Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.     

Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho 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-delta, CRYP-alpha and PTP-rho). 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-delta, and CRYP-alpha are also expressed in retinal ganglion cells during axonogenesis and during axon guidance from the retina to the optic tectum.     

Actin filament disruption blocks cerebellar granule neurons at the unipolar stage of differentiation in vitro

Cerebellar granule neurons developing in vitro initially extend a single axon, with the Golgi apparatus and centrosome positioned at the base of this axon and then begin the transition to a bipolar morphology by rotating the Golgi-centrosome to the opposite pole of the cell and extending a secondary axon; granule cells reach a mature, complex morphology by extending multiple, short dendrites by 5-6 days in vitro. (Zmuda and Rivas, 1998. Cell Motil Cytoskel 41:18-38). To test the effects of actin depolymerization on this characteristic pattern of granule cell axonogenesis, cultured granule cells were treated with either cytochalasin D (CD) or latrunculin A (Lat A) to depolymerize filamentous actin. Although actin depolymerization did not inhibit initial axon extension, it prevented the cells from proceeding on to the transitional, bipolar, or complex stages of differentiation, effectively blocking the cells at the unipolar stage of differentiation. Although the Golgi apparatus resided at the base of the axon in nontreated unipolar cells, or at the opposite pole of the cell body in nontreated transitional cells, the Golgi was randomly localized within the cytoplasm of cells that had been treated with either CD or Lat A. These results show that the transition from the unipolar to the bipolar stage and on to more mature stages of granule cell differentiation is dependent on an intact actin cytoskeleton and suggest that the characteristic pattern of granule cell differentiation may be dependent on the repositioning of the Golgi-centrosome during morphological development.     

Distribution of the small molecular weight GTP-binding proteins Rac1, Cdc42, RhoA and RhoB in the developing chick retina

Immunohistochemistry was used to determine the distribution of Rac1, Cdc42, RhoA and RhoB GTPases during development of the chick retina. All proteins appear as early as embryonic day 5 (E5) in cells of the vitreal margin, E7–8 in cells of the inner third of the inner nuclear layer and E9–10 in photoreceptors. From E10 until hatching, RhoA, Rac1 and Cdc42 were seen in perikarya and/or processes of amacrine, ganglion cells, and photoreceptors. Rho proteins were also observed in retinal Müller cells, with different distributions. RhoB showed a transient expression, being severely down regulated after E18. The distribution pattern of Rho proteins during the development of the chick retina suggests a concerted role in the differentiation of specific cell types, and probably during synaptogenesis.     

Early ganglion cell differentiation in the mouse retina: an electron microscopic analysis utilizing serial sections

The retina of a mouse embryo on day 13 of gestation, the first day when ganglion cells with axons are detectable, has been studied both qualitatively and quantitatively by reconstructing a large number of cells (more than 100) from an electron microscopic serial section series. Direct evidence has been obtained for migration of prophase nuclei of ventricular cells to the ventricle within an intact process which spans the thickness of the retinal wall. At metaphase most of the vitreal process appears to be pinched off, and the cell completely rounds up. After cytokinesis, cells take one two courses: (1) regrowth of their vitreal process to the vitreal surface while keeping their ventricular process attached at the ventricular surface by a junctional complex; these cells will undergo another round of DNA synthesis and division; (2) regrowth of their vitreal process only so far as the marginal layer with detachment of their ventricular process from the junctional complex and beginning migration of their centrioles and cilium away from the ventricle. These changes represent the earliest detectable quantitative or qualitative changes undergone by cells that will subsequently differentiate into ganglion cells. The sequence of events for the formation of unipolar ganglion cells from these early bipolar cells involves transformation of the simple vitreal process ending in the marginal layer into an axonal growth cone insinuating itself between the tangential axons of the marginal layer and growing toward the optic stalk; at the same time the Golgi complex and centrioles migrate to the perikaryon, and the ventricular process completely withdraws. Usually, but not always, both daughter cells of a mitotic division appear to have the same fate, either both remain ventricular cells or both become ganglion cells. This result is used to construct a simple hypothesis explaining some of the apparently contradictory results of neuronal development, both in the retina and in the rest of the central nervous system.     

