The proliferative and apoptotic activities of E2F1 in the mouse retina.

The E2F1 transcription factor controls cell proliferation and apoptosis. E2F1 activity is negatively regulated by the retinoblastoma (RB) protein. To study how inactivation of Rb and dysregulated E2F1 affects the developing retina, we analysed wild-type and Rb(-/-) embryonic retinas and retinal transplants and we established transgenic mice expressing human E2F1 in retinal photoreceptor cells under the regulation of the IRBP promoter (TgIRBPE2F1). A marked increase in cell proliferation and apoptosis was observed in the retinas of Rb(-/-) mice and TgIRBPE2F1 transgenic mice. In the transgenic mice, photoreceptor cells formed rosette-like arrangements at postnatal days 9 through 28. Complete loss of photoreceptors followed in the TgIRBPE2F1 mice but not in the Rb(-/-) retinal transplants. Both RB-deficient and E2F1-overexpressing photoreceptor cells expressed rhodopsin, a marker of terminal differentiation. Loss of p53 partially reduced the apoptosis and resulted in transient hyperplasia of multiple cell types in the TgIRBPE2F1 retinas at postnatal day 6. Our findings support the concept that cross-talk occurs between different retinal cell types and that multiple genetic pathways must become dysregulated for the full oncogenic transformation of neuronal retinal cells.

Transgenic zebrafish for studying nervous system development and regeneration.

Alpha1 tubulin gene expression is induced in the developing and regenerating CNS of vertebrates. Therefore, alpha1 tubulin gene expression may serve as a good probe for mechanisms underlying CNS development and regeneration. One approach to identify these mechanisms is to work backwards from the genome. This requires identification of alpha1 tubulin DNA sequences that mediate its developmental and regeneration-dependent expression pattern. Therefore, we generated transgenic zebrafish harboring a fragment of the alpha1 tubulin gene driving green fluorescent protein expression (GFP). In these fish, and similar to the endogenous gene, transgene expression was dramatically induced in the developing and regenerating nervous system. Although transgene expression generally declined during maturation of the nervous system, robust GFP expression was maintained in progenitor cells in the retinal periphery, lining brain ventricles and surrounding the central canal of the spinal cord. When these cells were cultured in vitro they divided and gave rise to new neurons. We also show that optic nerve crush in adult fish re-induced transgene expression in retinal ganglion cells. These studies identified a relatively small region of the alpha1 tubulin promoter that mediates its regulated expression pattern in developing and adult fish. This promoter will be extremely useful to investigators interested in targeting gene expression to the developing or regenerating nervous system. As adult transgenic fish maintain transgene expression in neural progenitors, these fish also provide a valuable resource of labeled adult neural progenitor cells that can be studied in vivo or in vitro. Finally, these fish should provide a unique in vivo system for investigating mechanisms mediating CNS development and regeneration.

Partial rescue of the ocular retardation phenotype by genetic modifiers.

The or(J) allele of the murine ocular retardation mutation is caused by a premature stop codon in the homeodomain of the Chx10 gene. When expressed on an inbred 129/Sv strain, the or(J) phenotype is characterized by microphthalmia and a thin, poorly differentiated retina in which the peripheral portion is affected to a greater extent than the central portion. Such mutant retinae lack differentiated bipolar cells and the optic nerve typically fails to form, leading to blindness. Here, we show that progeny from an outcrossed backcross between 129/Sv-or(J) /or(J) and Mus musculus castaneus produce animals that are homozygous for the or(J) mutation and exhibit a much ameliorated eye phenotype. Although not of normal size, such modified or(J) eyes are significantly larger than those in 129/Sv-or(J) /or(J) mice, and contain a better organized retina which includes bipolar cells. Furthermore, optic nerves are frequently present, and the eyes show a degree of function as reflected by electroretinogram and pupillary response. As in 129/Sv-or(J) /or(J) mice, however, modified or(J) eyes show incomplete growth and a lack of cell differentiation in the periphery of the retina. The selective, and apparently nonmodifiable, effect of the ocular retardation phenotype on the periphery of the retina indicates that Chx10 plays an important role in the central-to-peripheral gradient of retinal development. These findings demonstrate that the ocular retardation phenotype can be greatly modified by the genetic background, and help to define a role for Chx10 in ocular development.

