Xotch inhibits cell differentiation in the Xenopus retina.

The neurogenic gene Xotch acts to divert cellular determination during gastrulation in Xenopus embryos. We examined the role of Xotch in the developing retina, where cell signaling events are thought to affect differentiation. Xotch is expressed in undifferentiated precursor cells of the ciliary marginal zone and late embryonic central retina. It is not expressed in stem cells or in differentiated neurons and glia. Expression in the retina is spatially restricted even in the absence of cell division. The final Xotch-positive precursor cells in the central retina mostly differentiate as Müller glia, suggesting that this is the last available fate of cells in the frog retina. Transfection of an activated form of Xotch into isolated retinal cells causes them to retain a neuroepithelial morphology, indicating that the continued activation of Xotch inhibits cell differentiation.

A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

Multiple functions of fibroblast growth factor-8 (FGF-8) in chick eye development.

Fibroblast growth factor-8 (FGF-8) is an important signaling molecule in the generation and patterning of the midbrain, tooth, and limb. In this study we show that it is also involved in eye development. In the chick, Fgf-8 transcripts first appear in the distal optic vesicle when it contacts the head ectoderm. Subsequently Fgf-8 expression increases and becomes localized to the central area of the presumptive neural retina (NR) only. Application of FGF-8 has two main effects on the eye. First, it converts presumptive retinal pigment epithelium (RPE) into NR. This is apparent by the failure to express Bmp-7 and Mitf (a marker gene for the RPE) in the outer layer of the optic cup, coupled with the induction of NR genes, such as Rx, Sgx-1 and Fgf-8 itself. The induced retina displays the typical multilayered cytoarchitecture and expresses late neuronal differentiation markers such as synaptotagmin and islet-1. The second effect of FGF-8 exposure is the induction of both lens formation and lens fiber differentiation. This is apparent by the expression of a lens specific marker, L-Maf, and by morphological changes of lens cells. These results suggest that FGF-8 plays a role in the initiation and differentiation of neural retina and lens.

Retinal ganglion cell size groups projecting to the superior colliculus and the dorsal lateral geniculate nucleus in the North American opossum.

The retinal ganglion cells projecting to the superior colliculus (SC) and dorsal lateral geniculate nucleus (LGNd) of the North American opossum (Didelphis virginiana) were studied by using the retrograde transport of horseradish peroxidase (HRP). The four ganglion cell size groups recognized previously were found to project in systematically different ways. After injections of HRP into the superior colliculus, labeled cells were seen in nasal retina contralateral to the injection and in temporal retina both ipsilateral and contralateral to the injection. In contralateral nasal retina cells of all size classes were labeled, while in contralateral temporal retina small (8-14 micrometers diameter), small-medium (15-19 micrometers diameter), and large (greater than 24 micrometers diameter) cells were labeled but few, if any, large-medium (20-24 micrometers diameter) cells were labeled. In ipsilateral temporal retina, soma size groups labeled included small-medium, large-medium, and large cells, but very few small cells. A nasal-temporal difference in the soma size of ganglion cells projecting to the SC was found: Labeled cells in temporal retina were 1.7-4.2 micrometers larger than their counterparts in nasal retina. Following injection of HRP into the LGNd, label was seen in contralateral nasal and ipsilateral temporal retina with no label seen in contralateral temporal retina. The labeled cells were small-medium, large-medium, and large. No small ganglion cells were labeled from the LGNd. A small nasal-temporal soma size difference in retinal ganglion cells projecting to the LGNd was seen: labeled cells in temporal retina were 1.0-2.1 micrometers larger than in nasal. It is concluded that all four ganglion cell size groups in the opossum project to the SC, but that only the three largest project to the LGNd.

Time course of morphological differentiation of cat retinal ganglion cells: influences on soma size.

We have examined the growth of ganglion cell somas during development of the cat's retina. Until approximately E (embryonic day) 50, ganglion cell somas show no sign of the several variations in their size apparent in the adult. At about E50, the somas begin to accumulate granular cytoplasm. The accumulation proceeds first among area centralis cells which, for a few days, are the largest ganglion cells in the retina (whereas in the adult they are the smallest). By E57 three of the adult soma size trends have become apparent: the differentiation of soma size related to functional class, the nasal-temporal difference in soma size, and the small mean size of somas in the visual streak. The early appearance of these trends in soma size suggests that individual cells may be intrinsically programmed to develop as cells of a particular class, such as alpha-, beta-, or gamma-cells, even before their morphological differentiation begins. A fourth trend in soma size, the centro-peripheral difference, appears only after an initial period of ganglion cell growth; the small size of ganglion cells at the area centralis seems to be determined, at least partly, by a local "environmental" factor, the crowding of ganglion cells.

