We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups. We observed similarities but also unique features of the expression patterns of the zebrafish dlx genes. Among the three genes, dlx3 alone is expressed during gastrulation. Shortly after gastrulation, cells in the ventral forebrain rudiment express dlx2 and dlx4, but not dlx3, and hindbrain neural crest cells express only dlx2. Presumptive precursor cells of the olfactory placodes express dlx3 and dlx4 but not dlx2. Transcripts of dlx3 and dlx4 are present in overlapping subsets of cells in the auditory vesicle and in cells of the median fin fold, whereas dlx2 is never expressed in the auditory vesicle and only at low levels in localized regions of the median fin fold. Cells of the visceral arches and their primordia express all three dlx genes, but with different developmental time courses. We suggest that combinatorial expression of the dlx genes is part of a homeobox gene code specifying pattern formation or cell fate determination in the forebrain, in peripheral structures of the head, and in the fins.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2a: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2a: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2a, but presence of the ectodomain of usherin at the periciliary membrane of ush2a-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2a as well as ush2a larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

Homeo box-containing genes {Hox) are expressed in restricted regions of vertebrate embryos and may specify positional information. The organization and expression patterns of these genes are highly conserved among different species, suggesting that their regulation may also have been conserved. We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgene expression, and examined the activities of two mammalian Hox genes, mouse Hox-1.1 and human HOX-3.3. We found that these Hox promoters are activated in specific regions and tissues of developing zebrafish embryos and that this specificity depends upon the same regulatory elements within the promoters that specify the spatial expression of these genes in mice. Our results suggest that the promoter activities have been remarkably conserved from fish to mammals. To study the regulation of Hox expression in the developing nervous system, we analyzed the promoter activities in spt-1 mutants that have a mesodermal deficiency. Our results suggest that interactions, probably with the paraxial mesoderm, differentially regulate the activities of Hox promoters in the developing nervous system.

The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.