Insulin stimulates pp120 endocytosis in cells co-expressing insulin receptors.

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein that is expressed in the hepatocyte as two spliced isoforms differing by the presence (full-length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Co-expression of full-length pp120, but not its phosphorylation-defective isoforms, increased receptor-mediated insulin endocytosis and degradation in NIH 3T3 fibroblasts. We, herein, examined whether internalization of pp120 is required to mediate its effect on insulin endocytosis. The amount of full-length pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were co-expressed. In contrast, when phosphorylation-defective pp120 mutants were co-expressed, the amount of pp120 expressed at the cell surface did not decrease in response to insulin. Indirect immunofluorescence analysis revealed that upon insulin treatment of cells co-expressing insulin receptors, full-length, but not truncated, pp120 co-localized with alpha-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrin-coated pits. This suggests that full-length pp120 is part of a complex of proteins required for receptor-mediated insulin endocytosis and that formation of this complex is regulated by insulin-induced pp120 phosphorylation by the receptor tyrosine kinase. In vitro GST binding assays and co-immunoprecipitation experiments in intact cells further revealed that pp120 did not bind directly to the insulin receptor and that its association with the receptor may be mediated by other cellular proteins.

Maturational changes in cell surface antigen expression in the mouse retina and optic pathway.

The distribution of the cell surface molecules M6 and L1 was studied using the immunohistochemistry and in situ hybridization in the developing and adult mouse retina and optic nerve. L1 is a cell adhesion molecule while M6 is a cell surface molecule homologous to the myelin protein proteolipid protein (PLP/DM20). Although both molecules were expressed in retina and optic nerves of embryonic and neonatal mice, our studies show that their patterns of postnatal expression are quite different. While L1 continues to be expressed in optic axons throughout adulthood, expression of M6 on optic axons declines after birth and instead becomes strongly expressed on Müller glial endfeet and in the inner plexiform layer. The modulation of these molecules after birth could provide clues to changing cell-cell interactions occurring in the proximal portion of the optic pathway.

Axon-target interactions maintain synaptic gene expression in retinae transplanted to intracranial regions of the rat.

In the present study we examined the effects of optic axon-CNS target interactions on gene expression in the rat retina. These studies took advantage of a transplantation paradigm that allowed us to assay gene expression in retinae transplanted to different intracranial locations in the neonatal rat that either promoted (dorsal midbrain) or precluded (cerebral cortex) the formation of retino-collicular connections. Using in situ hybridization experiments, we observed that transplantation to the dorsal midbrain resulted in a relatively normal pattern of nicotinic acetylcholine receptor (nAChR) beta-3 subunit and glutamate receptor 3 (GluR3) gene expression. In contrast, retinae transplanted to the cerebral cortex (which did not result in normal retino-collicular interactions) showed a dramatic reduction in nAChR beta-3 subunit and GluR3 gene expression. These results agree with those obtained in the adult goldfish retina, where it has been demonstrated that an optic nerve-optic tectum interaction is responsible for the re-induction nAChR and NMDA receptor gene expression during optic nerve regeneration. Taken together, these results support the hypothesis that proper axon-target interactions are required for maintenance of nAChR and glutamate receptor gene expression in the mature vertebrate retina.

Opsin gene expression and regulation in retinal transplants.