Ontogeny of the retina and optic nerve of Xenopus laevis: IV. Ultrastructural evidence of early ganglion cell differentiation

Ultrastructural evidence indicates that Xenopus retinal ganglion cell axons differentiate early, between stages 28 and 32. Light microscope studies indicated the presence of argryophilic material in the ventral retina and optic stalk of early embryos. Ultrastructural analysis of this region confirmed the presence of axons in the stalk and interstices of ventral retinal cells. Axons containing aligned microtubules and neurofilaments and elongated mitochondria with a paucity of other cell inclusions are found with increasing frequency in the ventral retina from stages 28 through $\text{33}\text{34}$. Central and dorsal regions of the retinas examined show little or no evidence of axons. A discrete, small bundle of axons is found in the optic stalk of stage 28 embryos and by stage $\text{30}\text{31}$ the number of axons in bundles has increased, suggesting early fasciculation. Between stages 28 and $\text{33}\text{34}$ (± 12 hr) extracellular space surrounding early axons diminishes and processes from neuroretinal cells in contact with axons surround developing axon bundles. The evidence presented suggests that axon initiation occurs in stages much earlier than previously reported. Other investigators have failed to detect ganglion cell differentiation prior to stage 32 possibly because they examined regions of the retina with few axons. Thus, experiments which rotate the retina in the orbit may have to be reevaluated since regenerating axons may use previously established pathways to organize and “home in” on tectal target cells.     

Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

CALEB binds via its acidic stretch to the fibrinogen-like domain of tenascin-C or tenascin-R and its expression is dynamically regulated after optic nerve lesion

Recently, we described a novel chick neural transmembrane glycoprotein, which interacts with the extracellular matrix proteins tenascin-C and tenascin-R. This protein, termed CALEB, contains an epidermal growth factor-like domain and appears to be a novel member of the epidermal growth factor family of growth and differentiation factors. Here we analyze the interaction between CALEB and tenascin-C as well as tenascin-R in more detail, and we demonstrate that the central acidic peptide segment of CALEB is necessary to mediate this binding. The fibrinogen-like globe within tenascin-C or -R enables both proteins to bind to CALEB. We show that two isoforms of CALEB in chick and rodents exist that differed in their cytoplasmic segments. To begin to understand the in vivo function of CALEB and since in vitro antibody perturbation experiments indicated that CALEB might be important for neurite formation, we analyzed the expression pattern of the rat homolog of CALEB during development of retinal ganglion cells, after optic nerve lesion and during graft-assisted retinal ganglion cell axon regeneration by in situ hybridization. These investigations demonstrate that CALEB mRNA is dynamically regulated after optic nerve lesion and that this mRNA is expressed in most developing and in one-third of the few regenerating (GAP-43 expressing) retinal ganglion cells.     

The receptor tyrosine phosphatase CRYPalpha affects growth cone morphology

During development of the nervous system receptor tyrosine kinases and receptor protein tyrosine phosphatases act in a coordinate way during axon growth and guidance. In the developing avian retinotectal system, many different receptor protein tyrosine phosphatases are expressed. Most of them have unknown functions. Retinal ganglion cells express at least three different members of this receptor family on their axons and growth cones: CRYPalpha, CRYP-2 and PTPmu. CRYPalpha interacts heterophilically with at least two different ligands found in the basal membranes of the retina and the optic tectum. To analyze the role of the CRYPalpha-ligand interaction, retinal ganglion cell axons were grown on retinal basal membranes (inner limiting membrane) and the receptor-ligand interaction was blocked from both the receptor side (by receptor specific antibodies) and from the ligand side by using a receptor-alkaline phosphatase fusion protein. Both of these treatments reduced average retinal axon length and induced a dramatic change in morphology of retinal ganglion cell growth cones on basal membranes, but not on other substrates like laminin, N-cadherin, matrigel- and detergent-treated basal membranes. These results suggest that CRYPalpha and its ligand act as growth-promoting molecules during intraretinal axon growth.     

