Shannon Fisher

Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response

Primary cilia and basal bodies are evolutionarily conserved organelles that mediate communication between the intracellular and extracellular environments. Here we show that bbs1, bbs4 and mkks (also known as bbs6), which encode basal body proteins, are required for convergence and extension in zebrafish and interact with wnt11 and wnt5b. Suppression of bbs1, bbs4 and mkks transcripts results in stabilization of β-catenin with concomitant upregulation of T-cell factor (TCF)-dependent transcription in both zebrafish embryos and mammalian ciliated cells, a defect phenocopied by the silencing of the axonemal kinesin subunit KIF3A but not by chemical disruption of the cytoplasmic microtubule network. These observations are attributable partly to defective degradation by the proteasome; suppression of BBS4 leads to perturbed proteasomal targeting and concomitant accumulation of cytoplasmic β-catenin. Cumulatively, our data indicate that the basal body is an important regulator of Wnt signal interpretation through selective proteolysis and suggest that defects in this system may contribute to phenotypes pathognomonic of human ciliopathies.

Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish

Evaluating the biological relevance of the myriad putative regulatory noncoding sequences in vertebrate genomes represents a huge challenge. Functional analyses in vivo have typically relied on costly and labor-intensive transgenic strategies in mice. Transgenesis has also been applied in nonrodent vertebrates, such as zebrafish, but until recently these efforts have been hampered by significant mosaicism and poor rates of germline transmission. We have developed a transgenic strategy in zebrafish based on the Tol2 transposon, a mobile element that was recently identified in another teleost, Medaka. This method takes advantage of the increased efficiency of genome integration that is afforded by this intact DNA transposon, activity that is mediated by the corresponding transposase protein. The approach described in this protocol uses a universal vector system that permits rapid incorporation of DNA that is tagged with sequence targets for site-specific recombination. To evaluate the regulatory potential of a candidate sequence, the desired interval is PCR-amplified using sequence-specific primers that are flanked by the requisite target sites for cloning, and recombined into a universal expression plasmid (pGW_cfosEGFP). Purified recombinant DNAs are then injected into 1–2-cell zebrafish embryos and the resulting reporter expression patterns are analyzed at desired timepoints during development. This system is amenable to large-scale application, facilitating rapid functional analysis of noncoding sequences from both mammalian and teleost species.

Dissection of epistasis in oligogenic Bardet-Biedl syndrome.

Epistatic interactions have an important role in phenotypic variability, yet the genetic dissection of such phenomena remains challenging. Here we report the identification of a novel locus, MGC1203, that contributes epistatic alleles to Bardet–Biedl syndrome (BBS), a pleiotropic, oligogenic disorder. MGC1203 encodes a pericentriolar protein that interacts and colocalizes with the BBS proteins. Sequencing of two independent BBS cohorts revealed a significant enrichment of a heterozygous C430T mutation in patients, and a transmission disequilibrium test (TDT) showed strong over-transmission of this variant. Further analyses showed that the 430T allele enhances the use of a cryptic splice acceptor site, causing the introduction of a premature termination codon (PTC) and the reduction of steady-state MGC1203 messenger RNA levels. Finally, recapitulation of the human genotypes in zebrafish shows that modest suppression of mgc1203 exerts an epistatic effect on the developmental phenotype of BBS morphants. Our data demonstrate how the combined use of biochemical, genetic and in vivo tools can facilitate the dissection of epistatic phenomena, and enhance our appreciation of the genetic basis of phenotypic variability.

Bone Regenerates via Dedifferentiation of Osteoblasts in the Zebrafish Fin

While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.

Radiographic analysis of zebrafish skeletal defects

Systematic identification of skeletal dysplasias in model vertebrates provides insight into the pathogenesis of human skeletal disorders and can aid in the identification of orthologous human genes. We are undertaking a mutagenesis screen for skeletal dysplasias in adult zebrafish, using radiography to detect abnormalities in skeletal anatomy and bone morphology. We have isolated chihuahua, a dominant mutation causing a general defect in bone growth. Heterozygous chihuahua fish have phenotypic similarities to human osteogenesis imperfecta, a skeletal dysplasia caused by mutations in the type I collagen genes. Mapping and molecular characterization of the chihuahua mutation indicates that the defect resides in the gene encoding the collagen I(alpha1) chain. Thus, chihuahua accurately models osteogenesis imperfecta at the biologic and molecular levels, and will prove an important resource for studies on the disease pathophysiology. Radiography is a practical screening tool to detect subtle skeletal abnormalities in the adult zebrafish. The identification of chihuahua demonstrates that mutant phenotypes analogous to human skeletal dysplasias will be discovered.