In the present study we investigated the expression and regulation of the opsin gene in retinal transplants. Embryonic retinae were transplanted to intracranial locations in neonatal rodents in which they either reliably projected to the superior colliculus, or in locations (such as the cerebral cortex) in which they did not project to subcortical visual nuclei. Our results show that, regardless of the graft location, the developmental schedule of opsin gene expression in the outer nuclear layer was similar to normal, and that it was maintained in transplants for at least 6 months. To test if ambient light affected opsin gene expression, we dark-reared rats containing a retinal transplant for up to 26 days before assaying for opsin transcripts. In situ hybridization experiments showed that opsin gene expression in the transplants of these dark-reared recipients was not different either from transplants in animals reared in cyclic light conditions, or from the retina in situ. These observations support the hypothesis that the opsin gene is activated and maintained by molecular mechanisms intrinsic to the photoreceptor.

Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation.

Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

Pathfinding by retinal ganglion cell axons: transplantation studies in genetically and surgically blind mice.

Optic axons show a highly stereotypical intracranial course to attain the visual centers of the brainstem. Here we examine the course followed by axons arising from embryonic retinae implanted in neonatal ocular retardation mutant mice in which there had been no prior innervation of the visual centers. Retinae placed on the ventrolateral brainstem adjacent to the normal site of the optic tract send axons dorsolaterally toward the ipsilateral superior colliculus, which they innervate along with a number of other subcortical visual centers. Somewhat unexpectedly, axons also course ventrally to cross at the level of the suprachiasmatic nucleus or, less frequently, caudal to the mammillary body to follow the route of the optic tract and innervate contralateral visual centers. Retinae implanted along the course of the internal capsule emit axons that follow projection fibers through the striatum to innervate the lateral geniculate nucleus and other optic nuclei. These grafts also appear to project to the lateral part of the ventrobasal nucleus of the thalamus. The results show that prior existence of an optic projection is not necessary for axons derived from ectopic retinae to attain visual nuclei, not only on the side of implantation but also on the contralateral side of the brain. The cues that these growing axons follow appear to be stable temporally. The fact that axons can also follow highly anomalous routes, such as through the internal capsule, to attain target nuclei in the brainstem suggests that the normal optic pathway is not an obligatory route for optic outgrowth.

Patterning of the neocortical projections from the raphe nuclei in perinatal rats: investigation of potential organizational mechanisms.

Serotoninergic (5-HT) fibers in the cerebral cortex of perinatal rats have a pattern that coincides with the boundaries of primary sensory areas and within the primary somatosensory cortex form the rattunculus. This patterned immunoreactivity (IR) appears about 60 hours after birth and disappears between postnatal days (P-) 12 and 15. Three experiments were carried out to evaluate mechanisms that might underlie the precise patterning of the 5-HT-IR. Retrograde labelling with fluorescent tracers in perinatal rats revealed only a coarse rostrocaudal topography in the raphe-cortical projection and the existence of raphe cells projecting to multiple cortical locations. Thus, a precise point-to-point, raphe-cortical projection does not underlie the patterned cortical 5-HT-IR. Ablation of the thalamus prior to the age at which patterned 5-HT-IR could be seen in the developing cortex caused a complete loss of patterned immunoreactivity. This suggests that 5-HT fibers may require the presence of thalamocortical axons to achieve the pattern observed in normal animals. Serotoninergic raphe neurons transplanted to the cortices of newborn rats exhibited extensive axonal outgrowth, but did not form a somatotopic pattern. This result also suggests that specific spatiotemporal interactions between growing 5-HT and thalamocortical axons may be necessary for the somatotopic patterning of the former fibers.

Cell adhesion molecules in the early developing mouse retina: retinal neurons show preferential outgrowth in vitro on L1 but not N-CAM.

Both L1 and N-CAM are present on optic axons early in the developing mouse retina and optic nerve. In in vitro assays on substrates of purified cell adhesion molecules cells derived from E13 mouse retinae showed vigorous neurite extension on L1 but not on N-CAM. Although retinal neurons on N-CAM showed only limited attachment to the substrate, they were able to form lamellipodia immediately around the cell perimeter. In contrast, similarly derived cortical cells showed extensive neurite outgrowth on both substrates. Under these culture conditions, nearly all of the L1 and N-CAM present in the cell membrane appeared to be sequestered on the lower surface of the growth cones and neurites, indicating that most of these cell adhesion molecules were involved in homophilic interactions. Our results suggest differential roles for L1 and N-CAM in initiation and establishment of the optic pathway.

