Transgene-mediated skeletal phenotypic variation in zebrafish.

When considering relationships between genotype and phenotype we frequently ignore the fact that the genome of a typical animal, notably including that of a fish and a human, harbours a huge amount of foreign DNA. Such DNA, in the form of transposable elements, can affect genome function in a major way, and transgene biology needs to be included in our understanding of the genome. Here we examine an unexpected phenotypic effect of the chromosomally integrated transgene fli1a-F-hsp70l:Gal4VP16 that serves as a model for transgene function generally. We examine larval fras1 mutant zebrafish (Danio rerio). Gal4VP16 is a potent transcriptional activator that is already well known for toxicity and mediating unusual transcriptional effects. In the presence of the transgene, phenotypes in the neural crest-derived craniofacial skeleton, notably fusions and shape changes associated with loss of function fras1 mutations, are made more severe, as we quantify by scoring phenotypic penetrance, the fraction of mutants expressing the trait. A very interesting feature is that the enhancements are highly specific for fras1 mutant phenotypes, occurring in the apparent absence of more widespread changes. Except for the features due to the fras1 mutation, the transgene-bearing larvae appear generally healthy and to be developing normally. The transgene behaves as a genetic partial dominant: a single copy is sufficient for the enhancements, yet, for some traits, two copies may exert a stronger effect. We made new strains bearing independent insertions of the fli1a-F-hsp70l:Gal4VP16 transgene in new locations in the genome, and observed increased severities of the same phenotypes as observed for the original insertion. This finding suggests that sequences within the transgene, for example Gal4VP16, are responsible for the enhancements, rather than the effect on neighbouring host sequences (such as an insertional mutation). The specificity and biological action underlying the traits are subjects of considerable interest for further investigation, as we discuss. Our findings show that work with transgenes needs to be undertaken with caution and attention to detail.

Developmental tuning of mineralization drives morphological diversity of gill cover bones in sculpins and their relatives.

The role of osteoblast placement in skeletal morphological variation is relatively well understood, but alternative developmental mechanisms affecting bone shape remain largely unknown. Specifically, very little attention has been paid to variation in later mineralization stages of intramembranous ossification as a driver of morphological diversity. We discover the occurrence of specific, sometimes large, regions of nonmineralized osteoid within bones that also contain mineralized tissue. We show through a variety of histological, molecular, and tomographic tests that this "extended" osteoid material is most likely nonmineralized bone matrix. This tissue type is a significant determinant of gill cover bone shape in the teleostean suborder Cottoidei. We demonstrate repeated evolution of extended osteoid in Cottoidei through ancestral state reconstruction and test for an association between extended osteoid variation and habitat differences among species. Through measurement of extended osteoid at various stages of gill cover development in species across the phylogeny, we gain insight into possible evolutionary developmental origins of the trait. We conclude that this fine-tuned developmental regulation of bone matrix mineralization reflects heterochrony at multiple biological levels and is a novel mechanism for the evolution of diversity in skeletal morphology. This research lays the groundwork for a new model in which to study bone mineralization and evolutionary developmental processes, particularly as they may relate to adaptation during a prominent evolutionary radiation of fishes.

A rich diversity of opercle bone shape among teleost fishes.

The opercle is a prominent craniofacial bone supporting the gill cover in all bony fish and has been the subject of morphological, developmental, and genetic investigation. We surveyed the shapes of this bone among 110 families spanning the teleost tree and examined its pattern of occupancy in a principal component-based morphospace. Contrasting with expectations from the literature that suggest the local morphospace would be only sparsely occupied, we find primarily dense, broad filling of the morphological landscape, indicating rich diversity. Phylomorphospace plots suggest that dynamic evolution underlies the observed spatial patterning. Evolutionary transits through the morphospaces are sometimes long, and occur in a variety of directions. The trajectories seem to represent both evolutionary divergences and convergences, the latter supported by convevol analysis. We suggest that that this pattern of occupancy reflects the various adaptations of different groups of fishes, seemingly paralleling their diverse marine and freshwater ecologies and life histories. Opercle shape evolution within the acanthomorphs, spiny ray-finned fishes, appears to have been especially dynamic.

Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.

Heightened phenotypic variation among mutant animals is a well-known, but poorly understood phenomenon. One hypothetical mechanism accounting for mutant phenotypic variation is progenitor cells variably choosing between two alternative fates during development. Zebrafish mef2cab1086 mutants develop tremendously variable ectopic bone in their hyoid craniofacial skeleton. Here, we report evidence that a key component of this phenotype is variable fate switching from ligament to bone. We discover that a 'track' of tissue prone to become bone cells is a previously undescribed ligament. Fate-switch variability is heritable, and comparing mutant strains selectively bred to high and low penetrance revealed differential mef2ca mutant transcript expression between high and low penetrance strains. Consistent with this, experimental manipulation of mef2ca mutant transcripts modifies the penetrance of the fate switch. Furthermore, we discovered a transposable element that resides immediately upstream of the mef2ca locus and is differentially DNA methylated in the two strains, correlating with differential mef2ca expression. We propose that variable transposon epigenetic silencing underlies the variable mef2ca mutant bone phenotype, and could be a widespread mechanism of phenotypic variability in animals.

Pharyngeal morphogenesis requires fras1-itga8-dependent epithelial-mesenchymal interaction.

Both Fras1 and Itga8 connect mesenchymal cells to epithelia by way of an extracellular 'Fraser protein complex' that functions in signaling and adhesion; these proteins are vital to the development of several vertebrate organs. We previously found that zebrafish fras1 mutants have craniofacial defects, specifically, shortened symplectic cartilages and cartilage fusions that spare joint elements. During a forward mutagenesis screen, we identified a new zebrafish mutation, b1161, that we show here disrupts itga8, as confirmed using CRISPR-generated itga8 alleles. fras1 and itga8 single mutants and double mutants have similar craniofacial phenotypes, a result expected if loss of either gene disrupts function of the Fraser protein complex. Unlike fras1 mutants or other Fraser-related mutants, itga8 mutants do not show blistered tail fins. Thus, the function of the Fraser complex differs in the craniofacial skeleton and the tail fin. Focusing on the face, we find that itga8 mutants consistently show defective outpocketing of a late-forming portion of the first pharyngeal pouch, and variably express skeletal defects, matching previously characterized fras1 mutant phenotypes. In itga8 and fras1 mutants, skeletal severity varies markedly between sides, indicating that both mutants have increased developmental instability. Whereas fras1 is expressed in epithelia, we show that itga8 is expressed complementarily in facial mesenchyme. Paired with the observed phenotypic similarity, this expression indicates that the genes function in epithelial-mesenchymal interactions. Similar interactions between Fras1 and Itga8 have previously been found in mouse kidney, where these genes both regulate Nephronectin (Npnt) protein abundance. We find that zebrafish facial tissues express both npnt and the Fraser gene fibrillin2b (fbn2b), but their transcript levels do not depend on fras1 or itga8 function. Using a revertible fras1 allele, we find that the critical window for fras1 function in the craniofacial skeleton is between 1.5 and 3 days post fertilization, which coincides with the onset of fras1-dependent and itga8-dependent morphogenesis. We propose a model wherein Fras1 and Itga8 interact during late pharyngeal pouch morphogenesis to sculpt pharyngeal arches through epithelial-mesenchymal interactions, thereby stabilizing the developing craniofacial skeleton.

Epigenetic regulation of hematopoiesis by DNA methylation.

