The cellular expression of antiangiogenic factors in fetal primate macula.

PURPOSE: To characterize the cellular expression patterns of antiangiogenic factors differentially regulated in the fetal human macula. METHODS: RNA was extracted from macular, nasal, and surround biopsies of three human fetal retinas at midgestation. Relative levels of expression of pigment epithelium-derived factor (PEDF), brain natriuretic peptide (BNP), collagen type IValpha2 (COL4A2), and natriuretic peptide receptors A and C (NPRA and NPRC) were determined with quantitative PCR. Cellular expression of PEDF and BNP was investigated by in situ hybridization on retinal sections from monkeys aged between fetal day 55 and 11 years. BNP, COL4A2, and NPRA proteins were localized by immunohistochemistry. Labeling was imaged and quantified by confocal microscopy and optical densitometry. RESULTS: Quantitative PCR confirmed higher levels of PEDF and BNP and lower levels of COL4A2 in the macula at midgestation. PEDF mRNA was detected in ganglion cells (GCs) and the pigment epithelium (RPE). BNP mRNA was detected in GCs and macroglia, although BNP immunoreactivity (IR) was predominantly perivascular. COL4A2-IR was detected in large blood vessels and NPRA-IR on the retinal vascular endothelium, GC axons in fetal retinas, and cone axons at all ages. Optical densitometry showed a graded expression of PEDF and BNP at all ages, with highest levels of expression in GCs in the developing fovea. CONCLUSIONS: Because the retinal vessels initially form in the GC layer, it is likely that PEDF has a key role in defining and maintaining the foveal avascular area. The precise role of BNP is unclear, but it may include both antiangiogenic and natriuretic functions.

Tetrodotoxin-resistant voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 are expressed in the retina.

Voltage-gated sodium channels (VGSCs) are one of the fundamental building blocks of electrically excitable cells in the nervous system. These channels are responsible for the generation of action potentials that are required for the communication of neuronal signals over long distances within a cell. VGSCs are encoded by a family of nine genes whose products have widely varying biophysical properties. In this study, we have detected the expression of two atypical VGSCs (Na(v)1.8 and Na(v)1.9) in the retina. Compared with more common VGSCs, Na(v)1.8 and Na(v)1.9 have unusual biophysical and pharmacological properties, including persistent sodium currents and resistance to the canonical sodium channel blocker tetrodotoxin (TTX). Our molecular biological and immunohistochemical data derived from mouse (Mus musculus) retina demonstrate expression of Na(v)1.8 by retinal amacrine and ganglion cells, whereas Na(v)1.9 is expressed by photoreceptors and Müller glia. The fact that these channels exist in the central nervous system (CNS) and exhibit robust TTX resistance requires a re-evaluation of prior physiological, pharmacological, and developmental data in the visual system, in which the diversity of VGSCs has been previously underestimated.

Molecular mechanisms of vertebrate retina development: implications for ganglion cell and photoreceptor patterning.

Although the neural retina appears as a relatively uniform tissue when viewed from its surface, it is in fact highly patterned along its anterior-posterior and dorso-ventral axes. The question of how and when such patterns arise has been the subject of intensive investigations over several decades. Most studies aimed at understanding retinal pattern formation have used the retinotectal map, the ordered projections of retinal ganglion cells to the brain, as a functional readout of the pattern. However, other cell types are also topographically organized in the retina. The most commonly recognized example of such a topographic cellular organization is the differential distribution of photoreceptor types across the retina. Photoreceptor patterns are highly species-specific and may represent an important adaptation to the visual niche a given species occupies. Nevertheless, few studies have addressed this functional readout of pattern to date and our understanding of its development has remained superficial. Here, we review recent advances in understanding the molecular cascades that control regionalization of the eye anlage, relate these findings to the development of photoreceptor patterns and discuss common and unique strategies involved in both aspects of retinal pattern formation.

Emergence of cellular markers and functional ionotropic glutamate receptors on tangentially dispersed cells in the developing mouse retina.

Tangential cell dispersion in the retina is a spacing mechanism that establishes a regular mosaic organization among cell types and contributes to their final positioning. The present study has used the X-inactivation transgenic mouse expressing the lacZ reporter gene on one X chromosome. Due to X chromosome inactivation, 50% of early progenitor cells express beta-galactosidase (beta-Gal); therefore, all cells derived from a particular beta-Gal-expressing progenitor cell can be identified in labeled columns. The radial segregation of clonally related beta-Gal-positive and beta-Gal-negative cells can be used to determine whether single cells transgress a clonal boundary in the retina. We investigated the extent to which particular cell classes tangentially disperse by analyzing the placement of labeled cells expressing particular markers at several ages and quantifying their tangential displacement. Retinal neurons expressing cell markers at postnatal day (P) 1 have a greater degree of tangential dispersion compared with amacrine and bipolar cells at P5-6. We also studied whether there is a functional correlation with these dispersion patterns by investigating the emergence of functional ionotropic glutamate receptors. To determine the degree of functional glutamate receptor activation, agmatine (AGB) was used in combination with cell-specific labeling. AGB permeates functional glutamate receptor channels following activation with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate or N-methyl-D-aspartate (NMDA). Within these receptor groups, high concentrations of AMPA, kainate, and NMDA are associated with a high degree of tangential dispersion in the adult. Developmentally, functional kainate and AMPA receptors were detected by P1 and were associated with tangentially dispersed cells. Functional NMDA receptors were not detected as early as kainate and AMPA receptors. These results indicate that cells generated early during development are more likely to disperse tangentially compared with those generated later in development. Therefore, functional AMPA and kainate receptors may play a critical role in tangentially displacing cell types.