Transient outgrowth from retinae implanted in the neonatal rat cerebral cortex.

Retinae placed in the cortex of neonatal rodents show substantial outgrowth, both along the subpial margin and within the cortical white matter, which is not sustained beyond 6 days post-transplantation. These results, in combination with previous studies, suggest that optic axon outgrowth is sustained for a finite period, after which it requires target-derived influences for its long-term maintenance.

Cues intrinsic to the retina induce nAChR gene expression during development.

Recent studies of optic nerve regeneration in goldfish have indicated that the optic tectum plays an important role in modulating the induction of nicotinic acetylcholine receptor (nAChR) gene expression in regenerating retinal ganglion cells (Heiber, Agranoff, and Goldman, 1992, J. Neurochem. 58:1009-1015). These observations suggest that induction of these genes is regulated by brain target regions. The appearance of nAChR mRNA in the developing rat retina coincides with a time when ganglion cells are sending axons to their brain targets (Hoover and Goldman, 1992, Exp. Eye Res. 54:561-571). Might a mechanism similar to that seen during goldfish optic nerve regeneration also mediate induction of nAChR gene expression during development of the mammalian retina? This possibility was tested by either transplanting embryonic rat retina to different brain regions, or explanting it to organ culture and assaying for nAChR gene expression. These studies showed that induction of the nAChR genes in developing rat retina is independent of the environment in which the retina develops. These results indicate that either the retinal microenvironment or a signal intrinsic to the retinal ganglion cell is responsible for this induction.

Retinal transplants in congenitally blind mice: patterns of projection and synaptic connectivity.

Embryonic retinae were transplanted onto the midbrain of neonatal congenitally anophthalmic mice and neonatal mice from which both eyes had been removed. When donor mice of the AKR strain were used, the detailed patterns of the transplant projections to the host brain were demonstrated with an antibody to Thy-1.1, which specifically stains neural tissue derived from AKR donors. Many of the subcortical visual centers were innervated, and only small differences were encountered between anophthalmic and eye-enucleated mice. The terminal arbors of transplant-derived axons could not be classified as in normal animals, although several distinct arbor types were seen. In the superior colliculus, the laminar arrangements that characterize normal retinal arbors were disrupted. Despite this, the synaptic patterns formed by transplant-derived axons in the superior colliculus of anophthalmic mice compared very closely with those of retinal axons in normal, sighted animals. These observations indicate that the ability of a retinal transplant to innervate the host brain and to form the synaptic arrays characteristic of optic terminals are not dependent on prior innervation, nor do they appear to be influenced by the events that follow eye removal.

How do retinal axons find their targets in the developing brain?

Tissue culture studies show that cell survival and process outgrowth from retinal ganglion cells depend on the molecular composition of the substrates over which the neurites grow, and on diffusible factors present in the medium. Recent work has begun to show that at least some of these components might be interactive. Since the conditions in a culture dish, as well as the patterns of antigen expression on cells in vitro, can differ considerably from those encountered in vivo, it is important to design experiments in vivo that examine how growing neurites relate to their natural microenvironment. By the use of transplantation techniques, it has been possible to provide evidence for a comparable duality of substrate-dependent and target-derived controls of optic axon growth, which might provide insight into the normal developmental process.

Directed early axonal outgrowth from retinal transplants into host rat brains.