Spatiotemporal pattern of retinal ganglion cell differentiation revealed by the expression of neurolin in embryonic zebrafish

The expression of neurolin, the fish homologue of the cell adhesion molecule DM-GRASP/BEN/SC-1, is dynamically regulated. Here we demonstrate that the expression of neurolin correlates with early events of retinal ganglion cell (RGC) differentiation in zebrafish embryos. Neurolin mRNA first appears [28 h postfertilization, (PF)] in nasoventral cells, representing the first RGCs, then in dorsal, central (34 to 40 h PF) and temporal RGCs. After differentiation of RGCs in the central portion of the retina, RGCs exhibiting neurolin mRNA form rings. These rings move toward the retinal periphery and encompass older (central) RGCs. Thereafter, such as at 3.5 days PF, neurolin mRNA expressing RGCs are confined to the annular growth zone at the retinal peripheral margin. Two hours after onset of mRNA expression, RGCs acquire antineurolin immunoreactivity on the surface of their somata and on their axons as they extend to the tectum. The mRNA signal in RGCs decreases significantly within 20 h after its appearance, which correlates with the arrival of axons in the tectum. This is followed by weakening of neurolin immunoreactivity on RGCs and axons. This pattern of RGC differentiation in zebrafish revealed by the expression of neurolin is unique among vertebrates. The spatiotemporal expression pattern of neurolin suggests a functional significance of this cell adhesion molecule in RGC recognition and RGC axon growth. © 1996 John Wiley & Sons, Inc.     

Development of neurons in the abdominal ganglion of Aplysia californica. I. Axosomatic synaptic contacts.

The development of mariculture techniques for the raising of Aplysia californica in the laboratory from fertilized egg to reproductively mature adult permits the study of the developmental program whereby individual identified neurons in the abdominal ganglion acquire their specific adult properties. In this paper, we describe one of the early steps of this developmental program: the outgrowth of axonal processes by neurons of the abdominal ganglion. Axonal outgrowth is correlated with and may be triggered by the transient appearance of morphologically identifiable axosomatic contacts between the as yet undifferentiated cell body of specific neurons and an axon terminal from an incoming nerve fiber from the pleuroabdominal connective. The evidence that transient axosomatic contacts may signal neuronal differentiation is the following: (1) Axosomatic contacts have not been observed in the abdominal ganglion of adult animals, whereas they are commonly observed during the early stages of development. (2) Cells that receive axosomatic contacts are undifferentiated morphologically and do not as yet have axons. By contrast, cells with axons do not have soma contacts. (3) Individual cells that can be identified from animal to animal in the same and succeeding developmental stages receive axosomatic contacts on similar topographic postions of the cell body at one point in development. Axon outgrowth then occurs at the site of contact. Later in development, with further axon extension, these cells no longer have synaptic contacts on the cell body or axon.     

Intraretinal axon diameter: a single cell analysis in the marmoset (Callithrix jacchus)

We have labelled individual retinal ganglion cells of a New World primate, the common marmoset (Callithrix jacchus) with neurobiotin and then measured axon, soma and dendritic field diameter. A total of 111 cells were analysed (62 parasol cells, 22 midget cells, 16 hedge cells and 11 small bistratified cells). When all retinal ganglion cells were grouped together axon diameter was positively correlated to soma diameter. When analysed according to cell class only midget cells showed a positive correlation between soma size and mean axon diameter. Dendritic field diameter and mean axon diameter of both parasol and midget cells showed significant correlations. Axon diameter is not constant along the intraretinal length of the axon and the rate of change in diameter appears to be related to the cell class and the initial size of the axon. Midget cell axons showed a rapid increase of up to 20% over the first 200 μm in contrast to parasol cell axons which increased more slowly over this distance but then showed a marked increase in diameter of up to 40% over the next 450 μm. However, axon diameter did not remain at these increased diameters but decreased at greater distances from the soma. The degree to which an axon changes its diameter is related to retinal ganglion cell class and the initial size of the axon. We postulate that these variations in intraretinal axon diameter may have a direct influence on conduction velocity and reflect a compensatory mechanism to minimise spatiotemporal dispersion along the visual pathway.     

Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage

In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/- and Bax-/- mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/- or Bax-/- mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.