A thyroid hormone receptor that is required for the development of green cone photoreceptors.

Color vision is facilitated by distinct populations of cone photoreceptors in the retina. In rodents, cones expressing different opsin photopigments are sensitive to middle (M, 'green') and short (S, 'blue') wavelengths, and are differentially distributed across the retina. The mechanisms that control which opsin is expressed in a particular cone are poorly understood, but previous in vitro studies implicated thyroid hormone in cone differentiation. Thyroid hormone receptor beta 2 (TR beta 2) is a ligand-activated transcription factor that is expressed in the outer nuclear layer of the embryonic retina. Here we delete Thrb (encoding Tr beta 2) in mice, causing the selective loss of M-cones and a concomitant increase in S-opsin immunoreactive cones. Moreover, the gradient of cone distribution is disturbed, with S-cones becoming widespread across the retina. The results indicate that cone photoreceptors throughout the retina have the potential to follow a default S-cone pathway and reveal an essential role for Tr beta 2 in the commitment to an M-cone identity. Our findings raise the possibility that Thrb mutations may be associated with human cone disorders.

EGF and TGF-alpha stimulate retinal neuroepithelial cell proliferation in vitro.

Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transforming growth factor-alpha, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina.

Müller glia are a potential source of neural regeneration in the postnatal chicken retina.

The retina of warm-blooded vertebrates is believed to be incapable of neural regeneration. Here we provide evidence that the retina of postnatal chickens has the potential to generate new neurons. In response to acute damage, numerous Müller glia re-entered the cell cycle, and shortly thereafter, expressed CASH-1, Pax6 and Chx10, transcription factors expressed by embryonic retinal progenitors. These progenitor-like cells transiently expressed neurofilament. Newly formed cells became distributed throughout the inner and outer nuclear layers of the retina, and remained for at least three weeks after damage. Some of these newly formed cells differentiated into retinal neurons, a few formed Müller glia, and most remained undifferentiated, with continued expression of Pax6 and Chx10. These cells continued to proliferate when grown in culture, with some differentiating into retinal neurons or Müller glia. We propose that, in response to damage, Müller glia in the retina are a potential source of neural regeneration.

Expression of neurogenic basic helix-loop-helix genes in primitive neuroectodermal tumors.

The basic helix-loop-helix (bHLH) class of transcription factors plays a pivotal role in tissue-specific determination and differentiation. Moreover, dysregulated expression or loss of function of these factors contributes to leukemogenesis and solid tumor development. Neurogenesis is regulated by genes of the NEUROD/atonal and ACHAETE SCUTE families. We analyzed expression of human NEUROD1, NEUROD2, NEUROD3, and ACHAETE SCUTE 1 (HASH1) in cerebellar and cerebral primitive neuroectodermal tumors (PNETs), gliomas, and cell lines derived from a variety of neuroectodermal tumors by Northern analysis and in situ hybridization. NEUROD1 was expressed in each of the 12 medulloblastoma specimens, whereas NEUROD2 and NEUROD3/neurogenin were expressed in partly overlapping subsets of medulloblastomas. All of the tumors that presented with distant metastases expressed NEUROD3. The only other NEUROD3-positive tumor progressed early in treatment. Human ACHAETE SCUTE homologue (HASH1) was not expressed in medulloblastomas (infratentorial PNETs) but was expressed in three of five supratentorial PNETs. Neuroectodermal tumor cell lines derived from other sites (e.g., neuroblastoma and retinoblastoma) expressed NeuroD and ACHAETE SCUTE family members. No NEUROD message was detected in glial tumors or cell lines. Neurogenic bHLH transcription factor expression patterns suggest that specific family members may contribute to or reflect biological differences that arise during malignant transformation.

The nuclear receptor transcription factor, retinoid-related orphan receptor beta, regulates retinal progenitor proliferation.

