Mller Cell Determination

As previously described, cell determination in developing retina occurs through a lineage-independent mechanism involving multipotential pre-

Figure 2.5. Developmental pattern of 5All antigen (A,C,E) and carbonic anhydrase (B,D,F) in chick retina. Whereas the two antigens are present in many retinal cells at day 3 (A,B) and day 5 (C,D), by day 11 (E,F), their immunoreactivity is confined to the radial Müller cells (arrows). B, brain; 1, lens; and r, retina. Arrows point to Müller cells and arrowheads to the RPE (Linser, 1988). (Copyright 1988 Springer-Verlag New York, Inc., reprinted with permission.)

Figure 2.5. Developmental pattern of 5All antigen (A,C,E) and carbonic anhydrase (B,D,F) in chick retina. Whereas the two antigens are present in many retinal cells at day 3 (A,B) and day 5 (C,D), by day 11 (E,F), their immunoreactivity is confined to the radial Müller cells (arrows). B, brain; 1, lens; and r, retina. Arrows point to Müller cells and arrowheads to the RPE (Linser, 1988). (Copyright 1988 Springer-Verlag New York, Inc., reprinted with permission.)

cursors. Therefore, cell determination may be mediated either by a stochastic process in which a precursor gives rise to progeny with a given probability or by an instructive mechanism in which cell-cell interactions direct a precursor to generate a particular progeny (Cepko et al., 1996). Although retinal cells are generated in a reproducible order, it is not known how individual precursors choose specific cell fates. On the available evidence, it appears that both the intrinsic character of the precursor and extracellular signals in the environment are involved in influencing the developmental choice (Harris, 1997). Moreover, both of these features appear to change as the retina develops.

In their classic study, Reh and Tully (1987) observed that when dopaminergic amacrine cells were selectively destroyed in the frog retina, there was an overproduction of the dopaminergic cells in the regenerated retina by a process involving recruitment of uncommitted precursors (Reh, 1987). This observation and the results of related studies (Cepko, 1993), support the idea that cells decide their fate by interacting with more differentiated neighboring cells. It is likely that a signaling molecule secreted by a differentiated cell can instruct a neighboring precursor to commit to a specific cell fate.

It appears that both cell fate and cell number are largely regulated by instructive cell-cell interactions between differentiated cell types and precursors in the developing retina. Neither birthdate nor lineage appears to be a deciding factor. Indeed, more than one retinal cell type can be generated at any time (Cepko et al., 1996; Harris, 1997).

We have yet to learn when Müller cells become "determined" or what signals drive retinal precursors to differentiate into Müller cells in the developing retina. Indeed, Müller cell determination may depend on extracellular signals, or it might occur in their absence. Some experimental observations indicate neuronal signals can influence Müller cell differentiation. In chick retinal cultures, contact with neurons is required for induction of glutamine synthetase, a cell-specific marker for Müller cells (Linser and Moscona, 1979). Moreover, in retinal cell cultures, glial cell differentiation has been reported to occur in clumps containing neurons and glia, but not in isolated cells with no neuronal contact (Adler et al., 1982).

There is even more compelling evidence that extracellular signals such as growth and neurotrophic factors as well as cytokines, determine whether a precursor differentiates into a Müller cell or into another cell type (Lil-lien, 1995; Kirsch et al., 1996; Ezzeddine et al., 1997; McFarlane et al., 1998). In retinal cell cultures, high concentrations of TGF-a have been found to inhibit differentiation of retinal precursors into rods; since there is a concomitant increase in the number of Müller cells, it appears that TGF-a levels may decide the differentiation of precursors into Müller cells (Lillien, 1995). In accord with this data, overexpression of the EGF receptor increases the proportion of clones containing Müller cells in the rat retina (Fig. 2.6). These findings suggest that the levels of EGF receptor or its ligand, TGF-a, is naturally limiting and could serve as a potential determinant of Müller cell fate in the mammalian retina.

Growth and neurotrophic factors generally activate signaling pathways that lead to activation or expression of specific transcription factors (Segal and Greenberg, 1996). These molecules in turn direct the expression of cell-

Figure 2.6. Effect of EGF receptor expression on Müller cell development. A. Constructs used to infect cells contained the EGF-R gene and the ß-gal marker gene. B. A EGF-R-infected Müller cell. EGFR overexpression increased the number of Müller cells. C. Histogram shows proportion of Müller cells in rat retinas infected at birth. Each bar represents a retina (Lillien, 1995). (Copyright Macmillan Magazine Limited, reprinted with permission.)

Figure 2.6. Effect of EGF receptor expression on Müller cell development. A. Constructs used to infect cells contained the EGF-R gene and the ß-gal marker gene. B. A EGF-R-infected Müller cell. EGFR overexpression increased the number of Müller cells. C. Histogram shows proportion of Müller cells in rat retinas infected at birth. Each bar represents a retina (Lillien, 1995). (Copyright Macmillan Magazine Limited, reprinted with permission.)

specific proteins as well as restrict the choice of cell fate. The transcription factor MASH-1, for example, appears to be restricted to cells that are potential precursors for bipolar cells and Müller cells (Jasoni and Reh, 1996).

