Intellectual Merit: Vertebrate mesodermal patterning in the dorsoventral (D/V) axis depends on interactions between the dorsal region known as the organizer and non-axial mesoderm adjacent to it. Wnt8, which signals through the transcriptional coactivator b-catenin, is required to establish the normal balance between organizer and non-axial tissue: zebrafish wnt8 mutants have expanded organizers and reduced non-axial mesoderm. Since interactions between the organizer and non-axial mesoderm generate a ventral to dorsal gradient of Bone Morphogenetic Protein (Bmp) activity that determines different D/V fates, Wnt8 likely impacts Bmp activity and its regulation of mesodermal fates. Whether regulation of Bmp activity explains the wnt8 mutant phenotype is unclear. Preliminary studies in zebrafish show that Wnt8 and Bmp coregulate multiple non-axial mesodermal genes but Wnt8 is also essential for the expression of bmp2b in the embryonic margin, the site of mesendoderm involution. Further, Wnt8 activity is required to mediate the effects of overexpressing some Bmp ligands but not others. Clearly, understanding how Wnt8 impacts, controls and cooperates with Bmp signaling is essential for understanding how Wnt8 controls mesodermal development and patterning.
Objectives: The first objective of this project is to determine whether bmp2b is a primary transcriptional target of Wnt8/b-catenin. bmp2b expression disappears in the embryonic margin of zebrafish wnt8 mutants. The regulation of bmp2b in the margin could be a direct effect of Wnt8/b-catenin activity, or it could be secondarily affected by Bmp regulators that are under Wnt8 control. Which of these possibilities is correct will be distinguished using a combination of genetic, conditional overexpression and in vitro approaches.
The second objective is to determine whether Wnt8 induces mesodermal fates independently of Bmp. Current literature suggests that Wnt8 plays a role in D/V fate determination. The proposed experiments will test with genetic and conditional overexpression methods the hypothesis that Wnt8 plays a direct role in only one mesodermal fate decision: the establishment of non-axial vs. axial mesoderm domains. According to this hypothesis, mesodermal fate changes in wnt8 mutants during mid-late gastrulation are due to secondary changes in Bmp activity levels.
The third objective is to determine the reason different Bmp ligands behave differently in wnt8 mutants. That is, Radar (Rdr) and XBmp4 are inactive but Bmp2b is active in a wnt8 mutant background. This represents an unexpected and novel activity of Wnt8. What underlies their differential relationships? The proposed experiments will test the hypothesis that Rdr and XBmp4 stimulate a specific Wnt8-dependent Bmp signaling pathway.
Broader Impact: The proposed work will elucidate interactions between Wnt and Bmp signaling that orchestrate vertebrate embryonic patterning. The importance of this process to all vertebrates is underscored by the finding that misregulation of Wnt activity in mice, zebrafish and amphibians can lead to anencephaly and other birth defects and can lead to multiple cancer types in humans and model organisms. Additionally, this project will provide valuable undergraduate instructional material complementary to textbook material currently in use at Texas A&M University to illustrate embryogenesis, evolution and molecular biology. This project involves research training for two graduate and up to 8 undergraduate students.
