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A= Amacrine cell B= Bipolar cell G= Ganglion cell C=cone, R=rod H= Horizontal cell M=Müller cell forms across the retina. D, In the inner nuclear layer (LNL), bipolar cells and Müller glial cells are generated and differentiate. At this stage, both cone and rod photoreceptors comprising the outer nuclear layer (ONL) begin to mature, bearing few outer segments or discs. The outer plexiform layer (OPL) emerges, and connections between photoreceptors, horizontal cells, and bipolar cells are formed. Cell genesis (mainly rods) continues at this stage but to a greatly reduced degree. E, At maturity, all cell types are present, connections in both plexiform layers are established, and photore-ceptor outer segments are well developed.

Cell Fate Cell fate determination in the vertebrate retina is complex. Separate progenitors, each exclusively dedicated to the production of a single retinal cell type, do not exist. Instead, progenitor cells in the retina are regarded as multipotent (Holt et al., 1988; Turner and Cepko, 1987; Turner et al., 1990; Wetts and Fraser, 1988), that is, they are capable of producing more than one retinal cell class. How they adopt distinct fates during development has therefore been an area of intense research. The findings of numerous studies have resulted in a proposed model of retinal development in which cues both intrinsic and extrinsic to progenitors contribute to the determination of the cell fate (Cepko et al., 1996; Livesey and Cepko, 2001; Marquardt and Gruss, 2002).

In this model, progenitor cells are believed to move sequentially through a series of stages, during which they are capable of producing only a limited repertoire of cell types (Cepko et al., 1996). For example, progenitor cells taken from chick retina at an early age when ganglion cells are exclusively produced continue to produce this cell type alone even when transplanted into a later retinal environment when signals conducive to a rod cell fate are present (Austin et al., 1995). This limited competence of progenitor cells to produce only a small number of cell types at any given stage is thought to result from their genetic makeup. Progenitor cells destined for ganglion cell fates have been shown to express transcription factors such as Brain-3 (Brn-3) and retina-derived POU-domain factor-1 (RPF-1) (reviewed in Harris, 1997). Subsets of progenitors in the rat retina that express markers of mature amacrine and horizontal cells (syntaxin 1a and VC1.1) are biased toward producing these cell types (Alexiades and Cepko, 1997). Thus, the progenitor cell population is a heterogeneous one that is biased toward producing different cell fates. It is on this heterogeneous population of progenitors that extrinsic signals exert their influence.

Extrinsic signals that have been implied in cell fate determination include neurotrophic factors such as nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF), as well as other factors such as transforming growth factor a and b (TGF-a and -b), insulin-like growth factor (IGF), retinoic acid, thyroid hormone, and the amino acid taurine (reviewed in Harris, 1997). A direct role in cell fate determination is more evident for some factors such as CNTF (promotes bipolar cell fates) and retinoic acid and taurine [rod photoreceptor cell fate (Altshuler et al., 1993)], while for others it remains unclear whether they are also involved in neurogenesis, differentiation, and cell survival. Of course, extrinsic signals that promote a particular cell fate are only positively received by cells competent to respond to them.

