Presence of chondroitin sulphate and requirement for heparan sulphate biosynthesis in the developing zebrafish inner ear

Epithelial morphogenesis to form the semicircular canal ducts of the zebrafish inner ear depends on the production of the large glycosaminoglycan hyaluronan, which is thought to contribute to the driving force that pushes projections of epithelium into the lumen of the otic vesicle. Proteoglycans are also implicated in otic morphogenesis: several of the genes coding for proteoglycan core proteins, together with enzymes that synthesise and modify their polysaccharide chains, are expressed in the developing zebrafish inner ear. In this study, we demonstrate the highly specific localisation of chondroitin sulphate to the sites of epithelial projection outgrowth in the ear, present before any morphological deformation of the epithelium. Staining for chondroitin sulphate is also present in the otolithic membrane, whereas the otoliths are strongly positive for keratan sulphate. We show that heparan sulphate biosynthesis is critical for normal epithelial projection outgrowth, otolith growth and tethering. In the ext2 mutant ear, which has reduced heparan sulphate levels, but continues to produce hyaluronan, epithelial projections are rudimentary, and do not grow sufficiently to meet and fuse to form the pillars of tissue that normally span the otic lumen. Staining for chondroitin sulphate and expression of versican b, a chondroitin sulphate proteoglycan core protein gene, persist abnormally at high levels in the unfused projections of the ext2 mutant ear. We propose a model for wild-type epithelial projection outgrowth in which hyaluronan and proteoglycans are linked to form a hydrated gel that fills the projection core, with both classes of molecule playing essential roles in zebrafish semicircular canal morphogenesis

Olfactory rod cells : a rare cell type in the larval zebrafish olfactory epithelium with an actin-rich apical projection

We report the presence of a rare cell type, the olfactory rod cell, in the developing zebrafish olfactory epithelium. These cells each bear a single actin-rich rod-like apical projection extending about 10 μm from the epithelial surface. Live imaging with a ubiquitous Lifeact-RFP label indicates that the rods can oscillate. Olfactory rods arise within a few hours of the olfactory pit opening, increase in numbers and size during larval stages, and can develop in the absence of olfactory cilia. Olfactory rod cells differ in morphology from the known classes of olfactory sensory neuron, but express reporters driven by neuronal promoters. The cells also differ from secondary sensory cells such as hair cells of the inner ear or lateral line, or sensory cells in the taste bud, as they are not associated with established synaptic terminals. A sub-population of olfactory rod cells expresses a Lifeact-mRFPruby transgene driven by the sox10 promoter. Mosaic expression of this transgene reveals that olfactory rod cells have rounded cell bodies located apically in the olfactory epithelium

The adhesion GPCR Adgrg6 (Gpr126): Insights from the zebrafish model

Adhesion GPCRs are important regulators of conserved developmental processes and represent an untapped pool of potential targets for drug discovery. The adhesion GPCR Adgrg6 (Gpr126) has critical developmental roles in Schwann cell maturation and inner ear morphogenesis in the zebrafish embryo. Mutations in the human ADGRG6 gene can result in severe deficits in peripheral myelination, and variants have been associated with many other disease conditions. Here, we review work on the zebrafish Adgrg6 signaling pathway and its potential as a disease model. Recent advances have been made in the analysis of the structure of the Adgrg6 receptor, demonstrating alternative structural conformations and the presence of a conserved calcium‐binding site within the CUB domain of the extracellular region that is critical for receptor function. Homozygous zebrafish adgrg6 hypomorphic mutants have been used successfully as a whole‐animal screening platform, identifying candidate molecules that can influence signaling activity and rescue mutant phenotypes. These compounds offer promise for further development as small molecule modulators of Adgrg6 pathway activity

Expression of patched, prdm1 and engrailed in the lamprey somite reveals conserved responses to Hedgehog signaling

In the zebrafish embryo, expression of the prdm1 and patched1 genes in adaxial cells is indicative of their specification to give rise to slow twitch muscle fibers in response to Hedgehog (Hh) signaling. Subsets of these slow twitch muscle progenitors activate engrailed (eng) strongly in response to high-level Hh signaling, and differentiate into muscle pioneer cells, which are important for subsequent development of the horizontal myoseptum. In addition, eng is expressed more weakly in medial fast fibers in response to lower Hh levels. Somite morphology in the lamprey, an agnathan (jawless) vertebrate, differs significantly from that of teleosts. In particular, the lamprey does not have clear epaxial/hypaxial domains, lacks a horizontal myoseptum, and does not appear to possess distinct populations of fast and slow fibers in the embryonic somite. Nevertheless, Hh is expressed in the midline of the lamprey embryo, and we report here that, as in zebrafish, homologues of patched and prdm1 are expressed in adaxial regions of the lamprey somite, and an eng homologue is also expressed in the somite. However, the lamprey adaxial region does not exhibit the same distinct adaxial cell morphology as in the zebrafish. In addition, the expression of follistatin is not excluded from the adaxial region, and eng is not detected in discrete muscle pioneer-like cells. These data suggest the presence of conserved responses to Hh signaling in lamprey somites, although the full range of effects elicited by Hh in the zebrafish somite is not recapitulated

