Molecular Mechanisms Regulating Patterning Along the Animal-Vegetal Axis

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b-catenin and other components of the Wnt signaling pathway play an important role in the specification of cell fates along the animal vegetal axis of the sea urchin. b-catenin is required for the specification of the micromere lineage and for the propagation of the vegetal inducing signal that emanates from micromeres. Since members of the TCF/LEF family of HMG box DNA-binding proteins are potential nuclear partners of b-catenin, we will explore whether they are essential components of the molecular pathway that underlies cell fate specification along the animal vegetal axis. We will focus specifically on whether TCF mediates b-catenin's effects in skeletogenic specification and micromere signaling and address the following questions: 1) Does TCF mediate the specification of the micromere lineage? We will examine whether micromeres blocked in their ability to transduce the b-catenin signal by overexpressing dominant negative (DN TCF) differentiate as micromeres in situ. We will determine whether the autonomous expression of b-catenin in micromeres at the 16-cell stage and in dissociated embryos is altered in DN-TCF expressing embryos. We will also test whether micromere differentiation is potentiated in embryos overexpressing an activated TCF (Act-TCF).2) To determine whether a TCF/b-catenin complex is essential for the propagation and/or reception of the micromere signal that induces the vegetal territory, we will ask whether DN-TCF interferes with the ability of micromeres to induce a 2o archenteron. Micromeres from DN-TCF injected embryos will be transplanted to an uninjected host and 2o archenteron formation assessed with multiple markers of archenteron differentiation. To ask whether host blastomeres in which b-catenin signaling has been blocked can respond to the vegetal inducing signal, uninjected micromeres will be transplanted to DN-TCF injected hosts and similarly assessed. 3) Does the timing of TCF function coincide with the specification of the vegetal territory? We will determine whether the role of b-catenin in specifying vegetal cell fates is dependent on its interaction with TCF. We will examine the timing of TCF function and the competence of blastomeres to respond to Act-TCF or DN-TCF by overexpressing steroid inducible forms whose nuclear entry can be regulated.4) Since the differentiation of oral vs. aboral ectoderm has been shown to be effected by different concentrations of b-catenin we will test whether ectoderm specification is also mediated by TCF. We will examine the extent of oral and aboral ectoderm differentiation in embryos microinjected with different concentrations of DN- and Act-TCF. To further understand ectoderm specification we will also examine the effects of different concentrations of DN- and Act-TCF on the differentiation of oral vs. aboral ectoderm in isolated animal caps.