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P A Raymond

Publications and source records attributed to P A Raymond.

At least 19 recordsLinked to original sources
PubMed2001

Zebrafish genes rx1 and rx2 help define the region of forebrain that gives rise to retina.

Zebrafish retinal homeobox genes rx1 and rx2 are expressed exclusively in the optic primordia and then in cone photoreceptors of the differentiated neural retina. In this study, we show that the rx expression domain is coextensive with the region identified as the retinal field in published fate maps of the neural plate in zebrafish embryos. Analysis of the spatiotemporal relationships between retinal and forebrain precursors suggests that lateral movement of retinal precursors is responsible for evagination of the optic primordia. Overexpression of either rx1 or rx2 results in the loss of forebrain tissue and the ectopic formation of retinal tissue. We asked whether the deletion of forebrain and expansion of retinal tissue could be explained by the death of telencephalic precursors and enhanced proliferation of retinal precursors, and we found that it could not. Instead, our data are consistent with a change in cell fate of forebrain precursors associated with reduced expression of telencephalic markers (emx1 and BF-1) and ectopic expression of retinal markers (rx1/2/3, pax6, six6, and vsx2) at the neural keel stage. The rx homeodomain alone is sufficient to induce ectopic retinal tissue, although weakly so, and this observation, together with results from deletion constructs, suggests that interactions with unidentified transcriptional regulators are important for rx1 and rx2 function during early eye development. We conclude that regulated expression of zebrafish rx1 and rx2 helps to define the region of the forebrain fated to give rise to retinal tissue and may be involved in the cellular migrations that lead to splitting of the retinal field and formation of the optic primordia.

Animals↗
PubMed2001

Isolation and characterization of a zebrafish homologue of the cone rod homeobox gene.

PURPOSE: To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specifically in photoreceptors and pinealocytes, regulate photoreceptor gene expression, are necessary for normal photoreceptor differentiation, and when mutated cause a variety of photoreceptor degenerations. METHODS: A zebrafish retinal cDNA library was screened with a human CRX cDNA probe. Radiation hybrid mapping, Northern blot analysis, in situ hybridization, and transient transfection studies were performed using standard methods. RESULTS: Based on amino acid sequence comparisons, zebrafish crx shows 50% identity with human CRX, and 85% identity in the homeodomain. A phylogenetic analysis indicates that zebrafish crx is most closely related to the mammalian Crx proteins, and more distantly related to the Otx proteins. Zebrafish crx maps between 49.6 and 54.5 cm from the top of linkage group LG05C, a map position consistent with the location of the mouse and human CRX genes. Northern blot analysis and in situ hybridization indicate that zebrafish crx is expressed in the retina and pineal gland. In adult zebrafish, crx is expressed by both rods and cones in the outer nuclear layer, and in cells in the outer zone of the inner nuclear layer, in the region occupied by bipolar cells. Similar to mammalian Crx, zebrafish crx interacts with neural retinal leucine zipper (Nrl) to activate, although weakly, rhodopsin promoter activity. CONCLUSIONS: Based on molecular phylogeny, chromosomal location, expression pattern, and ability to activate rhodopsin promoter activity in transient transfection assays, zebrafish crx appears to be an orthologue and functional homologue of mammalian CRX:

Amino Acid Sequence↗
PubMed2000

Function for Hedgehog genes in zebrafish retinal development.

The hedgehog (hh) genes encode secreted signaling proteins that have important developmental functions in vertebrates and invertebrates. In Drosophila, expression of hh coordinates retinal development by propagating a wave of photoreceptor differentiation across the eye primordium. Here we report that two vertebrate hh genes, sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. A gene encoding a receptor for the hedgehog protein, ptc-2, is expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense (alphashh/alphatwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly exhibit retardation of photoreceptor differentiation. We propose that hedgehog genes may play a role in propagating photoreceptor differentiation across the developing eye of the zebrafish.

Animals↗
PubMed2000

Localization of cGMP-dependent protein kinase isoforms in mouse eye.