Identity of cells produced by two stages of cytogenesis in the postnatal cat retina.

Cytogenesis in the postnatal cat retina was studied with the aid of 3H-thymidine autoradiography to identify the cell classes generated. Cells proliferate in two stages, which are separate spatially and temporally. Previous studies have shown that during Stage 1, cytogenesis occurs at high density at the ventricular surface of the retina, whereas Stage 2 occurs at low density in the inner retinal layers. At the ages studied, the progeny of Stage 1 cytogenesis are distributed in an annulus toward the margin of the retina, and those of Stage 2 occur central to the annulus, indicating that Stage 2 follows Stage 1. Cell genesis in Stage 1 appears to cease by P16; genesis in Stage 2 persists until between P21 and P30. The same cell classes (amacrine cells, bipolar cells, Müller cells, and rod photoreceptors) are generated during both Stages 1 and 2, but there are significant changes in their proportions both within and between stages. The proportion of the Stage 1 mitoses that form bipolar cells increases from 31% at P0 to 62% at P14. A corresponding decrease is observed in the proportion of rods (from 60% at P0 to 32% at P14). The proportion of cells generated during Stage 2 that become rods increases from 39% at P0 to 70% at P21, whereas the proportion of bipolar cells decreases from 50% at P0 to 23% at P21. Müller cells form a relatively constant proportion (8 to 15%) of the cells generated during both Stage 1 and 2. Thus at the end of Stage 1, mostly bipolar cells are generated; at the end of Stage 2, mostly rods are generated.

Cytogenesis in the monkey retina.

Time of cell origin in the retina of the rhesus monkey (Macaca mulatta) was studied by plotting the number of heavily radiolabeled nuclei in autoradiograms prepared from 2- to 6-month-old animals, each of which was exposed to a pulse of 3H-thymidine (3H-TdR) on a single embryonic (E) or postnatal (P) day. Cell birth in the monkey retina begins just after E27, and approximately 96% of cells are generated by E120. The remaining cells are produced during the last (approximately 45) prenatal days and into the first several weeks after birth. Cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in the foveal and perifoveal regions by E56. Despite extensive overlap, a class-specific sequence of cell birth was observed. Ganglion and horizontal cells, which are born first, have largely congruent periods of cell genesis with the peak between E38 and E43, and termination around E70. The first labeled cones were apparent by E33, and their highest density was achieved between E43 and E56, tapering to low values at E70, although some cones are generated in the far periphery as late as E110. Amacrine cells are next in the cell birth sequence and begin genesis at E43, reach a peak production between E56 and E85, and cease by E110. Bipolar cell birth begins at the same time as amacrines, but appears to be separate from them temporally since their production reaches a peak between E56 and E102, and persists beyond the day of birth. Müller cells and rod photoreceptors, which begin to be generated at E45, achieve a peak, and decrease in density at the same time as bipolar cells, but continue genesis at low density on the day of birth. Thus, bipolar, Müller, and rod cells have a similar time of origin. The maximal temporal separation of cell birth is between cones and amacrine cells so that cell generation exhibits two relatively distinct phases: the first phase gives rise to ganglion, horizontal, and cone cells, and the second phase to amacrine, bipolar, rod, and Müller cells. In addition, cells of the first phase are generated faster than the second phase cells, and there are differences in the topography of spread of labeled cells between the two phases. Each cell class displays a central-to-peripheral gradient in genesis, although the spatiotemporal characteristics of the gradients differ between the classes.(ABSTRACT TRUNCATED AT 400 WORDS)

Genesis of neurons in the retinal ganglion cell layer of the monkey.

