Signaling From the Molluscan D Quadrant Organizer

University Of Arizona, Tucson AZ

Investigators

Abstract

PROJECT SUMMARY The mud snail Ilyanassa obsoleta has long been a model for the study of developmental mechanisms because it provides a spectacular example of localized cytoplasmic determinants. Ilyanassa has a distinctive pattern of cell division known as spiral cleavage, which it shares with several other phyla. In addition, it extrudes a cytoplasmic bulb - the polar lobe- during its first two cleavages. When the polar lobe is removed, the embryo lacks a dorsal-ventral axis and has radial symmetry. Not only are the direct physical progeny of the polar lobe missing in polar lobe ablated embryos, but lobeless embryos lack structures known to descend from other cells. Thus, through a process of cytoplasmic localization, the polar lobe provides one or more cells in the embryo with the ability to induce the fates other cells. The localization of polar lobe determinants thus specifies the organizer of the Ilyanassa dorsal-ventral axis. Although it has been more than 100 years since the discovery of the effects of the polar lobe (Crampton, 1896), the molecular mechanisms underlying the localization of the organizing activity, as well as the nature of the organizing activity itself, are only just being discovered. Dr. Nagy has identified the first known component of the molluscan organizer: a localized activation of mitogen-activated protein kinase (MAPK). MAPK activation is initially activated in the signaling cell, the 3D macromere. MAPK is secondarily activated in all cells that require the inductive signal for normal development and is functionally required for specification of second and third quartet micromeres. Based on our preliminary data, she has constructed the following model for the establishment and function of the Ilyanassa organizer: MAPK is activated cell autonomously in the signaling cell (3D) as a result of inheriting polar lobe determinants. Once activated, it triggers the release of at least two signals. The first signal is as yet unidentified, and functions to activate PKC and MAPK in a subset of the micromeres. MAPK in 3D also activates a second signal, DPP, by regulating dpp mRNA translation. Release of the DPP ligand functions to pattern the first quartet micromeres, whose progeny will become eyes. The aims of this grant are to test each of the steps of this model by probing the mechanisms that activate MAPK in the 3D macromere and the mechanisms by which the 3D signals to the rest of the embryo. More specifically, we propose the following aims: Aim 1) To determine whether the inheritance of the polar lobe is necessary and sufficient to activate MAPK in 3D, by performing a series of ablation experiments to assay the contribution of non-D quadrant cells to MAPK activation in 3D. Aim 2) To characterize the nature of the mechanism by which 3D signals to the micromeres, by analyzing the physical interactions between cells in the 24-cell embryo. Aim 3) To identify factors upstream of MAPK activation in both 3D and the micromeres by biochemically inhibiting components of the major pathways known to regulate PKC (micromeres) and MAPK activation (3D and micromeres). Aim 4). To determine whether DPP signaling functions downstream of MAPK in 3D to pattern a subset of the micromeres, by analyzing Dpp, tld and pSMAD expression in polar lobe ablated and MAPK inhibited embryos and assay their function with methods to over-express and knock-down function. Intellectual Merit: Dr. Nagy's findings on the molecular basis of Ilyanassa 3D signaling puts her in a position to integrate a century of embryological manipulations aimed at understanding the molluscan organizer into a molecular framework. Once completed, the proposed experiments will significantly increase our understanding of inductive signaling in molluscan development. As molluscan embryos share striking similarities of cell division and cell fates with embryos of a number of different phyla, insight into the patterning mechanism of Ilyanassa will have significant impact on our understanding of other molluscan and lophotrochozoan embryos. Broader impact: This project promotes education at multiple levels of inquiry. Some of the proposed work forms the basis of graduate training; other aspects will form undergraduate research projects. One of the graduate students currently working on the project is using her results in this system to design an online teaching module for developmental biology. The results of our research will be published in scientific journals and regularly presented at local, national and international meetings and discussed in undergraduate classes, seminars, and online discussion sections.