PURPOSE: To examine the expression of the major isoforms of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (cGK) in mouse eye. METHODS: Immunohistochemical localization of cGMP in mouse eye cryosections was performed using an anti-cGMP antibody, followed by visualization with indirect fluorescence microscopy. The presence of types Ialpha, Ibeta, and II cGK mRNAs in mouse eye extracts was determined initially by RNase protection analysis. Further localization of cGK I and II mRNAs on cryosections was accomplished by in situ hybridization using digoxigenin-labeled cRNA probes and an alkaline phosphatase-conjugated anti-digoxigenin antibody. Finally, cGK I protein was localized to subcellular areas within the retina using an anti-cGK I-specific primary antibody. RESULTS: In initial immunohistochemical experiments cGMP was present in numerous regions and layers within the eye and retina. Subsequent RNase protection studies demonstrated that cGK Ialpha, Ibeta, and II mRNAs were present in mouse eye and that type Ibeta mRNA were 6.6 and 30 times more abundant than type Ialpha and type II, respectively. By in situ hybridization, cGK I mRNA was localized to photoreceptor inner segments and the ganglion cell and inner nuclear layers of the retina, and lesser amounts were found in the ciliary epithelium, lens, and cornea. The cGK II mRNA expression pattern was similar but not identical with that of cGK I. Finally, within the retina, cGK I protein was most abundant in the inner plexiform layer, with significant amounts in ganglion cells and photoreceptor inner segments as well. CONCLUSIONS: The presence of these cGK isoforms in discrete areas throughout the eye suggests multiple roles for the cGMP-dependent signal transduction system in the regulation of physiologic and pathologic ocular processes.

Animals↗
PubMed1999

Spatial correspondence between R-cadherin expression domains and retinal ganglion cell axons in developing zebrafish.

Mechanisms underlying axonal pathfinding have been investigated for decades, and numerous molecules have been shown to play roles in this process, including members of the cadherin family of cell adhesion molecules. We showed in the companion paper that a member of the cadherin family (zebrafish R-cadherin) is expressed in retinal ganglion cells, and in presumptive visual structures in zebrafish brain, during periods when the axons were actively extending toward their targets. The present study extends the earlier work by using 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) anterograde tracing techniques to label retinal ganglion cell axons combined with R-cadherin in situ hybridization to explicitly examine the association ofretinal axons and brain regions expressing R-cadherin message. We found that in zebrafish embryos at 46-54 hours postfertilization, DiI-labeled retinal axons were closely associated with cells expressing R-cadherin message in the hypothalamus, the pretectum, and the anterolateral optic tectum. These results demonstrate that R-cadherin is appropriately distributed to play a role in regulating development of the zebrafish visual system, and in particular, pathfinding and synaptogenesis of retinal ganglion cell axons.

Animals↗
PubMed1999

R-cadherin expression in the developing and adult zebrafish visual system.

Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N-cadherin, paraxial protocadherin, and VN-cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E-cadherin. Here, we report a partial cDNA with extensive sequence homology to R-cadherin. Spatial and temporal expression of this putative zebrafish R-cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R-cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R-cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R-cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R-cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R-cadherin message. In the adult, R-cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R-cadherin-mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system.

Age Factors↗
PubMed1999

Expression of three Rx homeobox genes in embryonic and adult zebrafish.

The paired-class homeobox gene, Rx, is important in eye development. In this study we analyze expression patterns of three zebrafish Rx genes (Zrx1, 2, 3) in embryos and adults. All three genes show dynamic spatiotemporal patterns of expression. Zrx3 is expressed earliest, in the anteriormost region of the neural plate, in regions that give rise to ventral diencephalon and retinae. As development proceeds, Zrx3 expression is reduced in the lateral optic primordia, and is absent in the optic cup, but is retained at the ventral midline of the diencephalon, and is expressed in hypothalamus in the adult. As the neural retina begins to differentiate, Zrx3 is re-expressed in a subset of cells in the inner nuclear layer, presumably bipolar cells, and this expression is retained in the adult. In contrast, Zrx1/2 have a slightly later onset of expression, are initially coincident with Zrx3, but then become complementary, remaining on in the optic primordia but disappearing from the ventral midline of the diencephalon. Zrx1/2 are down-regulated as the retina differentiates, except in the outer nuclear layer where they continue to be expressed at high levels in cone, but not rod, photoreceptors. This is the first transcription factor described that distinguishes between cone and rod photoreceptors.

Animals↗
PubMed1998

Novel expression pattern of interphotoreceptor retinoid-binding protein (IRBP) in the adult and developing zebrafish retina and RPE.

PURPOSE: Interactions between the neural retina and retinal pigment epithelium (RPE) are mediated by the interphotoreceptor matrix (IPM). The transport of retinoids across the IPM is mediated by interphotoreceptor retinoid-binding protein (IRBP). To explore the possibility that IRBP is important during retinal development, we examined its spatiotemporal expression pattern in embryonic zebrafish. METHODS: IRBP mRNA expression was examined using RT-PCR and in situ hybridization. IRBP was localized using antiserum against recombinant zebrafish IRBP. IRBP synthesis and secretion were studied by in vitro metabolic labeling of retinas and RPE-eyecups. RESULTS: IRBP mRNA was first observed in the pineal at 24 hours post-fertilization (hpf) and in the ventral retina at 50 hpf. Immunoreactive IRBP was first observed at 72 hpf. Remarkably, IRBP was expressed not only by photoreceptors but also by the adult and embryonic RPE. In embryos, expression in both retina and RPE began in a ventronasal patch and spread to involve the entire eye. In general, early IRBP expression was dominated by photoreceptors, but then RPE expression spread beyond the limit of photoreceptor expression. Double in situ hybridizations suggests that cones express IRBP mRNA before they express a specific opsin, while rods may express rod opsin prior to IRBP. CONCLUSIONS: The temporal and spatial patterns of IRBP expression by the RPE and retina are consistent with a role in retinal development and suggest coordination of RPE and photoreceptor differentiation.

