Our long-term goal is to understand how signals from the microenvironment, or niche, affect hematopoietic stem cells during normal development and in malignancies. The proposed studies focus on the interplay between non-canonical and canonical Wnt signaling in niche cells during primitive hematopoiesis. Canonical and non-canonical Wnt pathways are often activated simultaneously and function to inhibit each other. This is important because imbalances in Wnt signaling lead to human birth defects and cancers; however the mechanisms that regulate this equilibrium are poorly understood. As described below, we have identified a novel activator of non-canonical Wnt signaling that is required for primitive hematopoiesis and likely contributes to maintaining the proper balance of Wnt signaling throughout development. The transcription factor GATA2 is required in the ectoderm of Xenopus embryos to induce expression of a protein that then signals to the mesoderm to enable it to form blood. Our preliminary data support the hypothesis that GATA2 induces expression of a novel protein, TRIL, which activates non- canonical Wnt signaling thereby enabling cells to be specified as blood progenitors. We will test this hypothesis and determine the mechanism by which TRIL signals, as follows (1) Determine whether ectodermal GATA2 represses canonical and activates non-canonical Wnt signaling, and whether this is necessary and sufficient for hematopoiesis. We will examine endogenous Wnt pathway activation in Xenopus embryos in which GATA2 function is perturbed, and will block non-canonical Wnt signaling in whole animals and tissue recombinants to ask whether Wnt signaling is necessary in ectoderm and/or mesoderm for blood formation. (2) Determine whether TRIL is required hematopoiesis and for activation of non-canonical Wnt signaling. We will analyze blood formation in Xenopus embryos in which TRIL expression is knocked down and will ask whether activation of non-canonical Wnt signaling is sufficient to rescue blood in these embryos. We will compare the level of activation of endogenous non-canonical Wnt signaling in embryos in which TRIL expression is perturbed. (3) Determine the molecular mechanism by which TRIL activates non-canonical Wnt signaling. We will use gain- and loss-of- function assays in Xenopus to test whether TRIL function is required upstream, downstream or in parallel with known components of non-canonical Wnt pathways. In addition, we will examine the subcellular localization of TRIL in Xenopus embryos relative to other Wnt pathway components and perform an vivo structure-function analysis of TRIL. Lastly, we will identify proteins that interact with TRIL in the context of Wnt signal transduction. )
Wnt proteins play critical roles in development and adult homeostasis. We have identified a novel protein that influences the balance of Wnt signaling, and the proposed studies will examine its functions and mechanism of action during embryonic development. Our results will set the stage for future studies of whether this gene product plays a protective and/or causal role in human cancers and congenital anomalies such as neural tube closure defects.