We examined the role of retinoid-related orphan receptor (ROR)beta, a member of the nuclear receptor family of transcription factors, in retinal neurogenesis. In situ hybridization studies showed that RORbeta is expressed in retinal progenitor cells in the embryonic rat retina. Further studies demonstrated that RORbeta colocalizes with Chx10, a transcription factor thought to influence retinal progenitor proliferation (Burmeister, M., Novak, J., Liang, M-Y., Basu, S., Ploder, L., Hawes, N.L. Vidgen, D., Hoover, F., Goldman, D. , Kalnins, V.I., Roderick, T.H., Taylor, B.A., Hankin, M.H. and McInnes, R.R., 1996. Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nature Genetics 12, 376-383). Northern analysis reveals that RORbeta expression is dramatically decreased in the ocular retardationJ mutant, which possesses a defect in the Chx10 gene. Overexpression of RORbeta in retinal progenitors by biolistic transfection results in an increase in the number of large cell clones. These data support a role for RORbeta in regulating retinal progenitor proliferation, possibly via the Chx10 gene.

Temporal and spatial pattern of MASH-1 expression in the developing rat retina demonstrates progenitor cell heterogeneity.

The temporal and spatial pattern of mammalian achaete-scute homolog 1 (MASH-1) expression in the developing rat retina was examined in an effort to correlate achaete-scute homolog expression with the generation of particular cell classes. The expression of MASH-1 was restricted to the latter portion of retinal neurogenesis and was most closely correlated with the appearance of bipolar cells and Müller glia, two cell classes that are generated late in retinogenesis. We also examined the proliferative nature of the MASH-1 -expressing cell type to confirm that MASH-1 is expressed by progenitor cells and to determine the proportion of the proliferating population that expresses MASH-1. MASH-1 was expressed by only 10-30% of the total proliferating population, depending on the age examined. Thus, MASH-1 expression provides a molecular marker of heterogeneity among retinal progenitor cells and may play a role in the commitment and/or differentiation of one or more of the late-appearing retinal phenotypes.

Immunocytochemical and morphological evidence for a retinopetal projection in anuran amphibians.

The centrifugal projection to the retina in anuran amphibians (Rana catesbeiana and Xenopus laevis) has been investigated by immunocytochemistry, HRP transport, and optic nerve lesionings. FMRFamide- and N-terminal substance P-immunoreactive (Fa-ir and SP(3-7)-ir) fibers were abundant in the normal retina and optic nerve but almost absent distal to an optic nerve section or crush after 7-14 days survival. Fa-ir and SP(3-7)-ir fibers were traced to the optic nerve from the lamina terminalis (or the septopreoptic junctional area), where there are many Fa-ir and SP(3-7)-ir perikarya. After application of HRP to the optic nerve and survival for 9-10 days, retrogradely labeled neurons were observed in the lamina terminalis. Conversely, following HRP injection into the septal and preoptic area, labeled fibers were observed in the optic nerve. These results suggest that Fa-ir and SP(3-7)-ir efferent fibers project from the lamina terminalis to the retina. But in anurans, unlike teleosts, these fibers are not gonadotropin-releasing-hormone (GnRH)-ir. The morphological relations of this retinopetal pathway with the GnRH-ir nervus terminalis are discussed.

The organization of the fibers in the optic nerve of normal and tectum-less Rana pipiens.

