The Role of Wnt Genes in Vertebrate Development and Cancer

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

The Yamaguchi laboratory is studying how the Wnt family of signaling molecules regulates the growth and development of embryonic and adult stem cells during embryogenesis and tumorigenesis. Wnt signaling has profound effects on stem cells - in the absence of a Wnt signal, stem cells often fail to self-renew, while aberrant activation of Wnt signaling arrests precursors in a progenitor state and can cause cancer. Thus, understanding how Wnt signaling regulates stem cell pathways may lead to new cancer therapies and new methods for cellular reprogramming for regenerative medicine applications. My laboratory is particularly interested in stem cell populations that form the spinal cord and musculoskeletal cells of the mammalian trunk. We are studying a unique bipotential progenitor known as the neuromesodermal progenitor (NMP) that resides in, and adjacent to, the primitive streak (PS) of the gastrulating embryo and gives rise to the spinal cord and musculoskeletal progenitors of the trunk and tail. Several Wnts, most notably Wnt3a, are expressed in the PS where they regulate the development of NMPs however the underlying mechanisms remain poorly understood. Wnts regulate cellular behavior by stabilizing beta(b)-catenin, which interacts with members of the Lef/Tcf family of DNA-binding transcription factors (TFs) to activate the transcription of hundreds of target genes. Although we know that the cellular response in the PS to Wnt signals occurs in an ordered and temporal fashion, how these robust and reproducible gene responses are orchestrated is not well understood. The Specific Aims of the laboratory are: 1) to define the Wnt-dependent gene regulatory networks (GRN) that control the specification, self-renewal and differentiation of NMPs, 2) to define the molecular mechanisms of Wnt target gene transcription. We have made significant progress in achieving our goals: 1) To understand the Wnt3a-dependent gene regulatory network (GRN) that controls NMP development, we previously transcriptionally profiled control and Wnt3a-/- embryos to identify differentially expressed genes. To screen for Wnt target genes that regulate NMP development we generated a series of ESCs carrying CRISPR generated LOF mutations, RNAi knockdowns, or Doxycycline (Dox)-inducible epitope-tagged GOF transgenes. Since stimulation of Epiblast stem cells (derived from ESCs) with Fgf and Wnt3a rapidly induces NMPs, we reasoned that the overexpression of transcriptional effectors of Wnt3a, in the absence of exogenous Wnt3a, should induce these cell types. These studies have led us to focus on several interesting downstream transcription factors, including T/Bra, Mesogenin (Msgn1), Sp5/Sp8, Lhx1, and Nkx1.2, as well as the poorly studied peptide hormones Apela and Apelin. Our work places the direct Wnt target gene, T/Bra, at the top of the GRN as it directly controls the expression of Sp5, Msgn1 and Tbx6 while suppressing the expression of the NMP and neural determinant Sox2. Although Tbox TFs have been studied for years, little is known about the molecular mechanisms of T/Bra transcriptional transactivation. We have generated an NMP library and performed a yeast 2-hybrid assay using the T-box domain of T/Bra to identify directly interacting protein partners of T/Bra and are currently characterizing their activity. We are particularly interested in identifying TFs that function as lineage selectors since harnessing these TFs is crucial for generating specific cell types for regenerative therapies. In this regard, we have previously shown that Msgn1, a bHLH transcription factor, functions as a master regulator of paraxial mesoderm specification (Chalamalasetty et al., 2011; 2014). Paraxial mesoderm is the precursor tissue to the somites, and ultimately to the vertebral column and muscle. We are currently studying how Msgn might suppress neural fates while eliciting paraxial mesoderm fates from the bipotent NMP. One question we are addressing is how the NMP, a bipotent stem cell, arises from the pluripotent epiblast stem cell. Preliminary studies using RNAi have shown that Lhx1 (LIM homeobox1) TF, is required for the expression of T/Bra and other NMP markers. Lhx1 is expressed in the primed epiblast and appears to depend upon Wnt3a for its maintenance. The requirement for Lhx1 to express NMP genes suggests that it may be required for the primed epiblast-to-NMP transition. Lhx1 appears to enable this transition by repressing the expression of Otx2, a TF that controls epiblast identity. Not only is it important to understand how stem cells self-renew but a major problem for stem cell biologists to resolve is how the embryo regulates stem cell pool size. Nkx1.2 (NK1 homeobox2 TF) is an early marker of the NMP that continues to be transiently expressed in NMP cells as they progress to a spinal cord fate. This pattern of expression suggests a role for Nkx1.2 in the maintenance of NMP or neural progenitors. To test this hypothesis, both CRISPR LOF and Dox-inducible Nkx1.2 GOF ESCs were generated. We reasoned that overexpression of Nkx1.2 in NMPs would lead to the precocious formation of NMP or neural progenitors. Our analysis reveals that paraxial mesoderm markers (Tbx6 and Msgn1), and Cyp26a1 (which degrades neural differentiation-promoting RA) are all suppressed while Sox2 is enhanced. Interestingly, Nkx1.2 GOF also suppressed epiblast, cardiac, and neuronal differentiation gene programs, suggesting that Nkx1.2 expands the NMP and neural progenitor pools by inhibiting their adoption of alternative cell fates or terminal differentiation programs. 2) In an effort to unravel the mechanisms of Wnt target gene transcription, the laboratory has focused on the Sp family of Zinc-finger transcription factors. Sp5 and Sp8 are expressed at sites of Wnt activity and when mutated display a Wnt3a-like phenotype (Dunty et al., 2014). Indeed, our demonstration that Wnt3a, Ctnnb1 (b-catenin), Tcf1;Lef1, and Sp5/8 define a syn-phenotype group suggests that Sp5/8 function in the Wnt signaling pathway. By combining molecular genetic, ChIP-seq and biochemistry, we have shown that Sp5/8 depend upon b-catenin-Tcf1/Lef1 for activity and are necessary to activate some, but not all, Wnt target genes. Intriguingly, Sp5/8 bind to GC boxes in select Wnt target gene promoters and bind directly to Tcf/Lef to facilitate b-catenin recruitment (Kennedy et al., 2016). Recent studies demonstrate that Sp5/8 mutants display phenotypes in many other Wnt-dependent tissues including the brain, limb and LR body axis, consistent with a role for Sp5/8 in Wnt signaling. We have recently found that Sp5/8 are necessary to maintain NMPs, ie. NMPs are specified but do not self-renew. This phenotype suggests that a Sp5/8 target gene(s) is a major regulator of NMP self-renewal. Identifying the genes that control self-renewal will be critical for understanding how the axial progenitors are maintained for sufficient lengths of time to populate the entire trunk and tail. Finally, we have found that the pluripotency TF Oct4 (Pou5f1) plays a critical role in the activation of Wnt and Sp5 target genes during NMP formation. We have shown that Oct4 is transiently expressed in the PS where NMPs reside, and that Oct4 is required for NMP formation. Examination of transcriptional complexes on Wnt target genes reveals that Oct4 is interacting with Sp5, Tcf1/Lef1 and b-catenin. We suggest that upon Wnt stimulation, Sp5/8 disrupts the pluripotency GRN by binding and repurposing Oct4 for activation of the NMP gene program.