We have analyzed the genesis of various neuronal classes and subclasses in the ganglion cell layer of the primate retina. Neurons were classified according to their size and the time of their origin was determined by pulse labeling with 3H-thymidine administered to female monkeys 38 to 70 days pregnant. All offspring were sacrificed postnatally, and their retinas processed for autoradiography. The somata of cells in the retinal ganglion cell layer generated on embryonic day (E) 38 ranged from 9 to 14 microns in diameter. Between E40 and E56, the minimum soma diameter remained around 8-9 microns, while the maximum gradually increased to 22 microns. As a consequence, the means of the distributions of labeled cells also increased with age, from 11.8 microns diameter for cells generated on E38 to 14.6 microns diameter at E56. Over this period the percentage of labeled cells in the 10.5-16.5 microns and greater than 16.5 microns diameter range gradually increased. The proportion of the labeled cells in the less than 10.5 microns diameter range decreased from E38 to E45, but subsequently increased rapidly. At the end of neurogenesis in the retinal ganglion cell layer, around E70, most labeled cells were considerably smaller (7-9 microns) than those generated earlier. Our results indicate that within the ganglion cell layer of the macaque, neurons of small caliber are generated first, followed successively by medium sized cells. Large, putative P alpha cells are generated late. The production between E56 and E70 of cells with the smallest somata suggests that the last-generated neurons in the ganglion cell layer are predominantly displaced amacrine cells. Within the same sector of retina, different classes of neurons in the ganglion cell layer of the rhesus monkey appear to have a sequential schedule of production.

Genesis of the retinal pigment epithelium in the macaque monkey.

The development of the retinal pigment epithelium (RPE) was studied in rhesus monkey (Macaca mulatta) fetuses, neonates, and juveniles exposed to a pulse of 3H-thymidine (3H-TdR) between embryonic day (E) 25 and postnatal day (P) 204 and examined at short and long intervals after the injection of the isotope. The RPE develops from the outer layer of the optic cup which by E45 consists of a multistratified epithelium. The outer layer appears immature near the retina's edge and gradually becomes monostratified and more mature centrally. Even at this early stage, all cells contain pigmented melanosomes, although peripherally the pigment is limited to the apical portion of the cells. Examination of autoradiograms from animals allowed to survive for several postnatal months shows that monkey RPE cell genesis begins just after E27, increasing to a peak frequency of 0.38 cells/mm at E43. Between E30 and E85 the density of radiolabelled cells varies within a restricted range of from 0.2 to 0.4 cells/mm (mean = 0.25 +/- 0.09). From the density of radiolabelled cells, and data on the overall density of RPE cells in the juvenile retina, we determined the labelling index. During the first half of gestation, between 0.38% and 0.99% (mean = 0.65 +/- 0.22) of RPE cells are generated during the short interval of isotope availability after pulse injection. Approximately 5% of RPE cells were generated by E33, and 50% by E71. After E85, RPE cytogenesis begins gradually to decrease, and 95% of the cells have been generated by the time of birth. Continued, very low density (0.01 cells/mm) cytogenesis in the RPE is seen at P17, and persists at least until seven months postnatally. RPE cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in both foveal and perifoveal regions by E56, at which time labelled cells first begin to appear peripheral to the equator. Besides the timing differences, RPE genesis in the central retina differs from that in the peripheral retina in that it proceeds at a higher rate, and lasts for a shorter time period. A prolonged postnatal period of low density RPE cell genesis persists in both central and peripheral retina. Comparison of the pattern of expansion of the area containing radiolabelled cells in the RPE and neuroretina demonstrates a remarkable spatial and temporal correspondence. Close analysis suggests that at any point on the retina, the last cells are generated in the neuroretina slightly before the last cells in the RPE.

Conduction of velocity groups in the optic nerve of the North American opossum (Didelphis virginiana): retinal origins and central projections.

The axonal conduction velocity groups in the optic nerve of the North American opossum were analyzed electrophysiologically and related to soma size groups of ganglion cells in terms of their retinal origin and laterality of projection. On the basis of analysis of field potentials and single unit responses recorded at the optic disc, three velocity groups were identified (d1, d2, and d3) and estimated to have average conduction velocities of 12, 8, and 5 meters/second. From recordings of the field potential around the perimeter of the optic disc, it was found that the d1 group was equally represented at all points around the disc, whereas the d2 group was largest in amplitude in superior temporal regions. Electrical stimulation of the optic tracts indicated that axons in the d1 group project either ipsilaterally or contralaterally, whereas the d2 group projects predominantly ipsilaterally, and the d3 group projects predominantly contralaterally. In order to relate these physiological data directly to soma size groups, horseradish peroxidase (HRP) was injected into one optic tract, and subsequently the retinae were processed for peroxidase reaction product in the ganglion cells. Labeled cells were seen in contralateral nasal, contralateral temporal, and ipsilateral temporal retina. Cells in all size classes were labeled in contralateral nasal retina. In contralateral temporal retina, labeled cells were either 10-17 micrometers diameter (small and medium) or 23-27 micrometer diameter (large), whereas in ipsilateral temporal retina, most labeled cells (94%) were 15-30 micrometer diameter (medium and large). The correspondence between these conduction velocity groups and the soma size groups described in the preceding paper (Rapaport et al., "81) is discussed.

