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.

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.

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.

Development of intersecting CNS fiber tracts: the corpus callosum and its perforating fiber pathway.

What are the mechanisms acting during development at points of intersection of central nervous system fiber tracts which influence the direction taken by a population of growing axons? In order to address this question, the ontogeny of the intersecting rostral corpus callosum and its perforating fiber pathway (PF), and the microenvironment through which these fiber systems grow, were examined in a series of mouse embryos and early postnates. Our results show that the perforating fibers are identifiable in silver-stained sections between embryonic days (E) 15 and 16, at least 1 day prior to the initial appearance of the callosal projection. Soon after the PF can be identified, a dense accumulation of subventricular cells surrounds the PF at a point just ventral to the location where the callosum and PF will intersect (i.e., at the corticoseptal boundary). Callosal axons, which are present at the point of intersection beginning on E17, do not joint the perforating fibers, nor do they appear to penetrate the underlying population of subventricular cells. Instead, the callosal fibers turn across the PF and enter the contralateral cerebral hemisphere. Thus, the intersection of the callosal and perforating fiber systems during development may be related both to the sequential development of each pathway and to the altered nonneuronal environment at the point of intersection.

Death of the subcallosal glial sling is correlated with formation of the cavum septi pellucidi.

In this study we have examined the developmental fate of a population of cells that is located beneath the rostral corpus callosum during the perinatal period. These cells form a distinct slinglike structure along the geographically defined corticoseptal boundary (CSB) and may play a role in guiding callosal axons across the midline. The sling is a transient structure present in fetal and neonatal animals but not in adults. Here we show that the CSB cells die and that this debris is removed by macrophages. The sequence of cell degeneration in the CSB is highly stereotyped and follows a spatiotemporal pattern that is correlated with fusion of the cerebral hemispheres and subsequent growth across the midline of the callosal axons. The subcallosal location of the resorbing CSB is found in the exact place in which a fluid-filled cavity (the cavum septi pellucidi) is transiently found during the perinatal period. The tight temporal and spatial correlation between callosal axon decussation, degeneration of the CSB, and cavum septi formation suggests that these three phenomena may be causally related.

Conditions for optic axon outgrowth.

A series of studies is described which show that retinal axon outgrowth can be stimulated by the close proximity of an appropriate target region. The suggestion is offered that, in normal development, optic axons first follow cues available at the surface of the brainstem, but once they reach a target region, ramify there in response to the presence of a target-derived factor.

Role of the target in directing the outgrowth of retinal axons: transplants reveal surface-related and surface-independent cues.

In the present investigation we have examined whether retinal axons can be directed to the superior colliculus via an alternate route when they do not have access to their normal substrates along the optic tract. To address this issue we transplanted embryonic mouse retinae into the midbrain parenchyma and to various positions around the outer surface of the midbrain of newborn rats and then examined the development of projections from the transplanted tissue. The projections from cortical grafts placed in the midbrain were studied to determine whether axons from different classes of neurons respond to the same cues for outgrowth. When retinae were placed within the midbrain close to the cerebral aqueduct, axons projected dorsally to the superficial layers of the superior colliculus. Directed outgrowth was seen from the earliest time a projection could be detected and was independent of whether the superior colliculus still received host optic afferents. In contrast, the major projection from similarly placed cortical transplants was directed toward the ventral part of the midbrain. Deafferentation of midbrain corticorecipient areas did not affect the projection patterns from either type of graft. Projections from retinae placed more ventrally in the midbrain tegmentum could not be detected. However, retinae placed on the surface of the midbrain, even as far ventral as the cerebral peduncle at the level of the inferior colliculus, always had a projection to the superior colliculus that ran along the brainstem surface. These observations suggest that the superior colliculus exerts a positive influence on the growth of optic axons to the midbrain. However, while target cues appear to be able to support retinal axon growth through the midbrain parenchyma, their range appears to be limited, and at distances beyond the extent of their influence, optic fiber outgrowth occurs only over the surface of the brain. It is suggested, therefore, that there are both local surface-related and target-derived surface-independent cues that guide optic axons to the tectum in developing mammals.