NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish.

neuroD is a member of the family of proneural genes, which function to regulate the cell cycle, cell fate determination and cellular differentiation. In the retinas of larval and adult teleosts, neuroD is expressed in two populations of post-mitotic cells, a subset of amacrine cells and nascent cone photoreceptors, and proliferating cells in the lineages that give rise exclusively to rod and cone photoreceptors. Based on previous studies of NeuroD function in vitro and the cellular pattern of neuroD expression in the zebrafish retina, we hypothesized that within the mitotic photoreceptor lineages NeuroD selectively regulates aspects of the cell cycle. To test this hypothesis, gain and loss-of-function approaches were employed, relying on the inducible expression of a NeuroD(EGFP) fusion protein and morpholino oligonucleotides to inhibit protein translation, respectively. Conditional expression of neuroD causes cells to withdraw from the cell cycle, upregulate the expression of the cell cycle inhibitors, p27 and p57, and downregulate the cell cycle progression factors, Cyclin B1, Cyclin D1, and Cyclin E2. In the absence of NeuroD, cells specific for the rod and cone photoreceptor lineage fail to exit the cell cycle, and the number of cells expressing Cyclin D1 is increased. When expression is ectopically induced in multipotent progenitors, neuroD promotes the genesis of rod photoreceptors and inhibits the genesis of Müller glia. These data show that in the teleost retina NeuroD plays a fundamental role in photoreceptor genesis by regulating mechanisms that promote rod and cone progenitors to withdraw from the cell cycle. This is the first in vivo demonstration in the retina of cell cycle regulation by NeuroD.

Persistent neurogenesis in the teleost retina: evidence for regulation by the growth-hormone/insulin-like growth factor-I axis.

Based on results from previous studies (J. Comp. Neurol. 394 (1998) 386, 395), it was hypothesized that the persistent neurogenesis in the retina of teleost fish is modulated by insulin-like growth factor-I (IGF-I), which, in turn, is regulated by growth hormone (GH). Two approaches were undertaken to test this hypothesis. First, a variety of techniques were used to determine if IGF-I and the IGF-I receptor (IGF-IR) are expressed in the retina. Second, GH was injected into animals to stimulate IGF-I synthesis in target tissues, and IGF-I expression and cell proliferation in the retina was quantitatively assayed. Reverse transcriptase-polymerase chain reaction, screening a retinal cDNA library and Northern analysis showed that genes encoding IGF-I and IGF-IR are expressed in the retina of goldfish. In situ hybridization showed that IGF-IR is expressed by retinal progenitors and all differentiated retinal neurons. Intraperitoneal injections of GH elevate IGF-I mRNA levels in the liver, brain and retina and produce a dose-dependent change in the proliferation of stem cells and progenitors in the retina. These data indicate that the principal components of the IGF-I signaling cascade are present in the retinas of teleosts, and we suggest these elements mediate the persistent, growth-associated neurogenesis in this tissue.

Expression of the insulin receptor in the retina of the goldfish.

PURPOSE: Insulin is a peptide growth factor that is active in most tissues, both during development and in adulthood. The action of insulin is through its specific membrane receptor. Previously retinal progenitors in the adult goldfish were shown to proliferate vigorously when exposed to insulin in vitro.(1) The present study was undertaken to clone and characterize partial cDNAs that encode the goldfish's insulin receptor (IR) and to establish the cellular pattern of expression of this gene in the retina. METHODS: Standard methods were used for RNA isolation, reverse transcription-polymerase chain reaction, Northern blot analysis, and in situ hybridization. RESULTS: Multiple clones were isolated that, based on sequence analysis, segregated into two groups, presumed to represent two genes that encode the IR. These clones were designated goldfish IR-1 (gfIR-1) and goldfish IR-2 (gfIR-2). Northern blot analysis showed that both genes are expressed in multiple tissues, including the retina. Both gfIR-1 and -2 give rise to a single, major transcript, but the sizes of the two transcripts are different. In situ hybridizations using digoxygenin-labeled riboprobes showed that gfIR-1 and -2 are expressed by all differentiated retinal neurons as well as neuronal progenitors in the circumferential germinal zone. CONCLUSIONS: These data demonstrate that the IR is expressed in the retina of the goldfish, and, on the basis of the cellular pattern of expression, suggest that insulin may act both to regulate neurogenesis and influence the function of differentiated neurons. The cellular coexpression of the receptors for both insulin-like growth factor (IGF) 1 and insulin suggests that neurons and/or neuronal progenitors in the retina of the goldfish may contain hybrid IGF-1/insulin receptors.

Putative stem cells and the lineage of rod photoreceptors in the mature retina of the goldfish.