Skeletogenic Fate of Zebrafish Cranial and Trunk Neural Crest

The neural crest (NC) is a major contributor to the vertebrate craniofacial skeleton, detailed in model organisms through embryological and genetic approaches, most notably in chick and mouse. Despite many similarities between these rather distant species, there are also distinct differences in the contribution of the NC, particularly to the calvariae of the skull. Lack of information about other vertebrate groups precludes an understanding of the evolutionary significance of these differences. Study of zebrafish craniofacial development has contributed substantially to understanding of cartilage and bone formation in teleosts, but there is currently little information on NC contribution to the zebrafish skeleton. Here, we employ a two–transgene system based on Cre recombinase to genetically label NC in the zebrafish. We demonstrate NC contribution to cells in the cranial ganglia and peripheral nervous system known to be NC–derived, as well as to a subset of myocardial cells. The indelible labeling also enables us to determine NC contribution to late–forming bones, including the calvariae. We confirm suspected NC origin of cartilage and bones of the viscerocranium, including cartilages such as the hyosymplectic and its replacement bones (hymandibula and symplectic) and membranous bones such as the opercle. The cleithrum develops at the border of NC and mesoderm, and as an ancestral component of the pectoral girdle was predicted to be a hybrid bone composed of both NC and mesoderm tissues. However, we find no evidence of a NC contribution to the cleithrum. Similarly, in the vault of the skull, the parietal bones and the caudal portion of the frontal bones show no evidence of NC contribution. We also determine a NC origin for caudal fin lepidotrichia; the presumption is that these are derived from trunk NC, demonstrating that these cells have the ability to form bone during normal vertebrate development.

Patterning the zebrafish axial skeleton requires early chordin function

Members of the bone morphogenetic protein (BMP) family actively promote ventral cell fates, such as epidermis and blood, in the vertebrate gastrula. More dorsally, the organizer region counteracts BMP signalling through secretion of BMP-binding antagonists chordin and noggin, allowing dorsally derived tissues such as neurectoderm and somitic muscle to develop. BMPs also function in skeletal development and regeneration of bone following injury. Noggin antagonism is thought to prevent osteogenesis at sites of joint formation, whereas chordin has not yet been implicated in skeletogenesis. Analyses of zebrafish mutants have confirmed the action of chordin (chd) in opposing ventralizing signals at gastrulation. Some ventralized mutants recover and develop into fertile adults, thereby revealing a requirement for chd function for the later processes of fin and caudal skeletal patterning. We observe in mutants the misexpression of genes encoding BMPs and putative downstream genes, and ectopic sclerotomal cells. Through injections of chd mRNA into the early embryo, we restored wild-type gene expression patterns, and the resultant fish, although genotypically mutant, developed normal axial skeletons and fins. Our results demonstrate that chordin function during gastrulation is important for the correct morphogenesis of the adult zebrafish skeleton.

Twisted gastrulation enhances BMP signaling through chordin dependent and independent mechanisms.

BMP signaling is modulated by a number of extracellular proteins, including the inhibitor Chordin, Tolloid-related enzymes (Tld), and the interacting protein Twisted Gastrulation (Tsg). Although in vitro studies have demonstrated Chordin cleavage by Tld enzymes, its significance as a regulatory mechanism in vivo has not been established in vertebrates. In addition, Tsg has been reported in different contexts to either enhance or inhibit BMP signaling through its interactions with Chordin. We have used the zebrafish gastrula to carry out structure/function studies on Chordin, by making versions of Chordin partially or wholly resistant to Tld cleavage and introducing them into chordin-deficient embryos. We examined the cleavage products generated in vivo from wild-type and altered Chordins, and tested their efficacy as BMP inhibitors in the embryo. We demonstrate that Tld cleavage is crucial in restricting Chordin function in vivo, and is carried out by redundant enzymes in the zebrafish gastrula. We also present evidence that partially cleaved Chordin is a stronger BMP inhibitor than the full-length protein, suggesting a positive role for Tld in regulating Chordin. We find that depletion of the embryo for Tsg leads to decreased BMP signaling, and to increased levels of Chordin. Finally, we show that Tsg also enhances BMP signaling in the absence of Chordin, and its depletion can partially rescue the chordin mutant phenotype, demonstrating that important components of the BMP signaling pathway remain unidentified.

Saltatory control of isometric growth in the zebrafish caudal fin is disrupted in long fin and rapunzel mutants.

Zebrafish fins grow by sequentially adding new segments of bone to the distal end of each fin ray. In wild type zebrafish, segment addition is regulated such that an isometric relationship is maintained between fin length and body length over the lifespan of the growing fish. Using a novel, surrogate marker for fin growth in conjunction with cell proliferation assays, we demonstrate here that segment addition is not continuous, but rather proceeds by saltation. Saltation is a fundamental growth mechanism shared by disparate vertebrates, including humans. We further demonstrate that segment addition proceeds in conjunction with cyclic bursts of cell proliferation in the distal fin ray mesenchyme. In contrast, cells in the distal fin epidermis proliferate at a constant rate throughout the fin ray growth cycle. Finally, we show that two separate fin overgrowth mutants, long fin and rapunzel, bypass the stasis phase of the fin ray growth cycle to develop asymmetrical and symmetrical fin overgrowth, respectively.

Developmental expression of the amyloid precursor protein, growth-associated protein 43, and somatostatin in normal and trisomy 16 mice

The expression during development of 3 genes located on mouse chromosome 16 (MMU 16) which are implicated in neurobiological processes was examined by blot hybridization beginning at early gestational ages in the mouse. The 3 genes, amyloid precursor protein (App), preprosomatostatin (Smst), and growth-associated protein 43 (Gap43), exhibited distinct profiles of expression. App expression increased steadily throughout fetal and postnatal development. Smst expression peaked during the third postnatal week, then reached a plateau at a slightly lower level in adults, and Gap43 expression was highest in the early postnatal period, declining in adults to levels below those seen at the earliest timepoints examined. Smst message levels exhibited a 1.5-fold increase in the brains of trisomy 16 (Ts16) mice as compared to normal littermates on day 15 of gestation, while Gap43 and App message levels were elevated approximately 2-fold.