During embryonic development, cell type-specific transcription factors promote cell identities, while epigenetic modifications are thought to contribute to maintain these cell fates. Our understanding of how genetic and epigenetic modes of regulation work together to establish and maintain cellular identity is still limited, however. Here, we show that DNA methyltransferase 3bb.1 (dnmt3bb.1) is essential for maintenance of hematopoietic stem and progenitor cell (HSPC) fate as part of an early Notch-runx1-cmyb HSPC specification pathway in the zebrafish. Dnmt3bb.1 is expressed in HSPC downstream from Notch1 and runx1, and loss of Dnmt3bb.1 activity leads to reduced cmyb locus methylation, reduced cmyb expression, and gradual reduction in HSPCs. Ectopic overexpression of dnmt3bb.1 in non-hematopoietic cells is sufficient to methylate the cmyb locus, promote cmyb expression, and promote hematopoietic development. Our results reveal an epigenetic mechanism supporting the maintenance of hematopoietic cell fate via DNA methylation-mediated perdurance of a key transcription factor in HSPCs.

Patterns of variation and covariation in the shapes of mandibular bones of juvenile salmonids in the genus Oncorhynchus.

What is the nature of evolutionary divergence of the jaw skeleton within the genus Oncorhynchus? How can two associated bones evolve new shapes and still maintain functional integration? Here, we introduce and test a "concordance" hypothesis, in which an extraordinary matching of the evolutionary shape changes of the dentary and angular articular serves to preserve their fitting together. To test this hypothesis, we examined morphologies of the dentary and angular articular at parr (juvenile) stage, and at three levels of biological organization­between salmon and trout, between sister species within both salmon and trout, and among three types differing in life histories within one species, Oncorhynchus mykiss. The comparisons show bone shape divergences among the groups at each level; morphological divergence between salmon and trout is marked even at this relatively early life history stage. We observed substantial matching between the two mandibular bones in both pattern and amount of shape variation, and in shape covariation across species. These findings strongly support the concordance hypothesis, and reflect functional and/or developmental constraint on morphological evolution. We present evidence for developmental modularity within both bones. The locations of module boundaries were predicted from the patterns of evolutionary divergences, and for the dentary, at least, would appear to facilitate its functional association with the angular articular. The modularity results suggest that development has biased the course of evolution.

Building the backbone: the development and evolution of vertebral patterning.

The segmented vertebral column comprises a repeat series of vertebrae, each consisting of two key components: the vertebral body (or centrum) and the vertebral arches. Despite being a defining feature of the vertebrates, much remains to be understood about vertebral development and evolution. Particular controversy surrounds whether vertebral component structures are homologous across vertebrates, how somite and vertebral patterning are connected, and the developmental origin of vertebral bone-mineralizing cells. Here, we assemble evidence from ichthyologists, palaeontologists and developmental biologists to consider these issues. Vertebral arch elements were present in early stem vertebrates, whereas centra arose later. We argue that centra are homologous among jawed vertebrates, and review evidence in teleosts that the notochord plays an instructive role in segmental patterning, alongside the somites, and contributes to mineralization. By clarifying the evolutionary relationship between centra and arches, and their varying modes of skeletal mineralization, we can better appreciate the detailed mechanisms that regulate and diversify vertebral patterning.

Skull developmental modularity: a view from a single bone - or two.

I review recent studies that connect development and evolution of skull bones in teleosts. Development uses genetic information to build a structured, modular phenotype, and since selection acts on the phenotype, developmental modularity may influence evolvability. Just how is a complex developing morphology spatially partitioned into modules? Here I briefly examine cellular, molecular genetic, and multivariate statistical approaches to the identification of developmental modules. Furthermore I review our evidence that developmental modularity provides evolutionarily labile regions within the skull and hence potentially biases evolutionary change in a positive manner. This view is rather different from early ones in the field of evolutionary developmental biology, in which developmental constraint due to patterns such as heterochronies were supposed to negatively impact evolution.

Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton.

Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca(b1086) loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal-ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.

edn1 and hand2 Interact in early regulation of pharyngeal arch outgrowth during zebrafish development.