The retinas of teleost fish grow continuously, in part, by neuronal hyperplasia and when lesioned will regenerate. Within the differentiated retina, the growth-associated hyperplasia results in the generation of new rod photoreceptors only, whereas injury-induced neurogenesis results in the regeneration of all retinal cell types. It is believed, however, that both new rod photoreceptors and regenerated neurons originate from the same populations of intrinsic progenitors. Experiments are described here that attempt to identify in the normal retina of goldfish neuronal progenitors intrinsic to the retina, particularly those which have remained cryptic because they divide infrequently. Long-term, systemic exposure to bromodeoxyuridine (BrdU) was used to label these cells. Five populations of proliferative cells were labeled: microglia, which are briefly described but not studied further; retinal progenitors in the circumferential germinal zone (CGZ); and rod precursors in the outer nuclear layer (ONL), both of which have been well characterized previously; and two populations of slowly-dividing cells in the inner nuclear layer (INL). The majority of these cells have a fusiform morphology, whereas the remaining ones are spherical. Longitudinal BrdU labeling suggests that the fusiform cells migrate to the ONL to replenish the pool of rod precursors. A subset of the spherical cells express pax6, although none are stained with markers of differentiated amacrine or bipolar cells. It is hypothesized that these rare, pax6-expressing cells are retinal stem cells, which give rise to the pax6-negative fusiform cells. Based on these data, two models are proposed: the first describes the lineage of rod photoreceptors in goldfish; the second is a consensus model of neurogenesis in the retinas of all teleosts.

Calcium channel beta 4 (CACNB4): human ortholog of the mouse epilepsy gene lethargic.

The mouse neurological mutant lethargic (lh) is characterized by ataxia, focal myoclonus, and absence epilepsy due to a loss-of-function mutation in the beta4 subunit of the voltage-gated calcium channel. To evaluate the role of this channel subunit in human neurological disease, we determined the chromosomal location and intron/exon structure of the human CACNB4 gene. The 1560-bp open reading frame of the CACNB4 cDNA predicts a 58-kDa protein with an amino acid sequence that is 99% identical to the rat protein. The 13 coding exons of CACNB4 span >55 kb of genomic DNA. Human cerebellar RNA contains one major CACNB4 transcript that is 9 kb in length. Expression of CACNB4 was detected in cerebellum, kidney, testis, retina, lymphoblasts, and circulating lymphocytes. Retinal transcripts were localized by in situ hybridization to ganglion cells and the inner nuclear layer. Analysis of the GeneBridge 4 radiation hybrid mapping panel localized CACNB4 to position 791 cR on human chromosome 2, in a conserved linkage group on human 2q22-q31 and mouse chromosome 2. We localized CACNB4 to the 1.3-Mb YAC clone 952F10 in Whitehead contig WC861, along with the polymorphic markers D2S2236 and D2S2299. The chromosomal linkage of three of the four beta subunit genes to homeobox gene clusters associates the evolutionary origin of the beta gene family with the events that generated the four HOX clusters early in vertebrate evolution.

Insulin-related growth factors stimulate proliferation of retinal progenitors in the goldfish.

The retina of the adult goldfish grows throughout the life of the animal, in part, by the continual addition of new neurons. Further, destruction of extant neurons in this tissue stimulates neuronal regeneration. In an attempt to identify growth factors that regulate both normal and injury-stimulated neurogenesis, we used organ culture techniques and tested nine peptide growth factors for their ability to modulate cell proliferation in both normal retinas and retinas with lesions. Of the growth factors tested, only the insulin-related peptides (insulin and insulin-like growth factors I and II) consistently stimulated proliferation, and this was restricted to the retinal progenitors within the circumferential germinal zone. None of the growth factors tested stimulated proliferation of rod precursors (cells in the mature retina whose progeny are exclusively rod photoreceptors) or the injury-stimulated retinal progenitors. Although the negative data are subject to multiple interpretations, these data suggest that in the retina of the adult goldfish, insulin-related peptides regulate proliferation of retinal progenitors within the circumferential germinal zone, but molecules that modulate the proliferation of the rod precursors or injury-induced retinal progenitors in the retina of the adult goldfish have yet to be identified.

Insulin-like growth factor-I binds in the inner plexiform layer and circumferential germinal zone in the retina of the goldfish.