The distribution of ganglion cells in the retina of the North American opossum (Didelphis virginiana).

The distribution of ganglion cells in the retina of the opossum was determined from whole-mounted retinae stained with cresyl violet. Isodensity lines were approximately circular with a peak density of 2,000 to 2,700 cells/mm2 in superior temporal retina (area centralis). The total number of retinal ganglion cells was estimated to be 72,000 to 135,000 (mean 101,026) in retinae ranging from 125 to 187 mm2 in total area. Three groups of ganglion cells were distinguished on the basis of soma size and retinal topography. Large cells (24 to 32 micrometer diameter) were fairly evenly distributed across the retina. Medium cells (12 to 23 micrometer diameter) were more numerous in the superior temporal quadrant than in other regions of the retina. Small cells (7 to 11 micrometer diameter) were prominent in all retinal regions, but particularly in nasal and inferior retina. An analysis of topographical differences in soma size distribution suggests that the medium size cells can be further subdivided into small-medium and large-medium groups.

The area centralis of the retina in the cat and other mammals: focal point for function and development of the visual system.

In many mammals, particularly species with frontalised eyes, a small region o retina is strongly specialised for high resolution, binocular vision. The region is typically located near the centre of the retina, a few millimetres temporal to the optic disc, and is termed the "area centralis" or, in some primates in which the specialisation is particularly well developed, the "fovea centralis". Where the specialisation is well developed, the area or fovea centralis dominates the organisation of the adult visual system. Studies of the histogenesis of the retina of the cat indicate that the process of retinal maturation is centred on the area centralis, which thus seems to be an organising focus in the ontogeny as well as the adult function of the visual system.

Sympathetic nervous system plays a role in postnatal eyeball enlargement in the rabbit.

PURPOSE: To examine the role of ocular sympathetic activity in the enlargement of the rabbit eyeball during postnatal growth. METHODS: Fourteen New Zealand albino rabbits aged 5 weeks underwent unilateral surgical transection of the cervical sympathetic trunk caudal to the superior cervical ganglion. Postoperative enlargement of both eyeballs was monitored by measuring the axial length and corneal diameters every 2 weeks for 22 weeks (7-27 weeks of age). Rabbits were housed under a 12-hour light/12-hour dark cycle, and the measurements were made in the middle of the light period. At a final age of 30 to 31 weeks, the refractive state of the whole eye was determined on both sides by measurement through the central cornea with a refractometer. Rabbits were then killed, eyeballs enucleated, and their ocular volumes determined. RESULTS: From 9 weeks of age the axial length and corneal diameters were significantly shorter (P < 0.05) in the decentralized eye (surgical side) compared with the intact eye. This reduction remained statistically significant throughout the study period. However, the final refractive states of the two eyes were found not to be different. The mean ocular volume determined after postmortem enucleation was 4.5% less in the decentralized eye than in the intact eye (P < 0.05). CONCLUSIONS: Sympathetic nervous system activity is involved in the normal enlargement of the rabbit eyeball during postnatal growth. However, removal of the ocular sympathetic tone at the age of 5 weeks does not significantly alter the refractive state of the eye when measured in young adulthood.

Epi-polarization and incident light microscopy readily resolve an autoradiographic or heavy metal label from an obscuring background or second label.

Difficulty encountered in resolving grains of exposed photographic emulsion in autoradiographs of the densely melanized retinal pigment epithelium was solved by using epi-polarized or incident light microscopy. The apparatus used included a metallurgical illuminator specifically designed for epi-polarization microscopy or, as a less expensive but only slightly less effective alternative, a modified fluorescence illuminator. The black melanin granules absorb incident light (as they do in vivo) while the silver grains reflect it producing a "darkfield-like" representation. Brightfield and darkfield-like images can be alternated easily and quickly, or both can be viewed simultaneously. Epi-polarization microscopy has wider application in resolving a reflective label over any opaque background staining or dark second label.