Axons from retinae transplanted to the brain stem of neonatal rats exhibit two patterns of outgrowth that can be experimentally uncoupled from each other depending upon the location of the graft. Retinae placed close to the surface of the rostral brain stem (as much as 5 mm from the tectum) emit axons that project toward the superior colliculus along the subpial margin of the rostral brain stem. In contrast, axons from grafts embedded deep within the midbrain parenchyma project through the neuropil directly to the overlying superior colliculus, as long as the retina is within about 1 mm of the tectal surface. The present study shows that, as long as the retina is located outside the superior colliculus, and regardless of whether the axons derive from grafts in subpial or intraparenchymal locations, the earliest projections are oriented towards the superior colliculus. We have also found, however, that axons from retinae transplanted directly onto the superior colliculus can form projections that extend along the subpial margin away from the tectum. There are several major conclusions that may be drawn from these observations. First, the final tectopetal, transplant-derived projection does not result from the reorganization of an initially random outgrowth but is directed from the start toward an appropriate region of termination. Second, it appears that the interaction of retinal axons with a primary target alters the ability of the growth cone to respond to directional cues along the optic tract. Thus, although adding support to the proposal that optic axons attain the superior colliculus through an interaction involving substrates distributed along the optic tract and diffusible factors originating in the target region, it is increasingly clear that such interactions are likely to be complex and hierarchical.

Fetal olfactory bulb transplants send projections to host olfactory cortex in the rat.

We are using the rat olfactory system to study developmental details of neurotransplantation. Tritiated [3H]thymidine-labeled fetal olfactory bulbs (OBs), were transplanted immediately into sites from which the neonatal host OB was removed. Subsequently, a small lesion was placed in the region of the transplanted OB and the tissue studied, using degeneration methods and autoradiography. Only OB's with extensive [3H]-label and precise lesions confined to the labeled areas were used. Degeneration was found mainly in the ipsilateral piriform cortex with lesser amounts at other nearby sites. The results demonstrate successfully transplanted donor OBs that send axons to specific and appropriate target areas of the host brain.

Proximity as a factor in the innervation of host brain regions by retinal transplants.

Embryonic mouse retinae transplanted to a variety of locations within the rostral midbrain of neonatal rats exhibit selective innervation of host visual nuclei when studied at maturity. Some of these nuclei (superior colliculus, nucleus of the optic tract, dorsal terminal nucleus) usually receive extensive transplant projections, others are innervated partially (dorsal division of the lateral geniculate nucleus, olivary pretectal nucleus, medial terminal nucleus), while a few (ventral division of the lateral geniculate nucleus, suprachiasmatic nucleus, intergeniculate leaflet) are not innervated at all. The selectivity of this innervation is largely independent of the transplant's position within the rostral brainstem, while the density of innervation of individual nuclei depends in part upon the proximity of the transplant to the nucleus and upon whether the host retinal projection to that nucleus is present or absent. These findings provide a foundation for further studies of the behavioral capabilities of retinal transplants, for developmental studies of factors responsible for the establishment of normal neural projections, and for examination of the immunological consequences of transplantation.

Induction of target-directed optic axon outgrowth: effect of retinae transplanted to anophthalmic mice.

In previous work using neural transplants (Hankin and Lund, 1987) we demonstrated two basic components of optic axon outgrowth in the mammalian retinotectal system: one category of outgrowth utilizes the subpial margin of the rostral brain stem as a preferential substrate (as do normal retinotectal axons); the other type of outgrowth, from retinae embedded deep within the midbrain parenchyma, is distance-dependent and highly target-oriented, but shows little apparent substrate specificity. One explanation for this directed outgrowth is that it is in response to a diffusible factor emanating from cells in the superior colliculus. In the present study we use congenitally anophthalmic mice as recipients for retinal transplants to test whether prior optic innervation of the superior colliculus plays a role in establishing either component of outgrowth. We show that outgrowth along the subpial pathway from a graft placed on the surface of the brain stem can take place in the absence of prior innervation of the superior colliculus. The target-directed outgrowth exhibited by embedded grafts only occurs if the tectum is also innervated by a second graft placed on the surface of the brain stem. It is proposed that tectal cells produce a factor in response to optic innervation and that this directs the growth patterns of embedded grafts. This suggests that optic innervation is a necessary prerequisite for the superior colliculus to produce the proposed diffusible chemotropic signal. In normal development such a factor could function to improve the efficiency of target-finding by later growing optic axons, but it might serve a quite different role, encouraging branching and trophic maintenance of the optic pathway once it has reached the tectum.