Plexina4 and cell survival in the developing zebrafish hindbrain.

BACKGROUND: Growth factors are important in the developing and mature nervous system to support the survival of neurons. Developmental signaling molecules are known for their roles in controlling neurogenesis and neural circuit formation. Whether or not these molecules also have roles in cell survival in the developing nervous system is poorly understood. Plexins are a family of transmembrane receptors that bind Semaphorin ligands and are known to function in the guidance of developing axons and blood vessels. RESULTS: In embryonic zebrafish, plexina4 is expressed widely in the brain, becoming largely restricted to the hindbrain as neurogenesis and differentiation proceed. Apoptosis is increased in the embryonic hindbrain of a plexina4ca307/ca307 CRISPR mutant. Based on the literature, we tested the secreted heat shock protein, Clusterin, as a candidate ligand to mediate cell survival through Plexina4. clusterin is expressed by the floor plate of the embryonic zebrafish hindbrain, in proximity to plexina4-expressing hindbrain cells. Morpholino-mediated knockdown of Clusterin increases cell apoptosis in the hindbrain, with additional cell death observed in epistasis experiments where Clusterin is knocked down in a plexina4 mutant background. CONCLUSIONS: Our data suggest that Plexina4 promotes cell survival in the developing zebrafish hindbrain, likely through a pathway independent of Clusterin.

Spatial regulation of amacrine cell genesis by Semaphorin 3f.

PURPOSE: The axonal projections of retinal ganglion cells (RGCs) of the eye are topographically organized so that spatial information from visual images is preserved. This retinotopic organization is established during development by secreted morphogens that pattern domains of transcription factor expression within naso-temporal and dorso-ventral quadrants of the embryonic eye. Poorly understood are the downstream signaling molecules that generate the topographically organized retinal cells and circuits. The secreted signaling molecule Semaphorin 3fa (Sema3fa) belongs to the Sema family of molecules that provide positional information to developing cells. Here, we test a role for Sema3fa in cell genesis of the temporal zebrafish retina. METHODS: We compare retinal cell genesis in wild type and sema3fa CRISPR zebrafish mutants by in situ hybridization and immunohistochemistry. RESULTS: We find that mRNAs for sema3fa and known receptors, neuropilin2b (nrp2b) and plexina1a (plxna1a), are expressed by progenitors of the temporal, but not nasal zebrafish embryonic retina. In the sema3faca304/ca304 embryo, initially the domains of expression for atoh7 and neurod4, transcription factors necessary for the specification of RGCs and amacrine cells, respectively, are disrupted. Yet, post-embryonically only amacrine cells of the temporal retina are reduced in numbers, with both GABAergic and glycinergic subtypes affected. CONCLUSIONS: These data suggest that Sema3fa acts early on embryonic temporal progenitors to control in a spatially-dependent manner the production of amacrine cells, possibly to allow the establishment of neural circuits with domain-specific functions. We propose that spatially restricted extrinsic signals in the neural retina control cell genesis in a domain-dependent manner.

Semaphorin3f as a cardiomyocyte derived regulator of heart chamber development.

BACKGROUND: During development a pool of precursors form a heart with atrial and ventricular chambers that exhibit distinct transcriptional and electrophysiological properties. Normal development of these chambers is essential for full term survival of the fetus, and deviations result in congenital heart defects. The large number of genes that may cause congenital heart defects when mutated, and the genetic variability and penetrance of the ensuing phenotypes, reveals a need to understand the molecular mechanisms that allow for the formation of chamber-specific cardiomyocyte differentiation. METHODS: We used in situ hybridization, immunohistochemistry and functional analyses to identify the consequences of the loss of the secreted semaphorin, Sema3fb, in the development of the zebrafish heart by using two sema3fb CRISPR mutant alleles. RESULTS: We find that in the developing zebrafish heart sema3fb mRNA is expressed by all cardiomyocytes, whereas mRNA for a known receptor Plexina3 (Plxna3) is expressed preferentially by ventricular cardiomyocytes. In sema3fb CRISPR zebrafish mutants, heart chamber development is impaired; the atria and ventricles of mutants are smaller in size than their wild type siblings, apparently because of differences in cell size and not cell numbers. Analysis of chamber differentiation indicates defects in chamber specific gene expression at the border between the ventricular and atrial chambers, with spillage of ventricular chamber genes into the atrium, and vice versa, and a failure to restrict specialized cardiomyocyte markers to the atrioventricular canal (AVC). The hypoplastic heart chambers are associated with decreased cardiac output and heart edema. CONCLUSIONS: Based on our data we propose a model whereby cardiomyocytes secrete a Sema cue that, because of spatially restricted expression of the receptor, signals in a ventricular chamber-specific manner to establish a distinct border between atrial and ventricular chambers that is important to produce a fully functional heart. Video abstract.

