Histological development and integration of the Zebrafish Weberian apparatus.

BACKGROUND: The Weberian apparatus enhances hearing in otophysan fishes, including Zebrafish (Danio rerio). Several studies have examined aspects of morphological development of the Weberian apparatus and hearing ability in Zebrafish. A comprehensive developmental description including both hard and soft tissues is lacking. This information is critical for both interpretation of genetic developmental analyses and to better understand the role of morphogenesis and integration on changes in hearing ability. RESULTS: Histological development of hard and soft tissues of the Weberian apparatus, including ossicles, ear, swim bladder, and ligaments are described from early larval stages (3.8 mm notochord length) through adult. Results show a strong relationship in developmental timing and maturation across all regions. All required auditory elements are present and morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and changes in shape continue throughout the examined developmental period. CONCLUSIONS: This study provides the first comprehensive histological description of Weberian apparatus development in Zebrafish. Morphological integration was found early, before increases in hearing ability were detected in functional studies (>10 mm total length), suggesting morphological integration precedes functional integration. Further research is needed to examine the nature of the functional delay, and how maturation of the Weberian apparatus influences functionality.

Histology and structural integration of the major morphologies of the Cypriniform Weberian apparatus.

The Weberian apparatus, a diagnostic feature of otophysan fishes, is a novel hearing adaptation integrating several developmental and morphological systems (ear-vertebral column-swim bladder). Otophysan fishes are one of the largest and most successful freshwater clades, with over 10,000 species across most continents. The largest otophysan order, Cypriniformes, dominates the freshwaters of Asia, Europe, North America, and Africa. Spanning such a wide variety of environments, the Weberian apparatus undergoes morphological modifications to maintain functionality. Within Cypriniformes, we propose three distinct morphological classes of the Weberian apparatus based on the level of skeletal expansion around the swim bladder: simple (typical of most Cyprinidae), anterior plate (found in families such as Gyrinocheilidae, Catostomidae, and Botiidae), and encapsulated (either single-capsule as found, e.g., in Gobionidae and Cobitidae, or double-capsule as found, e.g., in Nemacheilidae and Balitoridae). Little ontological or comparative data exists regarding the construction or integration of these different morphologies, and less is known about the tissue level integration and variation within these morphologies. We used paraffin histology to document the hard and soft tissue anatomy of the Weberian apparatus in six species representing all morphological classes. We found sites of similarity across the morphologies including size and structure of the saccule, aspects of ossicle ossification, and swim bladder tunica composition, indicating potential sites of developmental and functional constraint. In contrast, we found differences across both auditory and nonauditory features in otic chamber size, ossification within ossicles and other vertebral elements, and composition of ligaments, indicating likely sites of adaptability. Some of these changes are likely evolutionary (taxonomic), but may be influenced by the environmental niche occupied by the clade. These results show a clear need for increased ontological and comparative study of the complete cypriniform Weberian apparatus, particularly histologically, as well as increased auditory studies across morphological types.

Post-embryonic development of canal and superficial neuromasts and the generation of two cranial lateral line phenotypes.

The relatively simple structural organization of the cranial lateral line system of bony fishes provides a valuable context in which to explore the ways in which variation in post-embryonic development results in functionally distinct phenotypes, thus providing a link between development, evolution, and behavior. Vital fluorescent staining, histology, and scanning electron microscopy were used to describe the distribution, morphology, and ontogeny of the canal and superficial neuromasts on the head of two Lake Malawi cichlids with contrasting lateral line canal phenotypes (Tramitichromis sp. [narrow-simple, well-ossified canals with small pores] and Aulonocara stuartgranti [widened, more weakly ossified canals with large pores]). This work showed that: 1) the patterning (number, distribution) of canal neuromasts, and the process of canal morphogenesis typical of bony fishes was the same in the two species, 2) two sub-populations of neuromasts (presumptive canal neuromasts and superficial neuromasts) are already distinguishable in small larvae and demonstrate distinctive ontogenetic trajectories in both species, 3) canal neuromasts differ with respect to ontogenetic trends in size and proportions between canals and between species, 4) the size, shape, configuration, physiological orientation, and overall rate of proliferation varies among the nine series of superficial neuromasts, which are found in both species, and 5) in Aulonocara, in particular, a consistent number of canal neuromasts accompanied by variability in the formation of canal pores during canal morphogenesis demonstrates independence of early and late phases of lateral line development. This work provides a new perspective on the contributions of post-embryonic phases of lateral line development and to the generation of distinct phenotypes in the lateral line system of bony fishes. J. Morphol. 277:1273-1291, 2016. © 2016 Wiley Periodicals, Inc.

Heterochrony, modularity, and the functional evolution of the mechanosensory lateral line canal system of fishes.

