Developing Vertebrate Retina Research Articles

Expression of CRYP- α, LAR, PTP- δ, and PTP- ρ in the developing Xenopus visual system

Receptor protein tyrosine phosphatases (RPTPs), are involved in axon outgrowth and guidance not only in the Drosophila visual system ( Garrity et al., 1999. Neuron 22, 707–717) but also in the developing vertebrate retina ( Ledig et al., 1999a. J. Cell Biol. 147, 375–388). We have cloned a variety of Xenopus RPTPs, including four RPTPs expressed in the developing visual system (LAR, PTP- δ, CRYP- α and PTP- ρ). These four RPTPs are transcribed in the developing optic vesicle during differentiation and in overlapping but distinct patterns in the developing retina during retinal layer formation. LAR, PTP- δ, and CRYP- α are also expressed in retinal ganglion cells during axonogenesis and during axon guidance from the retina to the optic tectum.

Deciding cell fate

The control of cell fate vexes developmental biologists. The vertebrate retina is often used as a model system, because the neural retina contains seven different cell types, all derived from common precursor cells and with birth dates showing distinct temporal patterns. McFarlane et al.1xA role for fibroblast growth factor receptor in cell fate decisions in the developing vertebrate retina. McFarlane, S. Development. 1998; 125: 3967 3975PubMedSee all References1 demonstrate that several FGF receptors (FGFRs) are expressed in retinal precursor cells and that these FGFRs have a profound influence over specific cell fate decisions. By expressing a dominant-negative form of FGFR in the developing retina, a large increase in Muller cell numbers occurs at the expense of rod photoreceptors and amacrine neurons. This appears not to be an effect of selective cell death, or a temporal shift in cell birth dates or differentiation. Instead, it seems that the decision to become a rod or amacrine cell requires the receipt of an FGFR signal. Without the FGFR signal a Muller cell develops, either as a default or alongside additional, Muller-specific signals. In a further twist, retinal overexpression of HAVO, an FGFR that responds to FGF but not to some other non-FGF ligands, results in increased photoreceptors at the expense of amacrine and Muller cells. These novel findings indicate that specific cell fate decisions can be decided by a fine balance between agonistic and antagonistic signals acting through the same FGFR.

A role for the fibroblast growth factor receptor in cell fate decisions in the developing vertebrate retina.

The mature vertebrate retina contains seven major cell types that develop from an apparently homogenous population of precursor cells. Clonal analyses have suggested that environmental influences play a major role in specifying retinal cell identity. Fibroblast growth factor-2 is present in the developing retina and regulates the survival, proliferation and differentiation of developing retinal cells in culture. Here we have tested whether fibroblast growth factor receptor signaling biases retinal cell fate decisions in vivo. Fibroblast growth factor receptors were inhibited in retinal precursors in Xenopus embryos by expressing a dominant negative form of the receptor, XFD. Dorsal animal blastomeres that give rise to the retina were injected with cDNA expression constructs for XFD and a control non-functional mutant receptor, D48, and the cell fates of transgene-expressing cells in the mature retina determined. Fibroblast growth factor receptor blockade results in almost a 50% loss of photoreceptors and amacrine cells, and a concurrent 3.5-fold increase in Müller glia, suggesting a shift towards a Müller cell fate in the absence of a fibroblast growth factor receptor signal. Inhibition of non-fibroblast-growth-factor-mediated receptor signaling with a third mutant receptor, HAVO, alters cell fate in an opposite manner. These results suggest that it is the balance of fibroblast growth factor and non-fibroblast growth factor ligand signals that influences retinal cell genesis.

Ganglion cells influence the fate of dividing retinal cells in culture.

The different retinal cell types arise during vertebrate development from a common pool of progenitor cells. The mechanisms responsible for determining the fate of individual retinal cells are, as yet, poorly understood. Ganglion cells are one of the first cell types to be produced in the developing vertebrate retina and few ganglion cells are produced late in development. It is possible that, as the retina matures, the cellular environment changes such that it is not conducive to ganglion cell determination. The present study showed that older retinal cells secrete a factor that inhibits the production of ganglion cells. This was shown by culturing younger retinal cells, the test population, adjacent to various ages of older retinal cells. Increasingly older retinal cells, up to embryonic day 9, were more effective at inhibiting production of ganglion cells in the test cell population. Ganglion cell production was restored when ganglion cells were depleted from the older cell population. This suggests that ganglion cells secrete a factor that actively prevents cells from choosing the ganglion cell fate. This factor appeared to be active in medium conditioned by older retinal cells. Analysis of the conditioned medium established that the factor was heat stable and was present in the <3 kDa and >10 kDa fractions. Previous work showed that the neurogenic protein, Notch, might also be active in blocking production of ganglion cells. The present study showed that decreasing Notch expression with an antisense oligonucleotide increased the number of ganglion cells produced in a population of young retinal cells. Ganglion cell production, however, was still inhibited in cultures using antisense oligonucleotide to Notch in medium conditioned by older retinal cells. This suggests that the factor secreted by older retinal cells inhibits ganglion cell production through a different pathway than that mediated by Notch.