Olfactory rod cells : a rare cell type in the larval zebrafish olfactory epithelium with a large actin-rich apical projection

We report the presence of a rare cell type, the olfactory rod cell, in the developing zebrafish olfactory epithelium. These cells each bear a single actin-rich rod-like apical projection extending 5–10 μm from the epithelial surface. Live imaging with a ubiquitous Lifeact-RFP label indicates that the olfactory rods can oscillate. Olfactory rods arise within a few hours of the olfactory pit opening, increase in numbers and size during larval stages, and can develop in the absence of olfactory cilia. Olfactory rod cells differ in morphology from the known classes of olfactory sensory neuron, but express reporters driven by neuronal promoters. A sub-population of olfactory rod cells expresses a Lifeact-mRFPruby transgene driven by the sox10 promoter. Mosaic expression of this transgene reveals that olfactory rod cells have rounded cell bodies located apically in the olfactory epithelium and have no detectable axon. We offer speculation on the possible function of these cells in the Discussion

A screen of pharmacologically active compounds to identify modulators of the Adgrg6/Gpr126 signalling pathway in zebrafish embryos

Adhesion G protein-coupled receptors (GPCRs) are an underrepresented class of GPCRs in drug discovery. We previously developed an in vivo drug screening pipeline to identify compounds with agonist activity for Adgrg6 (Gpr126), an adhesion GPCR required for myelination of the peripheral nervous system in vertebrates. The screening assay tests for rescue of an ear defect found in adgrg6tb233c-/- hypomorphic homozygous mutant zebrafish, using the expression of versican b (vcanb) mRNA as an easily identifiable phenotype. In the current study, we used the same assay to screen a commercially available library of 1280 diverse bioactive compounds (Sigma LOPAC). Comparison with published hits from two partially overlapping compound collections (Spectrum, Tocris) confirms that the screening assay is robust and reproducible. Using a modified counter screen for myelin basic protein (mbp) gene expression, we have identified 17 LOPAC compounds that can rescue both inner ear and myelination defects in adgrg6tb233c-/- hypomorphic mutants, three of which (ebastine, S-methylisothiourea hemisulfate, and thapsigargin) are new hits. A further 25 LOPAC hit compounds were effective at rescuing the otic vcanb expression but not mbp. Together, these and previously identified hits provide a wealth of starting material for the development of novel and specific pharmacological modulators of Adgrg6 receptor activity

Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, and that otic patterning defects in hmx3a-/- mutants are a close phenocopy to those seen in fgf3-/- mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model

Enhancer trap lines with GFP driven by smad6b and frizzled1 regulatory sequences for the study of epithelial morphogenesis in the developing zebrafish inner ear

Live imaging in the zebrafish embryo using tissue-specific expression of fluorescent proteins can yield important insights into the mechanisms that drive sensory organ morphogenesis and cell differentiation. Morphogenesis of the semicircular canal ducts of the vertebrate inner ear requires a complex rearrangement of epithelial cells, including outgrowth, adhesion, fusion and perforation of epithelial projections to generate pillars of tissue that form the hubs of each canal. We report the insertion sites and expression patterns of two enhancer trap lines in the developing zebrafish embryo, each of which highlight different aspects of epithelial cell morphogenesis in the inner ear. A membrane-linked EGFP driven by smad6b regulatory sequences is expressed throughout the otic epithelium, most strongly on the lateral side of the ear and in the sensory cristae. A second enhancer trap line, with cytoplasmic EGFP driven by frizzled1 (fzd1) regulatory sequences, specifically marks cells of the ventral projection and pillar in the developing ear, and marginal cells in the sensory cristae, together with variable expression in the retina and epiphysis, and neurons elsewhere in the developing central nervous system. We have used a combination of methods to identify the insertion sites of these two transgenes, which were generated through random insertion, and show that Targeted Locus Amplification is a rapid and reliable method for the identification of insertion sites of randomly inserted transgenes

Morphological, behavioral and cellular analyses revealed different phenotypes in Wolfram syndrome wfs1a and wfs1b zebrafish mutant lines

Wolfram syndrome (WS) is a rare genetic disease characterized by diabetes, optic atrophy and deafness. Patients die at 35 years of age, mainly from respiratory failure or dysphagia. Unfortunately, there is no treatment to block the progression of symptoms and there is an urgent need for adequate research models. Here, we report on the phenotypical characterization of two loss-of-function zebrafish mutant lines: wfs1aC825X and wfs1bW493X. We observed that wfs1a deficiency altered the size of the ear and the retina of the fish. We also documented a decrease in the expression level of unfolded protein response (UPR) genes in basal condition and in stress condition, i.e. after tunicamycin treatment. Interestingly, both mutants lead to a decrease in their visual function measured behaviorally. These deficits were associated with a decrease in the expression level of UPR genes in basal and stress conditions. Interestingly, basal, ATP-linked and maximal mitochondrial respirations were transiently decreased in the wfs1b mutant. Taken together, these zebrafish lines highlight the critical role of wfs1a and wfs1b in UPR, mitochondrial function and visual physiology. These models will be useful tools to better understand the cellular function of Wfs1 and to develop novel therapeutic approaches for WS