Environmental signals could also act to suppress cell fates. Postmitotic neurons might be the source of such factors providing feedback to progenitors to cease the production of a particular cell type. This has been shown for ganglion cells; a still unidentified diffusible factor produced by ganglion cells limits the further production of this cell type (Waid and McLoon, 1998). While diffusible factors are a means of signaling between retinal cells during cell fate determination, communication also occurs by contact-mediated lateral inhibition. The receptor Notch and its ligand Delta have been shown to play a role in controlling cell fate. All cells start off with equal amounts of Notch and Delta. Activation of Notch blocks differentiation of the cell, at the same time leading to a reduction in its Delta levels. Expression of an activated form of Notch in progenitor cells from Xenopus and rat essentially inhibited cell differentiation and caused cells to remain in a progenitor-like state (Bao and Cepko, 1997; Dorsky et al., 1995). Conversely, when antisense oligonu-cleotides were used to reduce Notch activity in the retina, the number of ganglion cells produced was greatly increased (Austin et al., 1995). By misexpressing Delta in the Xenopus retina, Dorsky et al. (1997) demonstrated how the Notch-Delta pathway regulates cellular differentiation in the retina. At early stages of development, cells with high levels of Delta adopt a ganglion cell or cone photoreceptor fate when surrounded by wild-type cells because their neighbors failed to suppress their differentiation. Misexpression at older stages leads to a high proportion of photoreceptor fate. Delta overexpressors, however, failed to differentiate when they were surrounded by cells with similarly high Delta levels. In contrast, reduction of Delta levels by expression of a dominant negative form of Delta resulted in an increase in the percentage of cells with an earlier fate. Thus the Notch-Delta pathway is important for regulating the competence of progenitors to differentiate and respond to signals biasing their choice of fate at each stage of development.

Cell Migration All cells are effectively "born" at the outer surface of the retina, apposed to the pigment epithelium (Sidman, 1961). Postmitotic cells must therefore migrate some distance to occupy positions characteristic of their phenotype within the retina. Unlike the cerebral cortex, another highly laminated structure, cell positioning in the vertical dimension of the retina is not strictly related to cell genesis. Cells born at similar times, for example, horizontal cells and amacrine cells, can end up in different layers.

In the cerebral cortex, postmitotic neuroblasts migrate radially along radial glial fibers from the ventricular zone toward the pia to take up their final positions (Rakic, 1971, 1990). Retinal neuroblasts are also believed to disperse radially from their point of origin. However, whether such radially migrating neuroblasts use glial guides, like their counterparts in the cortex, remains contentious. The presence of a radial glial scaffold in the immature retina was suggested by studies based on electron microscopy and immunohistochemistry with glial-specific antibodies (Meller and Tetzlaff, 1976; Wolburg et al., 1991). However, the positive identification of such glial structures by electron microscopy remains unclear, and glial markers such as vimentin may not be specific to such cells during development (Bennett and DiLullo, 1985; Lemmon and Rieser, 1983).

Perikaryal translocation has been proposed as an alternative mechanism to account for radial migration within the retina (Book and Morest, 1990; Morest, 1970; Snow and Robson, 1994, 1995). This theory suggests that a newly post-mitotic cell, located at the scleral surface of the neuroep-ithelium, extends a process toward the vitreal surface, its nucleus moving to its final destination within this process. The cell maintains an attachment with the scleral side of the neuroepithelium as its nucleus translocates, losing this attachment and that from the vitreal surface only when migration is complete. Evidence in support of perikaryal translocation in the retina originally came from studies of the morphology of retinal neuroblasts by Golgi impregnations (Morest, 1970; Prada et al., 1981). Observations from the retrograde labeling of ganglion cells (Dunlop, 1990; Snow and Robson, 1994, 1995) and immunohistochemical studies using ganglion cell-specific antibodies (McLoon and Barnes, 1989) support this hypothesis. All these studies reported postmitotic cells with a bipolar morphology, attached by processes to both surfaces of the retinal epithelium, and somata located at various depths within the neu-roepithelium.

Whatever mechanism is employed, it is now well accepted that newborn retinal cells migrate to their final positions in a radial fashion. When single or small numbers of progenitor cells were marked early in development by the injection of retroviral constructs or fluorescent dyes (Fekete et al., 1994; Turner and Cepko, 1987; Turner et al., 1990), and the distribution of their progeny was examined in the mature retina, they were seen to be distributed radially across the entire depth of the retina in tightly organized columns. Such a cell dispersion pattern was suggestive of the way in which retinal neuroblasts move from their point of origin in the germinal zone. Not all cells move strictly in the radial axis, however. When larger numbers of progenitors were labeled using transgenic (Fig. 6.2; Reese et al., 1995, 1999) or chimeric mice (Williams and Goldowitz, 1992), a small

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