Results of the previous study suggest that insulin-related peptides regulate proliferation of retinal progenitors in the adult goldfish. Because of their known roles in retinal neurogenesis, we have chosen to focus future studies on insulin-like growth factor I (IGF-I) and the IGF-I receptor. In the study described here, we characterized the spatial distribution and specificity of IGF-I binding sites in the retina of the adult goldfish by performing receptor-binding autoradiography with [125I]-IGF-I alone and with unlabeled IGF-I-related molecules (IGF-I, IGF-II, insulin, and des-[1-3]-IGF-I) as competitive inhibitors of [125I]-IGF-I binding. The results of these experiments show that IGF-I binds in two locations in the retina of the adult goldfish, within the inner plexiform layer of the differentiated retina and the circumferential germinal zone. The competition experiments suggest that [125I]-IGF-I binds at sites specific for IGF-I, and that both IGF-I receptors and IGF-I binding proteins are present in the retina.

Pax2 expression and retinal morphogenesis in the normal and Krd mouse.

The Kidney and retinal defects (Krd) mouse carries a 7-cM transgene-induced deletion on chromosome 19 that includes the Pax2 locus. Adult mice heterozygous for the Krd deletion (Krd/+) are haploid for Pax2 and have a variable, semidominant phenotype characterized by structural defects of the kidney, retina, and optic disc. Renal and ocular anomalies present in heterozygous Pax2 mutants in both mice and humans support the hypothesis that haploinsufficiency of Pax2 underlies the Krd phenotype. To understand the embryonic basis of ocular defects observed in adult Krd/+ mice, we used immunohistochemistry, digital three-dimensional reconstructions, and quantitative morphometry to examine Pax2 protein distribution and ocular development in normal and Krd/+ mice from E10.5 to P2. In +/+ embryos, Pax2 immunopositive (Pax2+) cells demarcate the embryonic fissure as it forms in the ventral optic cup and optic stalk. After closure of the embryonic fissure, Pax2 immunostaining disappears from the ventral retina, but persists in a cuff of cells encircling the developing optic disc, the site where ganglion cell axons exit the retina. In Krd/+ embryos, Pax2+ cells in the posterior optic cup and the optic stalk undergo abnormal morphogenetic movements and the embryonic fissure fails to form normally. This results in an abnormal organization of the Pax2+ cells and ganglion cell axons at the nascent optic disc. The abnormal morphogenetic movements of the Pax2+ cells in the embryonic retina and optic stalk and the initial misrouting of the ganglion cell axons give rise to retinal and optic disc defects observed in the adult Krd/+ mice.

Vsx-1 and Vsx-2: two Chx10-like homeobox genes expressed in overlapping domains in the adult goldfish retina.

The genetic linkages of the murine ocular retardation mutation with the Chx10 gene and the murine small eye mutation with the Pax-6 gene has demonstrated the importance of Paired class homeobox genes in the development of the mammalian retina. Previously, we identified a Paired-class homeobox gene, Vsx-1, whose expression in the adult goldfish retina is restricted to the inner nuclear layer (INL) and to postmitotic, differentiating progenitor cells in the growth zone at the retinal peripheral margin, where neurogenesis continues throughout life. Here, we report the molecular cloning and expression pattern of a new Paired class homeobox gene, Vsx-2, in the adult goldfish retina. Like Vsx-1, Vsx-2 expression is highly restricted to the retina in the adult goldfish and overlaps with Vsx-1 expression in the mature INL. At the peripheral margin, Vsx-2 is expressed in mitotically active neuronal progenitors and is downregulated as these cells become postmitotic and begin to differentiate. Comparison of the amino acid sequences of Vsx-2, Vsx-1, Chx10, and C. elegans ceh-10 reveal a conserved homeodomain and a unique domain termed the CVC domain. The similarities of the Vsx-2, Vsx-1, and Chx10 expression patterns suggest that genes containing the CVC domain have conserved functions during retinal development in vertebrates.

Tracer coupling among regenerated amacrine cells in the retina of the goldfish.

This study sought to characterize the tracer coupling of regenerated amacrine cells in the retina of the goldfish and assess the integration of regenerated neurons into existing retinal circuits. Regeneration of new neurons from injury-induced progenitors was stimulated by surgically excising a small rectangular piece of retina. Several months after regeneration was complete, intracellular injections of Neurobiotin, a gap junction-permeant tracer, were made into single regenerated amacrine cells or nonregenerated (extant) amacrine cells lying outside the regenerated patch. Two groups of amacrine cells were injected: those that in normal retina are tracer coupled and a single type (the radiate amacrine cell) that is not. The data show that regenerated amacrine cells are tracer coupled to each other and to their homologous counterparts outside the patch of regenerated retina. Regenerated radiate cells possess morphologically abnormal dendrites, but these processes can extend out of regenerated retina into surrounding normal retina. Similarly, the dendrites of extant radiate cells, severed by the original lesion, can regenerate into the patch of regenerated retina. These results indicate that in the goldfish retina the cell-specific junctional circuitry present in normal retina is re-created in the regenerated retina, and suggest that regenerated neurons are functionally integrated into the existing retina.