We have examined the detailed order of retinal ganglion cell (RGC) axons in the optic nerve and tract of the frog, Rana pipiens. By using horseradish peroxidase (HRP) injections into small regions of the retina, the tectum, and at various points along the visual pathway, it has been possible to follow labelled fibers throughout their course in the nerve and tract. Several surprising features in the order of fibers in the visual pathway were discovered in our investigation. The fascicular pattern of RGC axons in the retina is similar to that described in other vertebrates; however, immediately central to their entry into the optic nerve head, approximately half of the fibers from the nasal or temporal retina cross over to the opposite side of the nerve. Although the axons from the dorsal and ventral regions of the retina generally remain in the dorsal and ventral regions of the nerve, some fiber crossing occurs in those axons as well. The result of this seemingly complex rearrangement is that the optic nerve of Rana pipiens contains mirror symmetric representations of the retinal surface on either side of the dorsal ventral midline of the nerve. The fibers in each of these representations are arranged as semicircles representing the full circumference of the retina. This precise fiber order is preserved in the nerve until immediately peripheral to the optic chiasm, at which point age-related axons from both sides of the nerve bundle together. Consequently, when a small pellet of HRP is placed in the chiasmic region of the nerve, an annulus of retinal ganglion cells and a corresponding annulus of RGC terminals in the tectum are labelled. As the age-related bundles of fibers emerge from the chiasm they split to form a medial bundle and a lateral bundle, which grow in the medial and lateral branches of the optic tract, respectively. Although the course followed by RGC axons in the visual pathway is complex, we propose a model in which the organization of fibers in the nerve and tract can arise from a few rules of axon guidance. To determine whether the optic tecta, the primary retinal targets, play a role in the development and organization of the optic nerve and tract, we removed the tectal primordia in Rana embryos and examined the order in the nerve when the animals had reached larval stages. We found that the order in the nerve and tract was well preserved in tectumless frogs. Therefore, we propose that guidance factors independent of the target direct axon growth in the frog visual system.

Immunocytochemical characterization of cysts in the peripheral retina and pars plana of the adult primate.

PURPOSE: To better characterize the cellular constituents of cysts in the peripheral retina and pars plana of the adult monkey. METHODS: Frozen sections of the peripheral retinal margin and pars plana from monkeys (Macaca nemestrina) between 1 and 15 years of age were stained with toluidine blue or immunolabeled with a variety of glia- and neuron-specific antibodies. RESULTS: In animals 1 to 2 years of age, the nonpigmented inner layer of the pars plana is a pseudostratified columnar epithelium. In these young animals, the peripheral retina had distinct layers and did not contain cysts. In animals 6 years of age or older, there were numerous cysts in the pars plana and in the peripheral retina. In the peripheral retina, neurons were randomly distributed and did not have a laminar organization. Cells surrounding cysts were immunoreactive for different types of markers for retinal neurons. Some of the cells surrounding cysts in the pars plana were also unexpectedly immunoreactive for antigens normally expressed only in retinal neurons and glia. CONCLUSIONS: Cysts form in the peripheral retina and pars plana in adult monkeys. The peripheral retinal cysts disrupt the normal lamination of the cells, but all types of retinal neurons are still present in the cysts. In an unexpected finding, cysts in the pars plana also contained cells immunoreactive for a few of the markers of retinal cells, suggesting that neurogenesis may occur in the pars plana of the adult primate.

Regulation of proliferation and photoreceptor differentiation in fetal human retinal cell cultures.

PURPOSE: To examine whether fetal human retinal cells can be maintained in vitro over long time periods and to determine whether exogenous growth factors can be used to generate large numbers of photoreceptors within these cultures. METHODS: Fetal human retinas (6 to 13 weeks after conception) were dissected, dissociated, and plated into culture wells. Specific growth factors and steroid/thyroid hormones, which have been shown to influence retinal progenitor cell proliferation and differentiation in rats, were added to the culture medium to determine whether any of these factors had similar effects on human retinal cells. RESULTS: Fetal human retinal cells survived and continued to proliferate for up to 300 days in vitro. Under control conditions, 15 million cells were generated from an initial plating of 100,000 cells; however, the addition of either epidermal growth factor or basic fibroblast growth factor stimulated proliferation and resulted in the generation of more than 100 million cells. A percentage of these cells was induced to differentiate as photoreceptors by adding either retinoic acid or triiodo-thyronine to the culture medium. CONCLUSIONS: Fetal human retinal cells can be maintained and expanded in vitro, indicating that this technique may be useful for generating large numbers of retinal cells. The number and types of cells generated can be influenced by adding exogenous factors to the culture medium. The response of human retinal cells to growth factors and hormones is similar to the response of rodent retinal cells to the same factors, suggesting that the effects of these factors are conserved across species.