At present, we do not know the identity or even the origin of neuronal signals for Müller cell determination. It is not clear whether instructions for Müller cell determination come from multiple neuronal types or are derived from a late-developing neuron, such as the rod or the bipolar. Alternatively, the signal for Müller cell determination could be derived from the

RPE. When perinatal, mouse retinal cultures are treated with Sonic Hedgehog (Shh) protein, which is known to be expressed by RPE in vivo, there is an increase in the number of rods and Müller cells suggesting that Shh stimulates proliferation of late retinal precursor cells (Levine et al., 1997; Jensen and Wallace, 1997).

An alternative suggestion is that Müller cell differentiation occurs by default rather than through an instructive pathway, i.e., if a retinal precursor does not differentiate into a neuron its only fate would be to become a Müller cell. The best experimental evidence for Müller cell genesis by a default pathway comes from developmental studies of Xotch (Dorsky et al., 1995), the Xenopus homolog of Notch, which encodes a well-known neuro-genic protein found in dividing neuronal precursors (Artavanis-Tsakonas et al., 1999). Dorsky and associates examined Xotch expression in developing Xenopus retinas in which cells had been prelabeled with BrdU to mark their time of birth. They observed that while most of the cells lost Xotch expression, a small number of Xotck-expressing, mitotic cells persisted in the inner nuclear layer of postembryonic central retina, even after the majority of cells had become postmitotic (Fig. 2.7). Immunostaining with a glia-specific marker suggested that the Xotck-expressing cells were indeed Müller cells. These data indicate that Müller cells are among the last cells to differenti

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Figure 2.7. Relationship between Xotch expression and cell differentiation in the Xenopus retina. The proportion of stem cells (BrdU+/Xotch-), dividing retinoblasts (BrdU+/Xotch+), differentiating cells (BrdU-/Xotch+), and mature cells (BrdU-/Xotch-) varies with the developmental stage. The few cells (located in the inner nuclear layer) that are mitotic and continue to express Xotch (BrdU+/Xotch+) in stage 50 retinas are likely to be Müller cells. The data support the idea that Müller cells may arise by a default pathway (Dorsky et al., 1995). (Copyright 1995 Cell Press, reprinted with permission.)

Figure 2.7. Relationship between Xotch expression and cell differentiation in the Xenopus retina. The proportion of stem cells (BrdU+/Xotch-), dividing retinoblasts (BrdU+/Xotch+), differentiating cells (BrdU-/Xotch+), and mature cells (BrdU-/Xotch-) varies with the developmental stage. The few cells (located in the inner nuclear layer) that are mitotic and continue to express Xotch (BrdU+/Xotch+) in stage 50 retinas are likely to be Müller cells. The data support the idea that Müller cells may arise by a default pathway (Dorsky et al., 1995). (Copyright 1995 Cell Press, reprinted with permission.)

ate, and therefore might arise by a default pathway in Xenopus retina. These findings are consistent with the general observation that among all retinal cells, Müller cells alone retain the capacity to become mitotic in the adult mammalian retina (see Chapter 6).

Although Müller cells may arise by a "default" pathway in Xenopus retina, it is not evident that they are also generated by default in other vertebrate retinas. There are known exceptions. In the goldfish, for example, where rod photoreceptors are the last cells to be generated (Johns, 1982), Müller cell differentiation must be determined by other mechanisms.

From a mechanistic point of view, one can argue whether Müller cell genesis can be called a "default" event. The results of cell culture experiments suggest that a set of genes that is active in progenitors drives them to become neurons of one type or another, when the progenitors are isolated. Therefore, to generate a Müller cell, the neurogenic pathway has to be inhibited. This can be achieved, for example, by continued expression of Xotch which activates Hairy and enhancer of split homologues and inhibits activity of Xash and other neurogenic genes. Based on this mechanism, the Müler cell can be considered to have been generated through the "default" pathway because it was formed in the absence of a neurogenic signal. However, it could also be argued that the formation of neurons occurs by "default" because the progenitors were primed to make neurons, unless Xotch was kept activated in them. Nonetheless, it is clear that the formation of Müller cells in the retina is not simplistic and must involve some kind of cell-cell interaction with neurons.

In summary, it is clear that Müller cells share lineage relationship with retinal neurons but not with retinal astrocytes or microglia. The majority of Müller cells withdraw from mitotic activity rather late in retinal development and may be the last cells to differentiate in the retina. It remains to be determined whether Müller cells arise by a default pathway in all vertebrate retinas or arise by an instructive mechanism in some cases (Fig. 2.8). At issue are questions such as which extrinsic factors influence Müller cell fate, what transcription factors determine Müller cell fate, and which retinal cell interactions are crucial. Finally, the signal involved in suppressing Müller cell mitotic activity in the adult retina needs to be identified.

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