Animals↗
PubMed1998

Vsx-2, a gene encoding a paired-type homeodomain, is expressed in the retina, hindbrain, and spinal cord during goldfish embryogenesis.

Vsx-2 encodes a paired-type homeodomain and is the goldfish ortholog of the murine Chx10 gene. During development, Vsx-2 is expressed at high levels in goldfish and zebrafish retina. In addition to the retina, in situ hybridization studies using whole mount and cryosection embryos demonstrate that Vsx-2 is also expressed in subsets of neurons in the hindbrain and in the spinal cord. Expression begins approximately at the metencephalon-myelencephalon border and continues in a restricted lateral zone along the rostral-caudal axis of the spinal cord. These observations suggest a potential requirement for Vsx-2 in the specification and/or the maintenance of neurons in specific CNS regions during embryogenesis. Also discussed are other transcription factors which may act combinatorially with Vsx-2 to regulate neuronal differentiation.

Animals↗
PubMed1998

Cloning of zebrafish vsx1: expression of a paired-like homeobox gene during CNS development.

vsx1 is a homeobox gene encoding a paired-type homeodomain and a CVC domain that was originally cloned from an adult goldfish retinal library. We previously reported the spatiotemporal expression pattern of vsx1 in the adult and developing retina of zebrafish and goldfish, and we suggested that vsx1 plays a role in determining the cell fate and maintenance of retinal interneurons. Other related genes encoding a CVC domain, such as vsx2 (alx) and chx10, are expressed both within and outside the retina during development. In this study, we report the cloning of zebrafish vsx1 and its developmental expression in both retinal and nonretinal regions of the CNS in zebrafish embryos. vsx1 expression was detected in a subset of hindbrain and spinal cord neurons before it was expressed in the retina. At about the same time that retinal expression began, the level of vsx1 was decreased in the spinal cord. The expression of vsx1 was progressively restricted, and eventually it was detected only in the inner nuclear layer (INL) of the developing retina. The combined expression patterns of teleost vsx1 and vsx2 (alx) during early zebrafish development encompasses the expression pattern observed for murine Chx10, and indicates a partitioning of function for CVC genes in lower vertebrates.

Amino Acid Sequence↗
PubMed1997

Vsx-1 and Vsx-2: differential expression of two paired-like homeobox genes during zebrafish and goldfish retinogenesis.

Vsx-1 and Vsx-2 are two homeobox genes that were cloned originally from an adult goldfish retinal library. They are members of the paired-like:CVC gene family, which is characterized by the presence of a paired homeodomain and an additional conserved region, termed the CVC domain. To analyze the possible roles for Vsx-1 and Vsx-2 in eye development, we used in situ hybridization to examine their expression patterns in zebrafish and goldfish embryos. Vsx-2 is initially expressed by proliferating neuroepithelial cells of the presumptive neural retina, then it is down-regulated as differentiation begins, and it is finally reexpressed at later stages of differentiation in a subset of cells, presumed to be bipolar cells, in the inner nuclear layer. In contrast, Vsx-1 is expressed only weakly in undifferentiated, presumptive neural retina and is then up-regulated selectively in presumptive bipolar cells at early stages of differentiation (when Vsx-2 is turned off), before decreasing to an intermediate level, which is maintained in the differentiated (adult) retina. The restricted expression patterns of Vsx-2 correspond to the observed phenotypes in mice with the ocular retardation mutation (orJ), further supporting the notion that Vsx-2 and Chx10 are homologues. The sequential complimentary and then corresponding expression patterns of Vsx-1 and Vsx-2 suggest that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during the development of the retina.

Amino Acid Sequence↗
PubMed1997

Spatiotemporal coordination of rod and cone photoreceptor differentiation in goldfish retina.