Involvement of Mash1 in EGF-Mediated Regulation of Differentiation in the Vertebrate Retina

It is believed that signaling through the epidermal growth factor (EGF) receptor plays a critical role in the development ofDrosophilaeyes. In the present study we have analyzed the role that EGF-mediated signaling plays in vertebrate retinal development. We have observed that during late retinal neurogenesis EGF delays rod photoreceptor differentiation and that this effect of EGF involves the modulation of expression of a homologue ofDrosophilaproneural genes,Mash1.EGF causes a significant decrease in Mash1 expression and an increase in the proportion of proliferating cells in the retinain vitro.The decrease in Mash1 expression is accompanied by a concomitant decrease in opsin expression, a marker for overt rod photoreceptor differentiation. Withdrawal of EGF leads to an increase in both Mash1 and opsin expression; however, the onset of expression of Mash1 precedes that of opsin. Our study identifies a proliferative intermediate precursor, characterized by Mash1 expression, that is the target of EGF-mediated suppression of rod photoreceptor differentiation. Based on the evolutionarily conserved roles of EGF- and Notch-mediated signaling in the delay of differentiation in proliferating precursors we propose that these distinct signaling mechanisms act in concert to ensure the fidelity of the strict temporal and spatial nature of cell fate determination in the retina.

Subsets of retinal progenitors display temporally regulated and distinct biases in the fates of their progeny.

Cell fate determination in the developing vertebrate retina is characterized by the sequential generation of seven classes of cells by multipotent progenitor cells. Despite this order of genesis, more than one cell type is generated at any time; for example, in the rat, several cell types are born during the prenatal period, while others are born postnatally. In order to examine whether there are classes of progenitor cells with distinct developmental properties contributing to this developmental progression, we examined antigen expression in progenitor cells during rat retinal development. Two markers of amacrine and horizontal cells, the VC1.1 epitope and syntaxin, were found to be expressed on a subset of progenitors in a temporally regulated manner that closely paralleled the birthdays of these cell types. In order to investigate which cell types were produced by the progenitors expressing these markers, fluorescent latex microspheres covalently coupled to VC1.1 antibodies were used to indelibly label VC1.1+ progenitor cells and their progeny. Early in retinal development, VC1.1+ progenitors generated a high percentage of amacrine and horizontal cells, but no cone photoreceptors. During this same period, a comparable number of cone photoreceptors were generated by VC1.1- progenitors. In the late embryonic and early postnatal period, VC1.1+ progenitors continued to generate predominantly amacrine cells, but also gave rise to an increasing number of rod photoreceptors. These findings demonstrate that expression of these two markers by progenitors is highly correlated with a bias towards the production of amacrine and horizontal cells. The fact that subsets of progenitors with temporally regulated and distinct biases are intermingled within the retinal neuroepithelium provides a basis for understanding how different cell types are generated both simultaneously and in a particular order by multipotent progenitors during retinal development.

Influence of spontaneous activity and visual experience on developing retinal receptive fields

Background The role played by early neural activity in shaping retinal functions has not yet been established. In the developing vertebrate retina, ganglion cells fire spontaneous bursts of action potentials before the onset of visual experience. This spontaneous bursting disappears shortly after birth or eye opening. In the present study, we have investigated whether the outgrowth of receptive fields in turtle retinal ganglion cells is affected by early spontaneous bursting or by early visual experience.Results Ganglion cells normally stop bursting spontaneously 2–4 weeks posthatching, the time when receptive-field areas reach adult size. When turtles are reared in the dark, the spontaneous bursting persists. Concomitantly, receptive-field areas expand to more than twice those observed in normal adults. To test whether chronic blockade of spontaneous bursting inhibits the expansion of developing receptive-field areas, we have exposed the retina to curare, a nicotinic cholinergic antagonist, because spontaneous bursting by ganglion cells requires acetylcholine. Curare was released from Elvax, a slow-release polymer that was implanted in the eye. When spontaneous bursting was chronically blocked with curare in hatchlings, dark-induced expansion of receptive fields was abolished. Moreover, receptive fields of ganglion cells exposed to curare in hatchlings reared in normal light and dark cycles were smaller than normal.Conclusions These results strongly suggest that early, acetylcholine-dependent spontaneous bursts of activity control the outgrowth of receptive-field areas in retinal ganglion cells. The onset of visual experience induces the disappearance of the immature spontaneous bursts, resulting in the stabilization of receptive-field areas to their mature size.

