Critical roles for calcium and beta-catenin in zebrafish left-right organ asymmetry.

摘要

Many aspects of animal development including fertilization as well as organ formation and function are dependent upon the dynamic release of calcium (Ca2+) ions. During embryonic development, specific cellular responses can be triggered by differences in the amplitude, frequency and duration of intracellular Ca2+ oscillations. Our hypothesis is that distinct Ca2+ outputs lead to specific cellular behaviors. To test this we searched for Ca2+ release activity on other developmental stages. In Chapter 2 we describe an aperiodic Ca2+ release activity during gastrulation in the dorsal forerunner cells (DFCs) and show that it is associated with antagonism of the Wnt/beta-catenin signaling network.;In vertebrates, internal organs are asymmetrically positioned across the left-right (LR) axis, and the establishment of this asymmetry is key for proper development and organ function. In zebrafish, molecular asymmetry is initiated by the Kupffer's vesicle (KV), a ciliated structure derived from the DFCs. Since we identified a Ca2+ release activity in these cells that impacted beta-catenin, we hypothesized that DFC regional Ca 2+ fluxes had a role in LR patterning. In Chapter 3, we show that manipulation of Ca2+ activity in the DFCs disrupts proper establishment of the KV and subsequent LR asymmetry in the zebrafish embryo. ha addition, we identify DFC-like cells and describe a Ca2+-sensitive event in Xenopus that also impacts beta-catenin activity and LR axis establishment.;The mechanism underlying the translation of Ca2+ fluxes in DFCs into LR cues is likely to require the function of a Ca2+ sensor. One potential link between Ca2+ and beta-catenin regulation is Nkd1, the zebrafish homologue of the Drosophila Wg antagonist nkd. Nkd1 contains a single EF-hand, which are conserved Ca2+-binding motifs. In Chapter 4, we show that targeted knockdown of Nkd1 in the DFCs results in LR defects. Furthermore, we show that Nkd1 promotes membrane localization and degradation of the cytoplasmic phosphoprotein disheveled (Dvl). Finally we demonstrate that residues in the EF-hand predicted to mediate ion binding are required for Nkd1-mediated Dvl degradation.