Pamela A. Raymond

Late-Stage Neuronal Progenitors in the Retina Are Radial Müller Glia That Function as Retinal Stem Cells

Neuronal progenitors in the mammalian brain derive from radial glia or specialized astrocytes. In developing neural retina, radial glia-like Müller cells are generated late in neurogenesis and are not considered to be neuronal progenitors, but they do proliferate after injury and can express neuronal markers, suggesting a latent neurogenic capacity. To examine the neurogenic capacity of retinal glial cells, we used lineage tracing in transgenic zebrafish with a glial-specific promoter (gfap, for glial fibrillary acid protein) driving green fluorescent protein in differentiated Müller glia. We found that all Müller glia in the zebrafish retina express low levels of the multipotent progenitor marker Pax6 (paired box gene 6), and they proliferate at a low frequency in the intact, uninjured retina. Müller glia-derived progenitors express Crx (cone rod homeobox) and are late retinal progenitors that generate the rod photoreceptor lineage in the postembryonic retina. These Müller glia-derived progenitors also remain competent to produce earlier neuronal lineages, in that they respond to loss of cone photoreceptors by specifically regenerating the missing neurons. We conclude that zebrafish Müller glia function as multipotent retinal stem cells that generate retinal neurons by homeostatic and regenerative developmental mechanisms.

Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin

Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1–4 (refs. 4–9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10–20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.

Improved method for obtaining 3-microns cryosections for immunocytochemistry.

The following describes a modified technique for obtaining 3-microns sections for light microscopic level immunocytochemistry. By combining 20% sucrose with Tissue-Tek OCT embedding compound in a ratio of 2:1, we produced a block that was suitable for cutting 3-microns sections on a conventional cryostat. The 3-microns sections were dramatically improved compared with 10-microns sections cut from tissue embedded in OCT alone, when viewed with both differential interference contrast microscopy (Nomarski optics) and indirect immunofluorescence. The method is simple, uses materials already available, and does not require training in a new technique.

Postembryonic growth of the optic tectum in goldfish. I. Location of germinal cells and numbers of neurons produced

The growth and morphology of the optic tectum of adult goldfish were studied with light and electron microscopy and with thymidine radioautography. The tectum is roughly hemispheric in shape, with a smaller radius of curvature rostrally than caudally. A narrow region containing proliferating cells (the germinal zone) is found along two- thirds of the rim of the tectal hemisphere but is absent rostrally, adjacent to the tectal region which receives input from the rostral visual field. New cells generated in the germinal zone are added to the tectum appositionally in crescent-shaped increments; these was no evidence of migration of new cells into the rostral region which lacks a germinal zone. Some of the new cells added to the adult tectum were shown to be neurons on the basis of cytological and ultrastructural features. Counts of tectal neurons likewise demonstrated that new cells were added with growth of the tectum; large goldfish (25 cm long) had 27% more tectal neurons than did small fish (4 cm long). Spreading apart of existing cells also contributed to overall growth of the tectum. These results confirm and extend those of R. L. Meyer ((1978) Exp. Neurol. 59: 99–111). The topological dissimilarity of the patterns of growth of retina (which adds cells appositionally around its entire perimeter) and tectum supports the suggestion that retinotectal terminals must continually move (Gaze R. M., M. J. Keating, A. Ostberg, and S. H. Chung (1979) J. Embryol. Exp. Morphol. 53: 103–143). Our estimates of cell numbers and tectal areas lead to predictions about the directions and magnitudes of these displacements.

Expression of rod and cone visual pigments in goldfish and zebrafish: A rhodopsin-like gene is expressed in cones

The primary purpose of the present study was to determine whether a rhodopsin-like gene, which has been postulated to represent the green cone pigment in several species, is in fact expressed in cone photoreceptors instead of rods. The expression patterns of rod opsin and blue and red cone opsins were also examined in both goldfish and zebrafish retinas using colorimetric in situ hybridization. The results demonstrate that the rhodopsin-like gene is expressed in green cones, as predicted. A subset of small cones that do not hybridize with these cRNA probes are tentatively identified as ultraviolet receptors. The results also demonstrate that opsin message in cones is restricted to the perinuclear region, whereas in rods, it is both perinuclear and adjacent to the ellipsoid.

Otx5 regulates genes that show circadian expression in the zebrafish pineal complex

The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus—the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. Our results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex.

Germinal cells in the goldfish retina that produce rod photoreceptors

Dividing cells and their progeny in retinae of young goldfish were labeled with [3H]thymidine, and selected cells were reconstructed from serial sections processed for electron microscopic autoradiography. Our goals were to characterize the cells that were identified as rod precursors in previous light microscopic autoradiographical studies and to determine their origin and fate. (In fish the population of rods increases several-fold postembryonically by proliferation of rod precursor cells scattered across the retina). Over 200 labeled cells taken from 11 retinas were examined, and 20 of these were reconstructed in their entirety. Some retinas were examined at short intervals (1 to 48 hr) after [3H]thymidine injection in order to study mitotically active cells, and others were examined after longer intervals (9 or 14 days) to discover the nature of the progeny of labeled dividing cells. Previous evidence from thymidine studies in larval goldfish suggested that proliferating cells destined to produce rods appear first in the inner nuclear layer and later in the outer nuclear layer, where they continue to divide and generate new rods (P.R. Johns, (1982) J. Neurosci. 2, 179). The present results provide morphological evidence in support of the suggestion that rod precursors migrate from inner to outer nuclear layer and, furthermore, show that the precursors are closely associated with, and perhaps guided by, the radial processes of Müller glial cells. Examination of EM autoradiographs of labeled cells at 9 and 14 days after a pulse label with thymidine confirms that the differentiated progeny of dividing precursor cells are exclusively rods. To our knowledge, rod precursors are the first example of a neuronal germinal cell in the vertebrate central nervous system that under normal conditions produces only one type of neuron.

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.

Development of the visual system

Many aspects of the retinal structure and visual function of fishes are typical of all vertebrates. Colour vision, based on the presence of multiple primary cone pigments (Marks, 1965) and on the colour-opponent organization of retinal ganglion cells (Wagner et al., 1960), is perhaps the best known example. While research on such typical properties has contributed greatly to our understanding of the neural basis of vertebrate vision, other properties deserve equal interest because they are not typical. The development of the visual system is an example of this kind of property.

Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish

In a microarray-based gene profiling analysis of Müller glia-derived retinal stem cells in light-damaged retinas from adult zebrafish, we found that 2 genes required for regeneration of fin and heart tissues in zebrafish, hspd1 (heat shock 60-kDa protein 1) and mps1 (monopolar spindle 1), were up-regulated. Expression of both genes in the neurogenic Müller glia and progenitors was independently verified by quantitative reverse transcriptase PCR and in situ hybridization. Functional analysis of temperature-sensitive mutants of hspd1 and mps1 revealed that both are necessary for Müller glia-based cone photoreceptor regeneration in adult zebrafish retina. In the amputated fin, hspd1 is required for the induction of mesenchymal stem cells and blastema formation, whereas mps1 is required at a later step for rapid cell proliferation and outgrowth. This temporal sequence of hspd1 and mps1 function is conserved in the regenerating retina. Comparison of gene expression profiles from regenerating zebrafish retina, caudal fin, and heart muscle revealed additional candidate genes potentially implicated in injury-induced epimorphic regeneration in diverse zebrafish tissues.