Retinoic acid promotes differentiation of photoreceptors in vitro.

The results of several recent studies have demonstrated that cell commitment and differentiation in the developing vertebrate retina are influenced by cell-cell interactions within the microenvironment. Retinoic acid has been shown to influence cell fates during development of the nervous system, and retinoic acid has been detected in the embryonic retina. To determine whether retinoic acid mediates the differentiation of specific neuronal phenotypes during retinal histogenesis, we treated dissociated cell cultures of embryonic and neonatal rat retina with varying concentrations of all-trans or 9-cis retinoic acid and analyzed the effects on cell fate using neuron and photoreceptor-specific antibodies. Addition of exogenous retinoic acid caused a dose-dependent, specific increase in the number of cells that developed as photoreceptors in culture throughout the period of retinal neurogenesis. In the same cultures, retinoic acid also caused a dose-dependent decrease in the number of cells that developed as amacrine cells. Also, results of double-labeled immunohistochemical studies using bromodeoxyuridine demonstrated that the primary effect of retinoic acid was to influence progenitor cells to develop as newly generated rod photoreceptors. Since retinoic acid and at least one of the retinoic acid receptors (RAR alpha) have been localized to the developing neural retina, these results suggest that retinoic acid may play a role in the normal development of photoreceptor cells in vivo.

Developmental expression of a novel murine homeobox gene ( Chx10): Evidence for roles in determination of the neuroretina and inner nuclear layer

Few potential regulatory proteins of vertebrate retinal development have been identified. We describe a 39 kDa murine polypeptide (Chx10) with a homeodomain 82% identical to that of the nematode protein ceh-10. In the developing mouse, the Chx10 transcript is expressed throughout the anterior optic vesicle and all neuroblasts of the optic cup. In the mature retina, the Chx10 protein is restricted to the inner nuclear layer, in which its expression decreases from the outer to the inner margin. Chx10 transcripts are also detected in regions of the developing thalamus, hindbrain, and ventral spinal cord. The data suggest that Chx10 plays critical roles in the formation of the neuroretina and in the development and maintenance of the inner nuclear layer.

Molecular cloning of the murine JAK1 protein tyrosine kinase and its expression in the mouse central nervous system

Degenerate oligonucleotide primers were employed in PCRs to clone protein tyrosine kinases that may play potential roles in the development of the mammalian CNS. Using one PCR clone to screen a mouse eye cDNA library, a full-length cDNA of a cytoplasmic tyrosine kinase, the homolog of human JAK1, was obtained. The murine JAK1 kinase belongs to a new family of cytoplasmic kinases that contain two tandem catalytic domains. Northern analyses indicated that murine JAK1 mRNA is expressed in a variety of tissues and cell lines. In the adult mouse eye, in situ hybridization and immunohistochemistry showed that JAK1 mRNA and protein were expressed in the retinal ganglion cell layer and the inner part of the inner nuclear layer, presumably in amacrine cells. JAK1 protein was also detected in horizontal cells and in the two synaptic layers of the adult retina. During retinal development, JAK1 protein was first detected in retinal ganglion cells and in their axons as early as embryonic day 14. Expression of JAK1 protein in amacrine cells and horizontal cells occurred only postnatally. This pattern of expression was also observed in the chick retina, suggesting an evolutionarily conserved function of JAK1 kinase in vertebrate retinal development and/or function. Immunohistochemical staining against JAK1 was detected in two areas of the adult mouse brain, the olfactory bulb and a group of cells in the hypothalamus. Together, these expression studies suggest a role for JAK1 kinase in the differentiation or function of a subset of CNS neurons.

Morphological patterns in the developing vertebrate retina

Changes in the morphology of the early optic cup were observed in embryos of two distantly-related vertebrate species, a teleost fish, northern pike (Esox lucius), and chicken (Gallus gallus). A similar morphological pattern was noted to appear in both species shortly after the involution of the optic vesicle and the formation of the inner retinal layer. At a gross level, three notches were observed in the retinal margin at approximately nasal, dorsal, and temporal positions, while in histological sections a sharp constriction was found in the thickness of the dorsal retinal layer. In both species, this dorsal constriction appeared to be continuous with the central or dorsal notch. The time of appearance and configuration of this morphological pattern is intriguingly similar to the specification and polarity of retinal positional markers, and suggest a segmentation hypothesis for the origin of retinal polarity.