The site of commencement of maturation in mammalian retina: observations in the cat.

The outer plexiform layer (OPL) of the retina has been studied in the developing cat, from E (embryonic day) 30. Prior to E51 the layer could not be detected, the retina comprising two cell layers, an inner layer which becomes the ganglion cell layer of the adult, and an outer 'neuroblast' layer. The OPL was first detected at E51 as a narrow gap separating the neuroblast layer into inner and outer parts, which will form the inner and outer nuclear layers of the adult. At E51 the OPL was present only over a small region at the area centralis. After E51 the OPL thickens and spreads, extending over the entire retina by P (postnatal day) 10. During development, the area over which the OPL is formed is horizontally elongated, resembling the visual streak specialization of the adult retina. This pattern of development seems distinct from the pattern of cell birth in non-mammals, in which the earliest-born cells are found at the optic disc, and later-born cells are found more peripherally, with a dorso-ventral asymmetry in their distribution. In the cat the maturation of the OPL begins simultaneously with the maturation of the ganglion cell layer reported previously. It is possible that the two processes are controlled by a single mechanism.

Lack of binocularity in cells of area 19 of cat visual cortex following monocular deprivation.

We have studied the visual receptive field properties of neurons in cortical area 19 of monocularly deprived cats. Almost all visually responsive units responded to stimulation of the non-deprived eye only. Receptive field properties assessed through the non-deprived eye were found to be normal. Monocular deprivation appears thus to have sharply reduced the normal binocularity of neurons in area 19. Since the W-cell component of the visual pathways provide the predominant input to area 19, our results suggest that W-cells are vulnerable to environmental manipulation.

Cell division in the developing cat retina occurs in two zones.

Cytogenesis in the kitten retina has been investigated with [3H]thymidine autoradiography and a stain for mitotic figures. The nuclei of cells in S-phase are located adjacent to the inner margin of the cytoblast layer, near the inner plexiform layer. The nuclei then migrate toward the outer limiting membrane (OLM) to divide. Evidence is presented that in a small population of mitotic cells, the nucleus does not migrate to the OLM, but divides in the inner part of the cytoblast layer or, after the outer plexiform layer has formed, in the inner nuclear layer. Cell division in this inner zone begins and ends later than at the OLM. Cells dividing there are fewer in number than those at the OLM, their mitotic spindles are oriented randomly rather than parallel to the retinal surface and their nuclei move little between S- and M-phases of the mitotic cycle. The zones of cytogenesis at the OLM and in the inner cytoblast layer resemble, respectively, the ventricular and subventricular zones of other areas of the developing central nervous system.

The site of commencement of retinal maturation in the rabbit.

In the developing retina of the rabbit the ganglion cell layer can first be identified between E(embryonic day) 20 and 24, but the regional variations found in the adult retina, particularly the visual streak, are not well developed until shortly before birth. At about E31, the last day of gestation, the laminar structure of the retina begins to mature, cytogenesis begins to cease and the outer plexiform layer starts to form. These processes commence in far temporal retina, at or near the site of the area centralis, and spread preferentially along the visual streak.

Quantitative aspects of synaptic ribbon formation in the outer plexiform layer of the developing cat retina.

The development of synaptic ribbons in rod and cone photoreceptor terminals of the cat retina was studied using quantitative electron microscopy. At the region of the area centralis, synaptic ribbon profiles are initially recognized at PCD (postconception day) 59. Synaptic ribbon density increases rapidly, reaching a peak of 0.55 ribbons/micron 3 at PCD 68 (postnatal day 3) and maintains approximately that value for an additional 8 d. Following PCD 76, ribbon density begins to decrease, to 0.37 ribbons/microns 3 at PCD 82 and 0.25 ribbons/microns 3 at PCD 102. Although ribbon density drops by approximately 50% during this 39-d period, the outer plexiform layer (OPL) volume at the area centralis increases by about 20%. Ribbon density continues to decrease gradually over a protracted period to reach a final adult value of 0.11-0.14 ribbons/microns 3. During the period of high ribbon density, rod spherules with two, or even three ribbon profiles, were routinely observed. In contrast, in the adult, spherules with more than one ribbon profile are only rarely encountered. During development, the length of synaptic ribbon profiles increases from a mean of 0.22 microns at PCD 62 to the 0.47 microns mean length found in the adult.