Retinoic acid promotes rod photoreceptor differentiation in rat retina in vivo.

Previous studies have indicated that retinoic acid (RA) promotes rod photoreceptor differentiation in dissociated cultures of rat retina and in zebrafish embryos. To determine whether RA will have the same affect in the mammalian retina in vivo, pregnant rats were given single i.p. injections of RA on the 18th and 20th days of gestation, and the retinas of the pups were analyzed for rods. HPLC showed that i.p. injections of RA substantially increased levels of retinal RA in the embryos. Embryonic exposure to RA caused an increase in the number of cells that differentiated as rod photoreceptors. There was a comparable decrease in the number of cells that differentiated as amacrine cells. These results demonstrate that RA promotes the differentiation of rods in vivo and further support the hypothesis that differentiation of rods is normally controlled partly by the RA concentration in the developing retina or RPE.

Qualitative and quantitative measures of plasticity during the normal development of the Rana pipiens retinotectal projection.

We have examined the following aspects of retinal development in the frog, Rana pipiens: (1) the overall pattern of cell addition to the retina; (2) the relative rate of retinal ganglion cell (RGC) accretion; and (3) the changes in RGC density during larval development. In addition, we have studied the development of the retinal projection onto the tectum by means of the anterograde transport of horseradish peroxidase (HRP) and measurements of the volume of tectal neuropil at several larval stages and in postmetamorphic frogs. We find that the addition of new cells to the retina of Rana pipiens larva is restricted to the ciliary margin and that this addition is concentric at all larval stages. Additionally, the morphometric measures of retinal and tectal growth, along with the HRP histochemistry, indicate that the retinal projection exhibits considerable plasticity during normal development. The plasticity we observe in normal development may explain why the retinotectal projection can compensate its area and volume in experimental paradigms that effect drastic changes in innervation density.

Axons of the pyramidal tract do not increase their transport of growth-associated proteins after axotomy.

We compared the effects of axotomy on the composition of fast axonally transported proteins in rat sciatic sensory axons and pyramidal tract axons, 7 days after axotomy. L-[35S]methionine was administered to dorsal root ganglia or sensorimotor cortex and after 6 or 4 h, respectively, labelled proteins were obtained from sciatic nerve or pyramidal tract. Transported proteins were characterized by one- and two-dimensional electrophoresis, followed by fluorography. As previously reported, sciatic axotomy produced changes in the labelling of fast-transported proteins, notably, increased labelling of a spot designated GAP43. In contrast axotomy of pyramidal axons produced no increased labelling of GAP43, although it could be identified in both normal and axotomized samples. Autoradiography of spinal cord distal to the lesion confirmed that our procedures did indeed label axons and that the axotomy did interrupt these axons. Our finding that axotomy does not stimulate changes in composition of fast-transported proteins in the corticospinal tract agrees with previous studies on mammalian retinal ganglion cell axons and leads us to conclude that this failure to respond to axotomy is a general feature of non-regenerating CNS axons.

Inhibition of retinal regeneration in larval Rana by an antibody directed against a laminin-heparan sulfate proteoglycan.

In vivo and in vitro experiments have demonstrated that the retinal pigmented epithelium can give rise to neuronal precursors during retinal regeneration in amphibians. In vitro experiments have further demonstrated that this process is greatly facilitated by two molecules associated with basement membranes: laminin and fibroblast growth factor. We now report that retinal regeneration can be blocked in vivo by an antibody that inhibits the interaction of cells with the laminin-heparan sulfate proteoglycan complex. These results further support a role for the extracellular matrix in regulating cellular phenotype during retinal regeneration.

A comparative study of neurogenesis in the retinal ciliary marginal zone of homeothermic vertebrates.

