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 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. Our work has shown that 7 of the 19 Wnt ligands are expressed in an overlapping fashion in the PS, and functional approaches in vivo have shown that these ligands have both redundant and unique activities. 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 target gene transcription. We have also shown that some of these Wnts, in particular Wnt5a, regulate gastrulation through a b-catenin independent pathway. How these ligands exert specific effects on PS cells is not well understood.
The Specific Aims of the laboratory are: 1) to understand how the fate of PS cells, and specifically NMPs, are regulated by Wnts, 2) to define the gene regulatory networks (GRN) that are activated by Wnt3a to control the differentiation of NMPs, 3) to define the molecular mechanisms of Wnt target gene transcription. We have made significant progress in achieving our goals: 1) We have previously utilized Cre-based approaches to trace the lineage of PS cells in the developing embryo. Using a subtractive approach with the T-CreERT2 transgenic mouse (collaboration with M. Lewandoski) to trace the lineage of epithelial and mesenchymal PS cells, and the Cited1-CreERT2 mouse to trace only mesenchymal PS cells, we showed that NMPs are primarily located in the epithelial PS (Garriock et al., 2015). One problem with the use of the T-CreERT2 transgene is that it is expressed broadly throughout the PS epithelium. To further refine our lineage tracing studies of the PS, we have recently collaborated with K. Storey (U. Dundee) to use the Nkx1.2-CreERT2 mouse to trace only anterior PS cells and to manipulate b-catenin activity in these cells. These data support a model that Sox2+T/Bra+Nkx1.2+ epithelial anterior PS cells are multipotent NMPs that give rise to the spinal cord and the axial skeleton of the trunk and tail in response to Wnt/b-catenin signals. 2) To understand the Wnt3a-dependent gene regulatory network (GRN) that controls NMP development, we generated genome-wide transcriptional profiles of wildtype (wt) and Wnt3a-/-PS and identified 729 differentially expressed genes (Dunty et al., 2014). 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 ESCs with Fgf and Wnt3a rapidly induces NMP-like cells, 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 (ie. Wnt3a-dependent) 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 works places the direct Wnt target gene, T/Brachyury, 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. 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, likely 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. 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 maintains constraints on 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 promotion of neural fates from the NMP. 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 neural progenitors. This is largely supported by our analysis as NMP and paraxial mesoderm markers (T/Bra, 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 gene programs, including Lhx1, Otx2 and Fgf5, suggesting that Nkx1.2 maintains NMPs and promotes their neural differentiation by inhibiting their adoption of alternative cell fates. 3) In an effort to unravel the mechanisms of Wnt target gene transcription, the laboratory has focused on the Sp1 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 and biochemical approaches, we have shown that Sp5/8 are necessary to activate Wnt target genes, but depend upon b-catenin-Tcf1/Lef1 for activity. Intriguingly, Sp5/8 bind to GC boxes in Wnt target gene enhancers 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 shown that Sp5/8 may play a key role in the epiblast-to-NMP transition. Sp5/8 are found in a transcriptional protein complex that includes the pluripotency TF Oct4. We have shown that Oct4 is transiently expressed in the PS where NMPs reside, and that Oct4 is required for NMP formation. We suggest that upon Wnt stimulation, Sp5/8 disrupts the pluripotency GRN by binding and repurposing Oct4 for activation of the NMP gene program.
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