BACKGROUND: The canals of the mechanosensory lateral line system are components of the dermatocranium, and demonstrate phenotypic variation in bony fishes. Widened lateral line canals evolved convergently in a limited number of families of teleost fishes and it had been hypothesized that they evolved from narrow canals via heterochrony and explore modularity in the lateral line system. Two species of cichlids with different canal phenotypes were used to test a hypothesis of heterochrony. Histological material prepared from ontogenetic series of Aulonocara stuartgranti (widened canals) and Tramitichromis sp. (narrow canals) was analyzed using ANCOVA to determine rates of increase in canal diameter and neuromast size (length, width) and to compare the timing of onset of critical stages in canal morphogenesis (enclosure, ossification). RESULTS: A faster rate of increase in canal diameter and neuromast width (but not length), and a delay in onset of canal morphogenesis were found in Aulonocara relative to Tramitichromis. However, rates of increase in canal diameter and neuromast size among canals, among canal portions and among canals segments reveal similar trends within both species. CONCLUSION: The evolution of widened lateral line canals is the result of dissociated heterochrony - acceleration in the rate of increase of both canal diameter and neuromast size, and delay in the onset of canal morphogenesis, in Aulonocara (widened canals) relative to Tramitichromis (narrow canals). Common rates of increase in canal diameter and neuromast size among canal portions in different dermatocranial bones and among canal segments reflect the absence of local heterochronies, and suggest modular integration among canals in each species. Thus, canal and neuromast morphology are more strongly influenced by their identities as features of the lateral line system than by the attributes of the dermatocranial bones in which the canals are found. Rate heterochrony manifested during the larval stage ensures that the widened canal phenotype, known to be associated with benthic prey detection in adult Aulonocara, is already present before feeding commences. Heterochrony can likely explain the convergent evolution of widened lateral line canals among diverse taxa. The lateral line system provides a valuable context for novel analyses of the relationship between developmental processes and the evolution of behaviorally and ecologically relevant phenotypes in fishes.

Comparative development and evolution of two lateral line phenotypes in lake Malawi cichlids.

A comparison of the pattern and timing of development of cranial lateral line canals and canal neuromasts in three species of Lake Malawi cichlids, Labeotropheus fuelleborni and Metriaclima zebra (narrow lateral line canals), and Aulonocara baenschi (widened lateral line canals) was used to test the hypothesis that the evolution of widened canals (thought to be an adaptive phenotype in the lateral line system) from narrow canals is the result of heterochrony. Using histological analysis and scanning electron microscopy, this study has provided the first detailed and quantitative description of the development of widened lateral line canals in a teleost, and has demonstrated that: 1) canal neuromast number and the pattern of canal morphogenesis are conserved among species with different adult canal phenotypes, 2) heterochrony ("dissociated heterochrony" in particular) can explain the evolution of widened canals and variation in morphology between canals within a species with respect to canal diameter and neuromast size, and 3) the morphology of the lateral line canals and the dermal bones in which they are found (e.g., the mandibular canal the dentary and anguloarticular bones of the mandible) can evolve independently of each other, thus requiring the addition of another level of complexity to discussions of modularity and integration in the skull of bony fishes.

Immunocytochemistry to study myogenesis in zebrafish.

During myogenesis, cells gradually transition from mesodermal precursors to myoblasts, myocytes, and then to muscle fibers. The molecular characterization of this process requires the ability to identify each of these cell types and the factors that regulate the transitions between them. The most versatile technique for assaying cell identities in situ is immunocytochemistry, because multiple independent molecular markers of differentiation can be assayed simultaneously. The zebrafish has developed into a popular model for the study of myogenesis, and immunocytochemical techniques have been critical. We have adapted existing protocols to optimize immunocytochemistry in zebrafish, and have tested many antibodies developed against mouse, chick, and frog muscle antigens for their cross-reactivity in zebrafish. Here, we present protocols for whole mount immunocytochemistry on both formaldehyde and Carnoy's fixed embryos as well as on sectioned zebrafish tissue. We include a table of antibodies useful for experiments on the molecular biology of myogenesis in zebrafish.

BMP regulation of myogenesis in zebrafish.

In amniotes, BMP signaling from lateral plate and dorsal neural tube inhibits differentiation of muscle precursors in the dermomyotome. Here, we show that BMPs are expressed adjacent to the dermomyotome during and after segmentation in zebrafish. In addition, downstream BMP pathway members are expressed within the somite during dermomyotome development. We also show that zebrafish dermomyotome is responsive to BMP throughout its development. Ectopic overexpression of Bmp2b increases expression of the muscle precursor marker pax3, and changes the time course of myoD expression. At later stages, overexpression increases the number of Pax7+ myogenic precursors, and delays muscle differentiation, as indicated by decreased numbers of MEF2+ nuclei, decreased number of multi-nucleated muscle fibers, and an increased myotome angle. In addition, we show that while BMP overexpression is sufficient to delay myogenic differentiation, inhibition of BMP does not detectably affect this process, suggesting that other factors redundantly inhibit myogenic differentiation.

Building an evolutionary innovation: differential growth in the modified vertebral elements of the zebrafish Weberian apparatus.