The retina of many fish and amphibians grows throughout life, roughly matching the overall growth of the animal. The new retinal cells are continually added at the anterior margin of the retina, in a circumferential zone of cells, known as the ciliary marginal zone, or CMZ. Recently, Fischer and Reh [Dev. Biol. 220 (2000) 197] have found that new neurons are added to the retina of the chicken via proliferation and subsequent differentiation of neurons and glia at the retinal margin in a zone highly reminiscent of the CMZ of lower vertebrates. In addition, other groups have reported that putative retinal stem cells could be isolated from the ciliary margin of the adult mouse. In light of these findings, we have re-investigated the eyes of three additional species to determine whether other homeothermic vertebrates also possess CMZ cells and whether we could detect evidence for addition of neurons at the retinal margin in mature animals. We examined one additional avian species, the quail, one marsupial, the opposum, and one mammal, the mouse. We find that the CMZ cells have been gradually diminished during vertebrate evolution. The quail has a reduced CMZ as compared to the chicken, while the opposum has only a few cells likely related to the CMZ and we failed to find evidence of CMZ cells at the margin of the mouse retina.

Independent regulation of primary dendritic and axonal growth by maturing astrocytes in vitro.

Whereas axon growth is limited in the mature mammalian central nervous system (CNS), dendritic remodeling occurs throughout life. Since glia can regulate neurite growth during development, we tested whether glia from the mature CNS differ in their ability to support axon and dendrite growth. Cortical neurons were co-cultured with neonatal or mature astroglial, and dendrite and axon outgrowth quantified after 5DIV using immunohistochemical techniques. Primary dendritic growth was supported equally well by mature and immature astrocytes. By contrast, mature astrocytes demonstrated a reduced ability to support axon growth. We propose that the independent regulation of axon and dendrite growth by astrocytes provides a means by which the mature nervous system may adjust its function to activity and the environment.

Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.

The sensory epithelium of the avian inner ear retains into adulthood progenitor cells for inner-ear hair cells and other cell types in the epithelium. Hair cells are produced normally on an ongoing basis in the vestibular sensory epithelium, and hair-cell production is increased after insult in both auditory and vestibular sensory epithelia. The details of postnatal hair-cell production are not understood. In particular, molecular factors involved in the initiation and regulation of hair-cell genesis and differentiation are not known. Studies of this phenomena have been hampered by the lack of cell culture models. An organ culture system was developed which encourages generation and differentiation of hair cells in mature inner-ear sensory epithelia. Continuous labeling with tritiated thymidine showed genesis of both supporting cells and hair cells in normal vestibular epithelia grown in culture, and an increase in hair-cell and supporting-cell proliferation in damaged sensory epithelia grown in culture as compared to undamaged controls. This demonstrates, in vitro, both the division and differentiation of hair-cell progenitor cells in normal vestibular epithelia, and the maintenance of the hair-cell regeneration process in damaged inner-ear epithelia. This culture system should be useful for studies of hair-cell genesis and differentiation as well as studies of hair-cell and supporting-cell functioning in general.

Regenerative medicine for retinal diseases: activating endogenous repair mechanisms.

The retina is subject to degenerative diseases that often lead to significant visual impairment. Non-mammalian vertebrates have the remarkable ability to replace neurons lost through damage. Fish, and to a limited extent birds, replace lost neurons by the dedifferentiation of Müller glia to a progenitor state followed by the replication of these neuronal progenitor cells. Over the past five years, studies have investigated whether regeneration can be stimulated in the mouse and rat retina. Several groups have reported that at least some types of neurons can be regenerated in the mammalian retina in vivo or in vitro, and that the regeneration of neurons can be stimulated using growth factors, transcription factors or subtoxic levels of excitatory amino acids. These recent results suggest that some part of the regenerative program that occurs in non-mammalian vertebrates remains in the mammalian retina, and could provide a basis to develop new strategies for retinal repair in patients with retinal degenerations.