David H. Rapaport

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.

Timing and Topography of Cell Genesis in the Rat Retina

To understand the mechanisms of cell fate determination in the vertebrate retina, the time course of the generation of the major cell types needs to be established. This will help define and interpret patterns of gene expression, waves of differentiation, timing and extent of competence, and many of the other developmental processes involved in fate acquisition. A thorough retinal cell “birthdating” study has not been performed for the laboratory rat, even though it is the species of choice for many contemporary developmental studies of the vertebrate retina. We investigated the timing and spatial pattern of cell genesis using 3H‐thymidine (3H‐TdR). A single injection of 3H‐TdR was administered to pregnant rats or rat pups between embryonic day (E) 8 and postnatal day (P) 13. The offspring of prenatally injected rats were delivered and all animals survived to maturity. Labeled cells were visualized by autoradiography of retinal sections. Rat retinal cell genesis commenced around E10, 50% of cells were born by approximately P1, and retinogenesis was complete near P12. The first postmitotic cells were found in the retinal ganglion cell layer and were 9–15 μm in diameter. This range includes small to medium diameter retinal ganglion cells and large displaced amacrine cells. The sequence of cell genesis was established by determining the age at which 5, 50, and 95% of the total population of cells of each phenotype became postmitotic. With few exceptions, the cell types reached these developmental landmarks in the following order: retinal ganglion cells, horizontal cells, cones, amacrine cells, rods, bipolar cells, and Müller glia. For each type, the first cells generated were located in the central retina and the last cells in the peripheral retina. Within the sequence of cell genesis, two or three phases could be detected based on differences in timing, kinetics, and topographic gradients of cell production. Our results show that retinal cells in the rat are generated in a sequence similar to that of the primate retina, in which retinogenesis spans more than 100 days. To the extent that sequences reflect underlying mechanisms of cell fate determination, they appear to be conserved. J. Comp. Neurol. 474:304–324, 2004.

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.

Defining retinal progenitor cell competence in Xenopus laevis by clonal analysis

Extrinsic cues and intrinsic competence act in concert for cell fate determination in the developing vertebrate retina. However, what controls competence and how precise is the control are largely unknown. We studied the regulation of competence by examining the order in which individual retinal progenitor cells (RPCs) generate daughters. Experiments were performed in Xenopus laevis, whose full complement of retinal cells is formed in 2 days. We lineage-labeled RPCs at the optic vesicle stage. Subsequently we administered a cell cycle marker, 5-bromodeoxyuridine (BrdU) at early, middle or late periods of retinogenesis. Under these conditions, and in this animal, BrdU is not cleared by the time of analysis, allowing cumulative labeling. All retinal cell types were generated throughout nearly the entire retinogenesis period. When we examined the order that individual RPCs generated daughters, we discovered a regular and consistent sequence according to phenotype: RGC, Ho, CPr, RPr, Am, BP, MG. The precision of the order between the clones supports a model in which RPCs proceed through stepwise changes in competence to make each cell type, and do so unidirectionally. Because every cell type can be generated simultaneously within the same retinal environment, the change in RPC competence is likely to be autonomous.