Endothelin-1 (Edn1) signaling provides a critical input to development of the embryonic pharygneal arches and their skeletal derivatives, particularly the articulating joints and the ventral skeleton including the lower jaw. Previous work in zebrafish has mostly focused on the role of Edn1 in dorsal-ventral (DV) patterning, but Edn1 signaling must also regulate tissue size, for with severe loss of the pathway the ventral skeleton is not only mispatterned, but is also prominently hypoplastic--reduced in size. Here we use mutational analyses to show that in the early pharyngeal arches, ventral-specific edn1-mediated proliferation of neural crest derived cells is required for DV expansion and outgrowth, and that this positive regulation is counterbalanced by a negative one exerted through a pivotal, ventrally expressed Edn1-target gene, hand2. We also describe a new morphogenetic cell movement in the ventral first arch, sweeping cells anterior in the arch to the region where the lower jaw forms. This movement is negatively regulated by hand2 in an apparently edn1-independent fashion. These findings point to complexity of regulation by edn1 and hand2 at the earliest stages of pharyngeal arch development, in which control of growth and morphogenesis can be genetically separated.

barx1 represses joints and promotes cartilage in the craniofacial skeleton.

The evolution of joints, which afford skeletal mobility, was instrumental in vertebrate success. Here, we explore the molecular genetics and cell biology that govern jaw joint development. Genetic manipulation experiments in zebrafish demonstrate that functional loss, or gain, of the homeobox-containing gene barx1 produces gain, or loss, of joints, respectively. Ectopic joints in barx1 mutant animals are present in every pharyngeal segment, and are associated with disrupted attachment of bone, muscles and teeth. We find that ectopic joints develop at the expense of cartilage. Time-lapse experiments suggest that barx1 controls the skeletal precursor cell choice between differentiating into cartilage versus joint cells. We discovered that barx1 functions in this choice, in part, by regulating the transcription factor hand2. We further show that hand2 feeds back to negatively regulate barx1 expression. We consider the possibility that changes in barx1 function in early vertebrates were among the key innovations fostering the evolution of skeletal joints.

FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution.

BACKGROUND: The vertebrate craniofacial skeleton may exhibit anatomical complexity and diversity, but its genesis and evolution can be understood through careful dissection of developmental programs at cellular resolution. Resources are lacking that include introductory overviews of skeletal anatomy coupled with descriptions of craniofacial development at cellular resolution. In addition to providing analytical guidelines for other studies, such an atlas would suggest cellular mechanisms underlying development. DESCRIPTION: We present the Fish Face Atlas, an online, 3D-interactive atlas of craniofacial development in the zebrafish Danio rerio. Alizarin red-stained skulls scanned by fluorescent optical projection tomography and segmented into individual elements provide a resource for understanding the 3D structure of the zebrafish craniofacial skeleton. These data provide the user an anatomical entry point to confocal images of Alizarin red-stained zebrafish with transgenically-labelled pharyngeal arch ectomesenchyme, chondrocytes, and osteoblasts, which illustrate the appearance, morphogenesis, and growth of the mandibular and hyoid cartilages and bones, as viewed in live, anesthetized zebrafish during embryonic and larval development. Confocal image stacks at high magnification during the same stages provide cellular detail and suggest developmental and evolutionary hypotheses. CONCLUSION: The FishFace Atlas is a novel learning tool for understanding craniofacial skeletal development, and can serve as a reference for a variety of studies, including comparative and mutational analyses.

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space.

fras1 shapes endodermal pouch 1 and stabilizes zebrafish pharyngeal skeletal development.

