This proposal describes a systematic effort to understand the development of left-right (L-R) asymmetric anatomy in vertebrate embryos in terms of (a) the molecules controlling """"""""leftness"""""""" vs. 'rightness"""""""", (b) their expression, movement, and interpretation within and between embryonic tissues, (c) the state of tissues during passage of the Nodal """"""""leftness-specifying"""""""" signal, and cell biological responses to this signal, (d) the mechanisms ensuring that a leftness signal is generated once and at the correct level, and (e) how L-R morphogenesis is integrated across the entire embryo. We identify major gaps in the field where we can leverage our expertise and the power of the model system to gain major new biological and mechanistic insights. Discoveries in Xenopus will be relevant to all vertebrate species including mammals, and Xenopus is an excellent choice for the studies because of the embryo's size, resilience to manipulation, and easily accessed time points of external development. Our studies include an understanding of how the Xnr1 expression pattern is transformed into a """"""""response-activity map"""""""" within L lateral plate mesoderm (LPM) at the level of intranuclear phospho-Smad2 (P-Smad2), and how this map correlates with the cell and tissue rearrangements of asymmetric morphogenesis. In contrast to the non-conserved mechanisms of L-R specification that operate in earlier embryogenesis, all vertebrates show conserved L LPM expression of the Nodal-Lefty-Pitx2 """"""""leftness-specifying"""""""" gene cassette. We will: (1) Address the mechanism of specification of L vs. R via the Nodal/Xnr1 expression wave;determine an LPM competence map for Xnrf expression and its mechanism of anterior propagation;test for a ventral midline separating the L &R compartments. We will convert Xnr1 expression into a P-Smad2 tissue-response map. (2) Create an LPM architecture atlas before, during and after its response to Xnr1 signaling, at the cytoskeletal and cell/tissue level (when is LPM epithelial and polarized? Does inter-tissue layer space allow freer movement of Xnr1/Xlefty? What are the cytoarchitectural and tissue responses to Xnr1 signaling for asymmetric morphogenesis? How do they precisely correlate with our P-Smad2 map?). As well as filling a major gap and being generally useful to the field, these results are the base for understanding constraints on Xnr1/Xlefty movement. (3) Assess the route, direction, and speed of movement of Xnr1/Xlefty (and their stabilities) within LPM and across the embryo, providing insight on the dynamic shift of Xnr1 expression in LPM, and our recently-detected long-range communication between both sides of the embryo. Unilateral Nodal expression is the prime L-R determinant leading to asymmetric anatomy of the viscera and cardiovascular system. Dysregulated Nodal signaling may occur in cancer. The proposal's significance to human health is high: (1) It explores the factors controlling strength, duration, and range of intercellular signaling in complex tissues. (2) The similar L-R anatomy amongst vertebrates raises the significance to human congenital L-R abnormalities, which are common (1 in 8500 births). Cardiovascular system formation involves coordinated anterior-posterior and L-R morphogenesis;defects cause severe anomalies, and human situs defects link to L-R pathway genes (e.g., nodal, lefty, Pitx2, Cryptic, and Zic3. (3) Nodal signaling may be a target for environmental toxic agents.
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