The acquisition of differentiated characteristics by individual cells is a critical problem of development. In many animals the program that transforms the fertilized egg into an organized multi-cellular animal involves several processes, including unequal distribution or expression of cytoplasmic factors from the oocytes, lineage-specified mechanisms, complex cascades of cellular interactions and region-, tissue- and cell-specific transcription of zygotic genes. Sorting out the relative importance of these steps during the development of the vertebrate retina is an important challenge because this information may provide the means for correcting congenital malformations and replacing populations lost during regenerative disease or trauma. We have focused on elucidating the mechanisms by which different subtypes of amacrine cell fates are determined. During the past application period, we demonstrated that: (a) vegetal embryonic lineages are repressed from making retina by maternal factors; (b) the D1.2.1 embryonic lineage is determined during cleavage stages to produce a specified subset of amacrine cells; (c) the descendants of one embryonic lineage are altered by genetically suppressing growth factor signaling; and (d) some embryonic lineages produce their specific retinal descendants in response to their position in a field of BMP/noggin signaling during neural induction. These findings lead us to investigate four critical issues regarding retinal fate determination.
In specific aim 1, we will investigate whether the depletion of two maternal factors, which play roles in endotherm induction, will allow vegetal lineages to express a retinal fate. We will also investigate which elements of the genetic pathway upstream and downstream of the gene cerebus restore retinal fate competence to this lineage.
In specific aim 2, we will test whether the temporally- and spatially-regulated expression of eye field transcription factors determine whether non-retinal blastomeres can be transformed to express a retinal fate.
In specific aim 3, we will test whether the blockade of activin or BMP signaling affects specific amacrine cell lineages.
In specific aim 4, we will test whether transcription factors that are expressed in the embryonic eye fields regulate the expression of specific amacrine subtypes and amacrine-based lineages. These studies will combine lineage, immunocytochemical and molecular genetic approaches to elucidate the fundamental steps in the early specification of retinal lineages. We will utilize the many experimental advantages of Xenopus and the abundant knowledge available regarding maternal factors, growth factor signaling and transcriptional factor expression to elucidate the cellular and molecular mechanisms by which different amacrine cell populations are determined.
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