Lesions in the epithelially expressed human gene FRAS1 cause Fraser syndrome, a complex disease with variable symptoms, including facial deformities and conductive hearing loss. The developmental basis of facial defects in Fraser syndrome has not been elucidated. Here we show that zebrafish fras1 mutants exhibit defects in facial epithelia and facial skeleton. Specifically, fras1 mutants fail to generate a late-forming portion of pharyngeal pouch 1 (termed late-p1) and skeletal elements adjacent to late-p1 are disrupted. Transplantation studies indicate that fras1 acts in endoderm to ensure normal morphology of both skeleton and endoderm, consistent with well-established epithelial expression of fras1. Late-p1 formation is concurrent with facial skeletal morphogenesis, and some skeletal defects in fras1 mutants arise during late-p1 morphogenesis, indicating a temporal connection between late-p1 and skeletal morphogenesis. Furthermore, fras1 mutants often show prominent second arch skeletal fusions through space occupied by late-p1 in wild type. Whereas every fras1 mutant shows defects in late-p1 formation, skeletal defects are less penetrant and often vary in severity, even between the left and right sides of the same individual. We interpret the fluctuating asymmetry in fras1 mutant skeleton and the changes in fras1 mutant skeletal defects through time as indicators that skeletal formation is destabilized. We propose a model wherein fras1 prompts late-p1 formation and thereby stabilizes skeletal formation during zebrafish facial development. Similar mechanisms of stochastic developmental instability might also account for the high phenotypic variation observed in human FRAS1 patients.

Developmental dissociation in morphological evolution of the stickleback opercle.

Oceanic threespine sticklebacks have repeatedly and independently evolved new morphologies upon invasions of freshwater habitats. A consistent derived feature of the freshwater form across populations and geography is a shape change of the opercle, a large early developing facial bone. We show that the principal multivariate axis describing opercle shape development from the young larva to the full adult stage of oceanic fish matches the principal axis of evolutionary change associated with relocation from the oceanic to freshwater habitat. The opercle phenotype of freshwater adults closely resembles the phenotype of the bone in juveniles. Thus, evolution to the freshwater condition is in large part by truncation of development; the freshwater fish do not achieve the full ancestral adult bone shape. Additionally, the derived state includes dissociated ontogenetic changes. Dissociability may reflect an underlying modular pattern of opercle development, and facilitate flexibility of morphological evolution.

Histone deacetylase-4 is required during early cranial neural crest development for generation of the zebrafish palatal skeleton.

BACKGROUND: Histone deacetylase-4 (Hdac4) is a class II histone deacetylase that inhibits the activity of transcription factors. In humans, HDAC4 deficiency is associated with non-syndromic oral clefts and brachydactyly mental retardation syndrome (BDMR) with craniofacial abnormalities. RESULTS: We identify hdac4 in zebrafish and characterize its function in craniofacial morphogenesis. The gene is present as a single copy, and the deduced Hdac4 protein sequence shares all known functional domains with human HDAC4. The zebrafish hdac4 transcript is widely present in migratory cranial neural crest (CNC) cells of the embryo, including populations migrating around the eye, which previously have been shown to contribute to the formation of the palatal skeleton of the early larva. Embryos injected with hdac4 morpholinos (MO) have reduced or absent CNC populations that normally migrate medial to the eye. CNC-derived palatal precursor cells do not recover at the post-migratory stage, and subsequently we found that defects in the developing cartilaginous palatal skeleton correlate with reduction or absence of early CNC cells. Palatal skeletal defects prominently include a shortened, clefted, or missing ethmoid plate, and are associated with a shortening of the face of young larvae. CONCLUSIONS: Our results demonstrate that Hdac4 is a regulator of CNC-derived palatal skeletal precursors during early embryogenesis. Cleft palate resulting from HDAC4 mutations in human patients may result from defects in a homologous CNC progenitor cell population.

Hedgehog-dependent proliferation drives modular growth during morphogenesis of a dermal bone.

