A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal

The outer segment of the vertebrate rod photoreceptor is a highly modified cilium composed of many discrete membranous discs that are filled with the protein machinery necessary for phototransduction. The unique outer segment structure is renewed daily with growth at the base of the outer segment where new discs are formed and shedding at the distal end where old discs are phagocytized by the retinal pigment epithelium. In order to understand how outer segment renewal is regulated to maintain outer segment length and function, we used a small molecule screening approach with the transgenic (hsp70:HA-mCherryTM) zebrafish, which expresses a genetically-encoded marker of outer segment renewal. We identified compounds with known bioactivity that affect five content areas: outer segment growth, outer segment shedding, clearance of shed outer segment tips, Rhodopsin mislocalization, and differentiation at the ciliary marginal zone. Signaling pathways that are targeted by the identified compounds include cyclooxygenase in outer segment growth, γ-Secretase in outer segment shedding, and mTor in RPE phagocytosis. The data generated by this screen provides a foundation for further investigation of the signaling pathways that regulate photoreceptor outer segment renewal

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes

AbstractPodocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell–cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell–cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains. Results We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size. Conclusions Our observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain

Hippocampal pyramidal cells: the reemergence of cortical lamination

The increasing resolution of tract-tracing studies has led to the definition of segments along the transverse axis of the hippocampal pyramidal cell layer, which may represent functionally defined elements. This review will summarize evidence for a morphological and functional differentiation of pyramidal cells along the radial (deep to superficial) axis of the cell layer. In many species, deep and superficial sublayers can be identified histologically throughout large parts of the septotemporal extent of the hippocampus. Neurons in these sublayers are generated during different periods of development. During development, deep and superficial cells express genes (Sox5, SatB2) that also specify the phenotypes of superficial and deep cells in the neocortex. Deep and superficial cells differ neurochemically (e.g. calbindin and zinc) and in their adult gene expression patterns. These markers also distinguish sublayers in the septal hippocampus, where they are not readily apparent histologically in rat or mouse. Deep and superficial pyramidal cells differ in septal, striatal, and neocortical efferent connections. Distributions of deep and superficial pyramidal cell dendrites and studies in reeler or sparsely GFP-expressing mice indicate that this also applies to afferent pathways. Histological, neurochemical, and connective differences between deep and superficial neurons may correlate with (patho-) physiological phenomena specific to pyramidal cells at different radial locations. We feel that an appreciation of radial subdivisions in the pyramidal cell layer reminiscent of lamination in other cortical areas may be critical in the interpretation of studies of hippocampal anatomy and function

Generation of a genetically encoded marker of rod photoreceptor outer segment growth and renewal

Summary Vertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability

Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development-5

In mutants, very little Crumbs protein is visible and ZO-1 labeling is disorganized. (C) In mRNA injected mutants, streaks of panCrb labeling are visible, but an organized OLM is absent. Ultrastructural transmission electron microscopic analysis at 6 dpf in wild-type (D), mutants (E) and mRNA injected mutants (F) retinas. Electron dense outer segments are seen in all individuals. Insets, higher magnifications of rod outer segments showing regular disc stacking is present in all individuals (100,000X). RPE, retinal pigmented epithelium; OS, outer segments; IS, inner Segments; CB, cell body. (A-C) are confocal z-projections, scale bar 10 μm (D-F). Scale bars, 5 μm (D-F), 100 nm (insets).<p><b>Copyright information:</b></p><p>Taken from "Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development"</p><p>http://www.biomedcentral.com/1471-213X/8/3</p><p>BMC Developmental Biology 2008;8():3-3.</p><p>Published online 11 Jan 2008</p><p>PMCID:PMC2266719.</p><p></p

Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development-3

N ventricles are reduced in size or absent, pericaridal edema is pronounced, and the RPE is patchy (inset). (C) In embryos injected with mRNA, brain ventricles are reduced in size or absent, pericaridal edema is pronounced, but the RPE is normal (inset). (D) embryos injected with mRNA, brain ventricles are absent or absent and pericardial edema is pronounced, and RPE defects are milder than those in uninjected embryos. (E) A magnified view of the RPE of a 60 hpf wild-type embryo shows that it is uniform and the cells are confluent. (F) In a wild-type retina at 4 dpf, GFProds localize next to the RPE and lamination is apparent. In 60 hpf mutants, the integrity of the RPE varies from mild (G), to moderate (I), to severe (H). However, GFProds are mislocalized in all mutants 4 dpf (H, J, L). The integrity of the RPE is improved and nearly normal in a 60 hpf mutant injected with mRNA (M) and most GFProds are adjacent to the RPE (N). (O) A wild-type embryo injected with showing brain ventricles that are reduced or absent. (P) At 30 hpf Epb4.1l5is cortically localized, upper inset is a 2× magnification of Epb4.1l5localization. Scale bars, 10 μm (F), 50 μm (lower insets in F).<p><b>Copyright information:</b></p><p>Taken from "Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development"</p><p>http://www.biomedcentral.com/1471-213X/8/3</p><p>BMC Developmental Biology 2008;8():3-3.</p><p>Published online 11 Jan 2008</p><p>PMCID:PMC2266719.</p><p></p