Electrophysiology and density of retinal neurons in mice with a mutation that includes the Pax2 locus.

PURPOSE: The Krd mouse has a deletion in chromosome 19 that includes the Pax2 gene locus. The aim of this study was to characterize in detail how these retinas differ from normal. METHODS: Both electroretinographic and anatomic methods were used to assess visual function. Full-field flash electroretinograms (ERGs) and planimetric densities were obtained from Krd and control animals. RESULTS: Measurements of the ERG show that in the Krd mice, both a- and b-wave amplitudes are attenuated relative to control by amounts that vary from animal to animal. The b-wave of the ERG generally is affected more severely than the a-wave. However, there is little or no shift of the curves relating the b-wave and a-wave amplitude to the intensity of the stimulus. Also, no change in the response kinetics seems to be associated with the attenuated responses. Estimates of planimetric cell density in the outer nuclear, inner nuclear, and ganglion cell layers show significant cell losses in affected animals that are more pronounced proportionally in the inner layers. Comparisons between electrophysiological and histologic measurements made on each eye show good correlation between the reduction in the ERG components and the magnitude of cell losses. CONCLUSIONS: These experiments show that the eyes of Krd mice have reduced ERGs and reduced cellular density. There is a loss of cells in all layers of the retina, but the inner layers are affected more severely. Consistent with this, the b-wave is reduced more than the a-wave. The normal functional dependency of the ERG on stimulus intensity and the normal response kinetics suggest the cellular losses are not associated with changes in cellular function.

Antibodies against Pax6 immunostain amacrine and ganglion cells and neuronal progenitors, but not rod precursors, in the normal and regenerating retina of the goldfish.

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration.

Expression of the bZIP transcription factor gene Nrl in the developing nervous system.

Proteins of the Maf/Nrl subfamily of bZIP transcription factors are involved in the regulation of tissue-specific gene expression. The Nrl gene, initially identified from a subtracted retinal library, is expressed in all cell layers of the adult retina, including photoreceptors. The Nrl protein has high sequence homology with Maf proteins, binds to an AP-1 like sequence element, and in photoreceptors appears to be involved in regulating the expression of rhodopsin. In the present study, we investigated the expression of Nrl in the developing and adult mouse using in situ hybridization and RT-PCR. We demonstrate that beginning at embryonic day 12.5 Nrl is expressed throughout the developing central and peripheral nervous system, with the exception of the nasal epithelium. The spatial pattern of hybridization suggests that Nrl is transcribed in post-mitotic, differentiating neurons, the developing cephalic mesenchyme and lens. Nrl expression is downregulated postnatally in the brain, and becomes restricted to neocortex and brainstem in the adult. High levels of Nrl transcripts, however, persist in the mature photoreceptors and other retinal neurons. Our studies suggest a role for the Nrl protein in neuronal differentiation and in mature neurons of the adult retina.

Plasticin, a newly identified neurofilament protein, is preferentially expressed in young retinal ganglion cells of adult goldfish.

The adult goldfish retina and optic nerve display continuous growth, plasticity, and the capacity to regenerate throughout the animal's life. The intermediate filament proteins in this pathway are different from those in adult mammalian nerves, which do not continuously grow or normally regenerate. One novel intermediate filament protein of the goldfish visual pathway is plasticin, which is synthesized in ganglion cells and transported into the optic nerve. Using specific polyclonal antibodies raised against a plasticin fusion protein, we investigated the distribution of this protein in the normal retina and nerve and in the retina and nerve following optic nerve crush. In the normal pathway, plasticin was localized predominantly to the axons of very young ganglion cells; however, there was considerable immunoreactivity in older axons as they approach the chiasm. In addition, following optic nerve crush, all ganglion cell somata and their axons proximal to the crush site became equally immunoreactive. The results suggest that plasticin may contribute to axonal growth, plasticity, and regeneration.

Restricted expression of a new paired-class homeobox gene in normal and regenerating adult goldfish retina.

