labriform swimming. Labriform propulsion has apparently evolved independently multiple times in coral reef fishes, providing an excellent system in which to study the evolution of pectoral fin propulsion.     

Pectoral fin morphology of batoid fishes (Chondrichthyes: Batoidea): Explaining phylogenetic variation with geometric morphometrics

The diverse cartilaginous fish lineage, Batoidea (rays, skates, and allies), sister taxon to sharks, comprises a huge range of morphological diversity which to date remains unquantified and unexplained in terms of evolution or locomotor style. A recent molecular phylogeny has enabled us to confidently assess broadscale aspects of morphology across Batoidea. Geometric morphometrics quantifies the major aspects of shape variation, focusing on the enlarged pectoral fins which characterize batoids, to explore relationships between ancestry, locomotion and habitat. A database of 253 specimens, encompassing 60 of the 72 batoid genera, reveals that the majority of morphological variation across Batoidea is attributable to fin aspect‐ratio and the chordwise location of fin apexes. Both aspect‐ratio and apex location exhibit significant phylogenetic signal. Standardized independent linear contrast analysis reveals that fin aspect‐ratio can predict locomotor style. This study provides the first evidence that low aspect‐ratio fins are correlated with undulatory‐style locomotion in batoids, whereas high aspect‐ratio fins are correlated with oscillatory locomotion. We also show that it is phylogeny that determines locomotor style. In addition, body‐ and caudal fin‐locomotors are shown to exhibit low aspect‐ratio fins, whereas a pelagic lifestyle correlates with high aspect‐ratio fins. These results emphasize the importance of phylogeny in determining batoid pectoral fin shape, however, interactions with other constraints, most notably locomotor style, are also highlighted as significant. J. Morphol. 275:1173–1186, 2014. © 2014 Wiley Periodicals, Inc.