The Weberian apparatus, a complex assemblage of greatly modified vertebral elements, significantly enhances hearing within Otophysi. Ultimately we are interested in investigating the genetic mechanisms responsible for the origin, development and morphological diversification of these vertebral elements in the Weberian apparatus of otophysan fishes. However, a necessary first step involves identifying changes in growth of this region as compared with the vertebrae from which these modified elements purportedly derive. Using an ontogenetic series of the zebrafish, Danio rerio, we collected growth data for specific elements within the Weberian apparatus, including neural arches, ribs, and parapophyses. These data are compared to both serially homologous structures in posterior thoracic vertebrae (which act as internal controls) and vertebral elements from the same axial levels in three other non-otophysan teleosts. Significant differences in growth rate were found among serially homologous structures, as well as at equivalent axial levels in different species. Uniform changes in growth rates (in which all structures derived from a specific somite were equally affected) were not found, suggesting precise targeting of morphological change to specific structures. The variation in growth of anterior vertebrae in and among species was greater than expected. This variation in growth rates created developmental patterns unique to each species. Such patterns of growth may help illuminate the specific heterochronic mechanisms required for the origin and subsequent morphological diversification of the Weberian apparatus. This morphological diversity is exemplified by the multitude of forms seen in the cypriniform Weberian apparatus. Understanding patterns of growth in discrete elements of the Weberian apparatus allows us to hypothesize as to the specific developmental changes, likely constituting differences in gene expression in pathways involved in bone and cartilage differentiation, responsible for this morphological diversity.

Morphological variation in the Weberian apparatus of Cypriniformes.

Cypriniformes (which includes the minnows, carps, loaches, algae-eaters, stone loaches, and suckers) is a morphologically diverse and incredibly speciose order of teleosts. It has been suggested that a number of evolutionary innovations, key to improved hearing and feeding, have played an important role in cypriniform fishes' success. One such innovation, the Weberian apparatus, is a novel assemblage of vertebral elements and modified ribs that relay and amplify sound pressure changes from the gas bladder to the inner ear. The Weberian apparatus unites Cypriniformes with other major orders into an extremely species-rich group of fishes, the Otophysi. Together, otophysan fishes comprise one of the largest groups of fishes in the world, as well as the majority of freshwater fishes. Here we present a detailed comparison of the Weberian apparatus in a number of cypriniform families using cleared and stained specimens. We present data regarding inter- and intrafamilial morphological variation within Cypriniformes. With few, but evolutionarily important, exceptions we find that diagnostic features of the Weberian apparatus characterize each family. Interspecific variation within each of the families Balitoridae, Gyrinocheilidae, and Catostomidae is only slight, whereas variation among subfamilies within Cyprinidae and Cobitidae is far more significant. This comparative study identifies a number of distinct morphologies, some of which appear highly correlated with ecological niche. For example, inhabiting swift-moving waters appears to be a key factor in the encapsulation of the anterior gas bladder in some cobitids, balitorids, and gobionin cyprinids.

Developmental morphology of the axial skeleton of the zebrafish, Danio rerio (Ostariophysi: Cyprinidae).

Before our rapidly increasing knowledge of gene interactions can be connected with the morphologic defects in mutant zebrafish, the normal course of skeletal development must be understood. Here, we describe the developmental morphology of the axial skeleton of zebrafish and review it in relation to the morphology of related species. The relative sequence of ossification in the skeleton is described. Two separate centers of development were found in the axial skeleton (Weberian apparatus and caudal fin) in contrast to tetrapods, which have a single anterior center. Slight variation was found in the overall relative timing of development. The extensive ichthyological literature on teleost anatomy and recent genetic data form the basis for our review and interpretation of homologies of various elements of the axial skeleton. Because homology forms the basis for all evolutionary comparisons, these data are critical for integration in evo-devo studies.

Evolution of median fin modules in the axial skeleton of fishes.

Detailed examples of how hierarchical assemblages of modules change over time are few. We found broadly conserved phylogenetic patterns in the directions of development within the median fins of fishes. From these, we identify four modules involved in their positioning and patterning. The evolutionary sequence of their hierarchical assembly and secondary dissociation is described. The changes in these modules during the evolution of fishes appear to be produced through dissociation, duplication and divergence, and co-option. Although the relationship between identified median fin modules and underlying mechanisms is unclear, Hox addresses may be correlated. Comparing homologous gene expression and function in various fishes may test these predictions.The earliest actinopterygians likely had dorsal and anal fins that were symmetrically positioned via a positioning module. The common patterning (differentiation) of skeletal elements within the dorsal and anal fins may have been set into motion by linkage to this positioning module. Frequent evolutionary changes in dorsal and anal fin position indicate a high level of dissociability of the positioning module from the patterning module. In contrast, the patterning of the dorsal and anal fins remains linked: In nearly all fishes, the endo- and exoskeletal elements of the two fins co-differentiate. In all fishes, the exoskeletal fin rays differentiate in the same directions as the endoskeletal supports, indicating complete developmental integration. In acanthopterygians, a new first dorsal fin module evolved via duplication and divergence. The median fins provide an example of how basic modularity is maintained over 400 million years of evolution.