Retinal Pigment Epithelium and Neural Retinal Progenitors Interact via Semaphorin 6D to Facilitate Optic Cup Morphogenesis.

Cell movement propels embryonic tissues to acquire shapes required for mature function. The movements are driven both by acto-myosin signaling and by cells interacting with the extracellular matrix (ECM). Unknown is whether cell-cell interactions within a tissue are also required, and the molecular mechanisms by which such communication might occur. Here, we use the developing visual system of zebrafish as a model to understand the role cell-cell communication plays in tissue morphogenesis in the embryonic nervous system. We identify that cell-cell-mediated contact between two distinct cell populations, progenitors of the neural retina and retinal pigment epithelium (RPE), facilitates epithelial flow to produce the mature cupped retina. We identify for the first time the need in eye morphogenesis for distinct populations of progenitors to interact, and suggest a novel role for a member of a key developmental signaling family, the transmembrane Semaphorin6d, as mediating communication between distinct cell types to control tissue morphogenesis.

Semaphorin 3fa Controls Ocular Vascularization From the Embryo Through to the Adult.

Purpose: Pathological blood vessel growth in the eye is implicated in several diseases that result in vision loss, including age-related macular degeneration and diabetic retinopathy. The limits of current disease therapies have created the need to identify and characterize new antiangiogenic drugs. Here, we identify the secreted chemorepellent semaphorin-3fa (Sema3fa) as an endogenous anti-angiogenic in the eye. Methods: We generated a CRISPR/Cas9 sema3fa zebrafish mutant line, sema3faca304/304. We assessed the retinal and choroidal vasculature in both larval and adult wild-type and sema3fa mutant zebrafish. Results: We find sema3fa mRNA is expressed by the ciliary marginal zone, neural retina, and retinal pigment epithelium of zebrafish larvae as choroidal vascularization emerges and the hyaloid/retinal vasculature is remodeled. The hyaloid vessels of sema3fa mutants develop appropriately but fail to remodel during the larval period, with adult mutants exhibiting a denser network of capillaries in the retinal periphery than seen in wild-type. The choroid vasculature is also defective in that it develops precociously, and aberrant, leaky sprouts are present in the normally avascular outer retina of both sema3faca304/304 larvae and adult fish. Conclusions: Sema3fa is a key endogenous signal for maintaining an avascular retina and preventing pathologic vascularization. Furthermore, we provide a new experimentally accessible model for studying choroid neovascularization (CNV) resulting from primary changes in the retinal environment that lead to downstream vessel infiltration.

Polarity and morphogenesis of the eye epithelium requires the adhesion junction associated adaptor protein Traf4.

During development, neuroepithelial progenitors acquire apico-basal polarity and adhere to one another via apically located tight and adherens junction complexes. This polarized neuroepithelium must continue to integrate cells arising through cell divisions and intercalation, and allow for cell movements, at the same time as undergoing morphogenesis. Cell proliferation, migration and intercalation all occur in the morphing embryonic eye. To understand how eye development might depend on dynamic epithelial adhesion, we investigated the function of a known regulator of junctional plasticity, Tumour necrosis factor receptor-associated factor 4 (Traf4). traf4a mRNA is expressed in the developing eye vesicle over the period of optic cup morphogenesis, and Traf4a loss leads to disrupted evagination and elongation of the eye vesicles, and aberrant organization and apico-basal polarity of the eye epithelium. We propose a model whereby Traf4a regulates apical junction plasticity in nascent eye epithelium, allowing for its polarization and morphogenesis. Symbols and Abbreviations: AB: apico-basal; aPKC: atypical protein kinase-C; CRISPR: clustered regularly-interspaced short palindromic repeats; GFP: green fluorescent protein; hpf: hours post-fertilization; MO: antisense morpholino oligonucleotide; pHH3: phospho histone H3; ss: somite stage; Traf4: Tumour necrosis factor receptor-associated factor 4; ZO-1: zona occludens-1.