We describe the cloning and expression pattern of a new paired-class homeobox gene, Vsx-1, in the continuously growing retina of the goldfish. Vsx-1 belongs to a subset of paired-class homeobox genes that lack a second DNA binding domain, the paired-domain, and is closely related to the C. elegans ceh-10 gene. In the adult goldfish, Vsx-1 expression is restricted to the neural retina. In the central, mature retina, Vsx-1 mRNA is synthesized in a subset of differentiated cells in the inner nuclear layer in a pattern suggestive of bipolar cells. In immature retina, adjacent to the retinal margin, Vsx-1 is expressed in a relatively broader subset of newly postmitotic cells but is downregulated in some of these cells to form the mature expression pattern. Following retinal injury, during the early phase of regeneration, Vsx-1 mRNA synthesis appears to be upregulated in cells in the inner nuclear layer and is expressed de novo in cells outside this layer. By virtue of its identity as a transcriptional regulatory gene and its patterns of expression, we speculate that Vsx-1 may stabilize the differentiated state of a subset of cells in the inner nuclear layer and may be involved in cellular differentiation during retinal development and regeneration.

Kidney and retinal defects (Krd), a transgene-induced mutation with a deletion of mouse chromosome 19 that includes the Pax2 locus.

The semidominant mutation Krd (kidney and retinal defects) was identified in transgenic line Tg8052. Krd/+ mice have a high incidence of kidney defects including aplastic, hypoplastic, and cystic kidneys. Retinal defects in Krd/+ mice include abnormal electroretinograms and a reduction of cell numbers that is most extreme in the inner cell and ganglion layers. Viability of Krd/+ mice is strongly influenced by genetic background, and growth retardation is observed in young animals. Homozygosity results in early embryonic lethality. Fluorescence in situ hybridization of a transgene-specific probe localized the insertion site to the distal region of mouse Chromosome 19. The sequence of the insertion site revealed transgene insertion into a LINE element with deletion of a single nucleotide from the 3' terminus of the transgene. A polymorphic microsatellite, D19Umi1, was identified in a junction clone and mapped in several large crosses. D19Umi1 is located 1.7 +/- 1.0 cM distal to Pax2, which encodes a paired type transcription factor expressed in embryonic kidney and eye. Deletion of Pax2 from the transgenic chromosome was demonstrated by Southern analysis of genomic DNA from (Krd/+ x SPRET/Ei)F1 mice. Additional genetic and molecular data are consistent with an approximately 7-cM deletion that includes the loci stearoyl CoA desaturase (Scd1), pale ear (ep), D19Mit17, D19Mit24, D19Mit27, D19Mit11, and Pax2. This deletion, Del(19)TgN8052Mm, will be useful for genetic and functional studies of this region of mouse Chromosome 19.

Synaptic organization of regenerated retina in the goldfish.

In the adult goldfish, any manipulation that significantly depletes retinal neurons stimulates neurogenesis and the regeneration of nearly normal retina. We sought to determine the extent to which the regenerated neurons formed normal synaptic connections. We used qualitative and quantitative electron microscopy to compare the organization of the synaptic layers in regenerated and normal retinas. In eight eyes, a small patch of retina was surgically excised, stimulating regeneration of new retina in its place. Animals were killed 16-20 weeks after surgery. Qualitative comparisons of the synaptic architecture of photoreceptor terminals in the outer plexiform layer and quantitative comparisons of the synaptic organization in the inner plexiform layer were made between the patch of regenerated retina and an adjacent intact site. In the regenerated outer plexiform layer, cone pedicles and rod spherules were not arranged as regularly as normal, but they formed normal-appearing synaptic contacts. In the regenerated inner plexiform layer, with one exception, the quantitative descriptors of the synaptic organization in the normal and regenerate were not significantly different: The planimetric and numerical densities of the synapses, number of synapses/inner retinal neuron, and, with the exception of the bipolar terminals in the inner plexiform layer, and synapse depth profiles were similar. These data suggest that 1) relatively normal synaptic connections are recreated during regeneration, 2) the cellular mechanisms that guide synaptogenesis during development act during retinal regeneration, and 3) the physiological response properties of regenerated neurons should be comparable to that found in the normal retina.

Regeneration of the dopamine-cell mosaic in the retina of the goldfish.

A fundamental anatomical feature of retinal neurons is that they form planar mosaics. Each mosaic can be described by its density, pattern, and regularity (non-randomness). As part of ongoing studies to quantitatively describe the anatomy of regenerated retina in the goldfish, we determined the planimetric density and regularity of the mosaic of dopaminergic interplexiform cells in patches of regenerated retina and compared this to the mosaic generated de novo. In addition, we selectively ablated dopaminergic neurons with the neurotoxin 6-hydroxydopamine (6-OHDA) before inducing local regeneration and determined whether or not the absence of the extant dopaminergic neurons modulated the planimetric density or number of regenerated ones. The results showed that dopaminergic neurons are regenerated at higher planimetric densities and in less orderly arrays than normal. Furthermore, there was no statistical difference in the density or number of regenerated cells in normal retinas and retinas treated with 6-OHDA.