The determination of neuronal phenotypes is a protracted, multistep event. The steps that are well documented include primary neural induction and interactions with the surrounding microenvironments in the immature nervous system. However, there is good evidence that neuronal fate determination begins during cleavage stages to bias embryonic precursor cells toward the production of neurons. Very little is known about the nature of these early steps, but with the use of lineage tracing techniques we have identified some specific blastomere interactions that are necessary for the normal production of neuronal progeny. The proposed experiments will greatly enhance our understanding of these important earliest steps in neuronal lineage determination and begin a molecular characterization of the underlying cellular events. Using intracellular lineage markers in frog blastomeres, I have mapped the lineages (neural and non-neural) of the early cleavage stages. using single cell ablation and transplantation techniques, we studied whether these lineages are influenced by early blastomere interactions. At cleavage stages some blastomeres are committed to their normal neuronal lineages. Others respond to loss of their neighbors by regulating, by producing unique progeny, or by ceasing to produce CNS progeny. These studies indicate that neighbor cell interactions at early cleavage stages are important for the expression of normal neuronal lineages. Of particular interest are those blastomeres that are committed to their neuronal fate, and those blastomeres that require neighbor interactions in order to express their neuronal fates. The former provide an opportunity to study the nature of cell commitment, and the latter provide an opportunity to study the nature of a very early neural induction. This application describes experiments that will identify the signals that instruct blastomeres to participate in neuronal lineages, and will investigate the cellular and molecular events involved in these signals.
The specific aims of this proposal are to : 1) identify the nature of the cell-cell interaction that induces tier-3 blastomeres to produce neuronal lineages. This will be accomplished by; a) separating these cells from their neighbors with Nucleopore filters, and thus determine whether they require direct cellular contact or depend upon diffusible signals (EXP IA), and b) injecting antibodies made against a Xenopus maternally-derived gap junction protein (connexin 38) into neighboring blastomeres (EXP IB). 2) Determine whether altered patterns of clone migration during gastrulation account for: a) the loss of neuronal lineages by tier-3 blastomeres that do not interact with their neighbors (EXP IIA), and b) the way that the clone of transplanted D1.1 regains its normal position in host CNS (EXP IIB). 3) Determine whether the determined state of blastomere D1.1, a major progenitor of the CNS, is enhanced by diffusible neurals from its vegetal neighbors (EXP IIIA), and if so, by which candidate growth factors (EXP IIIB). 4) Determine the molecular nature of blastomere D1.1's state of determination. We will test whether the cytoplasmic information contained in D1.1 is a protein or an mRNA (EXP IVA), and whether D1.1 cytoplasm induces dorsal-specific protein synthesis in recipient blastomeres (EXP IVB). These experiments will lead to a future molecular dissection of these earliest events in neural development.
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