In the developing skeleton, dermal bone morphogenesis includes the balanced proliferation, recruitment and differentiation of osteoblast precursors, yet how bones acquire unique morphologies is unknown. We show that Hedgehog (Hh) signaling mediates bone shaping during early morphogenesis of the opercle (Op), a well characterized dermal bone of the zebrafish craniofacial skeleton. ihha is specifically expressed in a local population of active osteoblasts along the principal growing edge of the bone. Mutational studies show that Hh signaling by this osteoblast population is both necessary and sufficient for full recruitment of pre-osteoblasts into the signaling population. Loss of ihha function results in locally reduced proliferation of pre-osteoblasts and consequent reductions in recruitment into the osteoblast pool, reduced bone edge length and reduced outgrowth. Conversely, hyperactive Hh signaling in ptch1 mutants causes opposite defects in proliferation and growth. Time-lapse microscopy of early Op morphogenesis using transgenically labeled osteoblasts demonstrates that ihha-dependent bone development is not only region specific, but also begins exactly at the onset of a second phase of morphogenesis, when the early bone begins to reshape into a more complex form. These features strongly support a hypothesis that dermal bone development is modular, with different gene sets functioning at specific times and locations to pattern growth. The Hh-dependent module is not limited to this second phase of bone growth: during later larval development, the Op is fused along the dysmorphic edge to adjacent dermal bones. Hence, patterning within a module may include adjacent regions of functionally related bones and might require that signaling pathways function over an extended period of development.

Independent axes of genetic variation and parallel evolutionary divergence of opercle bone shape in threespine stickleback.

Evolution of similar phenotypes in independent populations is often taken as evidence of adaptation to the same fitness optimum. However, the genetic architecture of traits might cause evolution to proceed more often toward particular phenotypes, and less often toward others, independently of the adaptive value of the traits. Freshwater populations of Alaskan threespine stickleback have repeatedly evolved the same distinctive opercle shape after divergence from an oceanic ancestor. Here we demonstrate that this pattern of parallel evolution is widespread, distinguishing oceanic and freshwater populations across the Pacific Coast of North America and Iceland. We test whether this parallel evolution reflects genetic bias by estimating the additive genetic variance-covariance matrix (G) of opercle shape in an Alaskan oceanic (putative ancestral) population. We find significant additive genetic variance for opercle shape and that G has the potential to be biasing, because of the existence of regions of phenotypic space with low additive genetic variation. However, evolution did not occur along major eigenvectors of G, rather it occurred repeatedly in the same directions of high evolvability. We conclude that the parallel opercle evolution is most likely due to selection during adaptation to freshwater habitats, rather than due to biasing effects of opercle genetic architecture.

Mutations in fam20b and xylt1 reveal that cartilage matrix controls timing of endochondral ossification by inhibiting chondrocyte maturation.

Differentiating cells interact with their extracellular environment over time. Chondrocytes embed themselves in a proteoglycan (PG)-rich matrix, then undergo a developmental transition, termed "maturation," when they express ihh to induce bone in the overlying tissue, the perichondrium. Here, we ask whether PGs regulate interactions between chondrocytes and perichondrium, using zebrafish mutants to reveal that cartilage PGs inhibit chondrocyte maturation, which ultimately dictates the timing of perichondral bone development. In a mutagenesis screen, we isolated a class of mutants with decreased cartilage matrix and increased perichondral bone. Positional cloning identified lesions in two genes, fam20b and xylosyltransferase1 (xylt1), both of which encode PG synthesis enzymes. Mutants failed to produce wild-type levels of chondroitin sulfate PGs, which are normally abundant in cartilage matrix, and initiated perichondral bone formation earlier than their wild-type siblings. Primary chondrocyte defects might induce the bone phenotype secondarily, because mutant chondrocytes precociously initiated maturation, showing increased and early expression of such markers as runx2b, collagen type 10a1, and ihh co-orthologs, and ihha mutation suppressed early perichondral bone in PG mutants. Ultrastructural analyses demonstrated aberrant matrix organization and also early cellular features of chondrocyte hypertrophy in mutants. Refining previous in vitro reports, which demonstrated that fam20b and xylt1 were involved in PG synthesis, our in vivo analyses reveal that these genes function in cartilage matrix production and ultimately regulate the timing of skeletal development.