In this study, we have compared spatial and temporal aspects of development of new rods and cones in the adult goldfish by using a combination of bromodeoxyuridine immunocytochemistry and opsin in situ hybridization to determine the intervals between terminal mitosis (cell "birth") and expression of opsin mRNA for each photoreceptor cell type. The goldfish opsins include rod opsin and four different cone opsins: red, green, blue, and ultraviolet. In a cohort of photoreceptors born at the same time, rods expressed opsin mRNA within 3 days of cell birth, while expression of cone opsin mRNA required at least 7 days. This temporal discrepancy in differentiation, coupled with a discordance in the site of cell genesis of rods and cones, allowed opsin expression to commence in both cell types in approximately the same retinal location. Commitment to the generic cone phenotype occurred within approximately 6 days throughout the cone cohort, as indicated by expression of interphotoreceptor retinoid-binding protein (IRBP) mRNA, but expression of a specific spectral phenotype was delayed until rods differentiated nearby. Onset of expression of cone opsin mRNA followed a phenotype-specific sequence: red, then green, then blue, and finally ultraviolet; in situ hybridization with two opsin probes confirmed that individual photoreceptors expressed only one type of opsin as they differentiated. This stepwise process of cone differentiation is consistent with the hypothesis that cell-cell interactions among developing photoreceptors may coordinate selection of specific photoreceptor phenotypes.

Animals↗
PubMed1997

A goldfish Notch-3 homologue is expressed in neurogenic regions of embryonic, adult, and regenerating brain and retina.

Members of the Notch gene family are thought to be involved in the regulation of cell fate decisions in a variety of embryonic tissues, particularly in the developing central nervous system (CNS) in Drosophila and vertebrates. In goldfish the CNS continues to develop and add neurons well into adulthood and has the capacity to regenerate new neurons. Using probes derived from Xenopus Notch to screen an adult goldfish retinal cDNA library, followed by 5' RACE, we isolated a partial cDNA for a goldfish Notch homologue, G-Notch. Sequence alignment supported assignment of G-Notch to the Notch-3 class. Northern blot analysis revealed a single transcript of > 8 kb, and RNase protection assays indicated that G-Notch is expressed in eye and brain but not muscle of adult goldfish. The spatiotemporal pattern of expression of G-Notch was defined from early embryonic stages to adulthood by in situ hybridization. Expression in the embryonic CNS was localized to neurogenic regions and was downregulated in differentiated cell populations. In adult goldfish, expression persisted in and adjacent to the germinal zones in the retina and the brain. Weak expression was seen in scattered cells in the inner nuclear layer of the retina, which might include neurogenic stem cells. Following retinal lesions (puncture wounds or laser lesions restricted to photoreceptors in the outer nuclear layer), G-Notch was upregulated in proliferating cell populations throughout the retina, in association with a generalized mitogenic response. In the region of the laser lesion, where earlier studies have demonstrated that photoreceptors are regenerating at 1-3 weeks following the lesion, G-Notch expressing cells were abundant in the outer nuclear layer. These observations suggest that retinal regeneration involves the re-expression of an important developmental signaling molecule in neuroepithelial cells resident in the differentiated retina.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and characterization of the putative ultraviolet-sensitive visual pigment of goldfish.

A cDNA full length encoding a putative ultraviolet (UV)-sensitive visual pigment of goldfish was isolated. The deduced amino acid sequence shows 64% identity to those of human blue and chicken violet, and less identity (40-49%) to those of other vertebrate visual pigment. The mRNA is localized in the miniature short single cone cells, which are known to have a sensitivity maximum in the near UV-region.

Amino Acid Sequence↗
PubMed1996

Zebrafish interphotoreceptor retinoid-binding protein: differential circadian expression among cone subtypes.

Retinoid trafficking between the photoreceptors and pigmented epithelium is probably mediated by interphotoreceptor retinoid-binding protein (IRBP), a 124-145 kDa glycolipoprotein in mammals and amphibians. In these animals, IRBP is composed of four homologous regions (modules) 300 amino acids in length. We have determined the primary structure of zebrafish IRBP and its expression pattern by northern analysis, reverse transcriptase-polymerase chain reaction and in situ hybridization under a variety of lighting conditions. Zebrafish IRBP is half the size (66.3 kDa) of mammalian IRBP because it is composed of only two modules, similar to goldfish IRBP. The first half of the zebrafish protein is most similar to the first module of mammalian IRBP and the second half to the fourth module of mammalian IRBP. This suggests that during the evolution of the ray-finned fish (Actinopterygii), the middle two modules were lost. Each of the modules contains conserved hydrophobic domains which may form the ligand-binding pocket. The expression of zebrafish IRBP mRNA is sevenfold higher in the middle of the light period (at mid-light) than in the middle of the dark period (at mid-dark). This rhythm persists for 2 days under conditions of constant light or constant darkness, then dampens to an intermediate level by 8 days of constant conditions. At mid-light, IRBP mRNA is expressed by all cone types and to a lesser extent by the rods. At mid-dark, the mRNA is restricted to the ultraviolet-sensitive short single cones. These data suggest that IRBP expression is regulated by circadian and light-driven mechanisms that act differentially on the various photoreceptor subtypes in the zebrafish retina.

Amino Acid Sequence↗