A molecular view of vertebrate retinal development.

Immunological probes have begun to identify molecules that delineate cell layers and cell types during the formation of the retina and other parts of the optic cup. Within the developing retina, cell-type-specific monoclonal antibodies have been used to show that differentiation occurs before cells reach their final laminar position. Cell surface molecules have been found expressed in position-dependent gradients across the retina. These molecules may convey positional information to the retinal cells and their topographic connections. One such molecule is a modified carbohydrate group on a ganglioside, suggesting that such groups may play a role in neural development. A variety of molecules that are expressed by rod photoreceptors at defined stages of their differentiation have been characterized. These molecules have been used to show the development of subcellular compartments within rods. In vitro studies have suggested that photoreceptor molecules expressed at different times are under different forms of regulation. Some of these cell-specific molecules have been shown to be under transcriptional control and thus defined cell interactions seem to be linked to changes in gene expression during retinal development.

The rhodopsin cycle in the developing vertebrate retina: II. Correlative study in normal mice and in mice with hereditary retinal degeneration

A comparative study was made of the developmental aspects of the rhodopsin cycle in normal mice (DBA strain) and in mice with hereditary retinal degeneration (C 3 H strain) 5–50 days after birth. Rhodopsin was first detected 7 days after birth; its content in the DBA mice increased rapidly from 8 to 23 days, thereafter remaining constant. The rhodopsin content in the C 3 H mice began to fall below that of the DBA mice at 11 days, reached a maximum at 12 days, and then decreased to almost zero over the next seven days. The difference spectra for the two strains had the same maximum (500 mμ) and shape, suggesting that the visual pigment in the C 3 H mice was not grossly different from that in the DBA mice. The rhodopsin content and the outer segment length paralleled each other very closely over the entire age span studied. The width of the outer nuclear layer decreased sharply in the C 3 H mice after 11 days and, from 21 days on, it consisted of a single row of nuclei. The outer nuclear layer reached maximum widths in both strains at 8–9 days, when the rapid increase of outer segment length and rhodopsin level started. No obvious differences in development were noted in the other retinal layers. The close parallelism of the degenerative changes in the rhodopsin level, outer segment length and electroretinogram amplitudes, as well as the localization of the earliest morphological change in the inner segment, seemed to rule out lack of rhodopsin as the cause of retinal degeneration in the C 3 H mouse and suggested that the primary defect is an inborn error of the inner segment metabolism. The findings in the DBA mouse were, in most respects, similar to those in the normal rat. The degenerative pattern in the C 3 H mouse was considerably different from that in the rat with hereditary retinal degeneration.

The rhodopsin cycle in the developing vertebrate retina: I. Relation of rhodopsin content, electroretinogram and rod structure in the rat

An integrated study was made of the developmental aspects of the rhodopsin cycle in the albino rat, including measurements of rhodopsin content, electroretinogram, and photoreceptor growth. A simplified determination of rhodopsin is described which avoids isolation of the retina, or the rod outer segments. Rhodopsin was first detected 7 days after birth; it increased rapidly until 22 days, after which the concentration remained constant, while the content per eye oontinued to inorease. The electroretinogram appeared at 12 days; the amplitudes of the a- and b-waves increased rapidly until the 21st day and slowly thereafter, paralleling the rhodopsin concentration. The spectral sensitivity curve of the electroretinogram showed a maximum, coinciding with that of the difference spectrum of rhodopsin. The length of the rods and of the outer segments in both the central and peripheral areas was determined from birth to maturity. Growth of all of these structures slowed abruptly at 14 days. Closest parallelism existed between the growth of the central outer segments and the rhodopsin concentration up to 14 days. Between 14 and 22 days both the rhodopsin concentration and the electroretinogram amplitude more than doubled, although the outer segment length and total outer segment volume increased only slightly. The development of rhodopsin, the electroretinogram and the length of the photoreceptors followed typical growth curves. Consideration of these findings in the light of recent electron-microscopic observations on rod outer segments shows that rhodopsin appears concomitantly with the primitive cilium and increases rapidly with the development of the rod sacs; the electroretinogram appears only after a considerable degree of rod sac orientation has occurred.