The first step selecting competent retinal progenitors

Retinal development can be divided into several steps, some of which occur even before overt morphogenesis of the eye cup (Fig. 5.1). First, a subset of the pluripotent embryonic cells acquires the competence to contribute to the retina. From this competent pool, a smaller subset is biased to become the retina-forming cells. Later, a specified retinal stem cell population emerges from the descendants of the biased embryonic cells to form the eye field in the anterior neural plate. Further interactions during neural tube morphogenesis segregate this population into three major com-

Selection by Selection of Specification Regionalization compctencc biased lineages of stem cclls of progenitors

Figure 5.1. Early steps of retinogenesis. Retinal development begins with several steps that set aside embryonic precursors. First, a subset of embryonic cells (green) becomes competent to contribute to the retina. The blue-hatched cell is inhibited from forming retina. Next, a smaller subset (yellow) is biased to become the retina-forming cells. Then a specified retinal stem cell population (red) emerges from the descendants of the biased embryonic cells (yellow) to form the eye field in the anterior neural plate (green). Finally, during neural tube morphogenesis, this specified population is segregated into three major compartments: neural retina (red), pigmented retina (black), and optic stalk (blue). (See color plate 1.)

partments: neural retina, pigmented retina, and optic stalk. Each of these compartments goes on to produce different subsets of specialized cells with distinct functions in the mature eye.

An initial question is, how and when during development do pluripotent embryonic cells acquire the differential competence to contribute to the retina? Is this a partly stochastic process, or do early factors bias or restrict the selection of retina-forming embryonic progenitors? Xenopus embryos have been crucial for studying the roles of maternal determinants, lineage effects, and cell-cell inductions at this early stage (even before gastrulation) because eggs and blas-tomeres are large and easily manipulated (Moody, 1999). Each retina in Xenopus descends from a stereotypic subset of 9 animal blastomeres at the 32-cell stage (Fig. 5.2), and each of these blastomeres produces characteristic proportions of the cells that make up the mature retina (Huang and Moody, 1993). Are these nine cells the only cells that are competent to form the retina, and just how fixed is their commitment to a retinal fate?

These questions have been largely answered by transplanting single cells to novel positions at a very early stage when only maternally inherited transcripts are expressed. The first key finding is that even at this stage, not all blastomeres are equally competent to contribute to the retina. For example, vegetal blastomeres transplanted to the most retinogenic coordinates never contribute progeny to the retina (Huang and Moody, 1993). This developmental restriction could not be overcome by providing components of the known maternal signaling pathways involved in neural and dorsal fate specification (see below), even in situations in which ectopic heads and eyes were induced successfully (Moore and Moody, 1999).

Figure 5.2. Retina competent and biased blastomeres in Xenopus laevis embryos. Left: animal pole view of a 32-cell embryo showing the major (orange) and minor (yellow) blastomeres that contribute to the retina. The numbers within each cell indicate the percentage of retinal cells that derive on average from each blastomere. Right: side view of a 32-cell embryo showing the retina-forming blastomeres (yellow), the blastomeres that do not make retina but are competent to do so (green), and the vegetal blastomeres that are inhibited by maternal factors from making retina (blue). Data are from Huang and Moody (1993). An, animal pole; D, dorsal; V, ventral; Veg, vegetal pole. (See color plate 2.)

Figure 5.2. Retina competent and biased blastomeres in Xenopus laevis embryos. Left: animal pole view of a 32-cell embryo showing the major (orange) and minor (yellow) blastomeres that contribute to the retina. The numbers within each cell indicate the percentage of retinal cells that derive on average from each blastomere. Right: side view of a 32-cell embryo showing the retina-forming blastomeres (yellow), the blastomeres that do not make retina but are competent to do so (green), and the vegetal blastomeres that are inhibited by maternal factors from making retina (blue). Data are from Huang and Moody (1993). An, animal pole; D, dorsal; V, ventral; Veg, vegetal pole. (See color plate 2.)

These results indicate that vegetal blastomeres contain one or more maternal molecules that repress the transcription of genes that are critical in the initial steps of retinal differentiation.

Other blastomeres are not so refractive. Both ventral animal blastomeres and equatorial blastomeres that normally do not contribute progeny to the retina can reprogram when transplanted to the center of the retinogenic zone (Huang and Moody, 1993). Furthermore, if the most retino-genic blastomere is deleted, a ventral implant will be respec-ified in response to interactions with neighboring cells to help produce a normal-sized retina (Huang and Moody, 1993).

Complementing these findings, the retina-forming blastomeres are biased but not completely committed. When transplanted to a ventral vegetal site that normally produces gut and tail, they retain their neural fate but fail to make retina (Gallagher et al., 1991). Collectively, these embryonic manipulations demonstrate that the correct cellular coordinates within the animal hemisphere are necessary from the earliest stage for a blastomere to produce its normal cohort of mature retinal cells.

The position-specific selection of retina-forming blas-tomeres from the competent pool appears to be mediated by the local signaling environment within the blastula. The ectopic expression of components of several growth factor pathways involved in embryo patterning, such as activin, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP), demonstrates that competent blastomeres acquire the ability to express a retinal fate by being located in an environment in which BMP signaling is repressed (Moore and Moody, 1999). Expression of BMP4 in a blastomere that normally is a major contributor to the retina inhibits its retinal fate, whereas repressing BMP signaling in a blastomere that normally gives rise to epidermis induces retinal cells within that lineage. Consistent with this model in frog, Bmp7 null mice are anophthalmic (Dudley et al., 1995) and rat embryos cultured in anti-BMP7 antibodies have reduced or absent eyes (Solursh et al., 1996). The inhibition of BMP signaling in the dorsal part of the embryo defines the domain of presumptive retina, just as it defines the domain of presumptive neural plate (Harland, 2000).

Although fate maps show that there are nine retina-forming blastomeres in Xenopus (Fig. 5.2), not every one of these cells contributes to retina in every embryo. In the initial map (Moody, 1987), the major progenitors contributed to retina 85-100% of the time, whereas the minor progenitors contributed only 20-50% of the time. Thus, there is surprising individual variation in the number of embryonic cells that produce the retinal lineage. There also is individual variation in the number of retinal cells descended from each retina-forming blastomere (Huang and Moody, 1993). For the major progenitor, there is a 2-fold range in descendants between embryos, but for the others there is as much as a 10-fold range. These data have been verified in many subsequent studies using similar techniques. They demonstrate that the ultimate numbers of progeny produced by each lineage are not predetermined and that individual variation is well tolerated within consistent limits. This is apparently just as true in mammals as in Xenopus (Goldowitz et al., 1996; Williams and Goldowitz, 1992a).

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