Expression of the homeodomain transcription factor Meis2 in the embryonic and postnatal retina.

Members of the Meis subfamily of homeodomain-containing transcription factors play important roles during development and disease. Here we report that the Meis family protein Meis2 is expressed by a subpopulation of gamma-aminobutyric acid (GABA)ergic amacrine (AM) cells in the adult and embryonic retina of different vertebrate species. In mice, Meis2-expressing (Meis2+) AM cells are not cholinergic or dopaminergic, but some are immunoreactive for neuronal nitric oxide synthase (bNOS). About 50% of the mouse Meis2+ AM cell population expresses the calcium-binding protein calretinin, and some Meis2+ AM cells show characteristics of Type II CD-15+ cells. AM cell expression of Meis2 is lost in a conditional knockout mouse model for Pax6, indicating a dependency upon Pax6. Bromodeoxyuridine pulse labeling experiments and immunohistochemical staining for the neuronal marker NeuN in embryonic mouse retinae indicate that Meis2 is an early marker for newly postmitotic AM cells. In addition, taking advantage of the protracted retinal development in humans, we show that newly generated AM cells express Meis2 before adopting the GABAergic or glycinergic neurotransmitter phenotype. As development proceeds, some AM cells lose Meis2 expression concomitantly with the appearance of glycine, while other AM cells retain Meis2 expression after they express GABA. These data identify Meis2 as a suitable marker for the study of AM cell diversity and development in addition to providing evidence for the stepwise specification of the glycinergic and GABAergic neurotransmitter phenotypes during AM cell differentiation.

Chick receptor protein tyrosine phosphatase lambda/psi (cRPTPlambda/cRPTPpsi) is dynamically expressed at the midbrain-hindbrain boundary and in the embryonic neural retina.

The isthmic organizer, located near the boundary between the developing midbrain and hindbrain, controls the patterning of adjacent brain regions. Here we describe the spatial and temporal expression of chick receptor-like protein tyrosine phosphatase lambda (cRPTPlambda, also known as cRPTPpsi) during the development of this structure. After an initial widespread expression throughout the caudal forebrain and midbrain region, expression of cRPTPlambda is confined to the ventral midline of the neural tube, the future neural retina and lens, and a sharp ring at the isthmic constriction, overlying the molecular mid-hindbrain boundary (MHB). MHB expression of cRPTPlambda borders at the caudal limit of the expression domain of the transcription factor Otx2, appears to partially overlap with that of the secreted protein Wnt1 and is similar to that of the Ig-CAM CEPU-1. In the neural retina, expression is restricted to the ventricular zone, where the cell bodies of retinal progenitor cells reside.

Expression of photoreceptor-specific nuclear receptor NR2E3 in rod photoreceptors of fetal human retina.

PURPOSE: To study the physiological function of NR2E3 and possible molecular mechanisms underlying enhanced short-wavelength cone syndrome (ESCS) pathogenesis in developing human retina, and to compare its expression to that of Neural Retina Leucine zipper (NRL), a transcription factor essential for rod differentiation. METHODS: Expression of NR2E3, a photoreceptor-specific orphan nuclear receptor, was examined in human retinas between fetal weeks (Fwk) 9 to 22 by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Both NR2E3 and NRL expression patterns were followed by immunocytochemistry. The human retina develops in a central to peripheral pattern, in which a protein may take weeks to be expressed throughout the entire retina. This allowed a detailed temporal analysis of NR2E3 and NRL expression. RESULTS: NR2E3 expression was detected shortly after the appearance of NRL in putative immature rods on the foveal edge at Fwk 11.7. Expression of both markers was maintained in rod opsin expressing fetal photoreceptors. NR2E3 expression was not detected in either long/medium- or short-wavelength cones. Its absence from cones was also supported by the position of labeled nuclei deep in the outer nuclear layer, and by the absence of NR2E3 from the fovea. CONCLUSIONS: A role for NR2E3 in the rod developmental pathway is suggested. The closely related expression patterns of NRL and NR2E3 supported an interactive function, where both transcription factors determine the rod fate and suppress immature rods from adopting the S-cone fate.