A dorsal-ventral gradient of Wnt3a/ß-catenin signals controls mouse hindgut extension and colon formation.

Despite the importance of Wnt signaling for adult intestinal stem cell homeostasis and colorectal cancer, relatively little is known about its role in colon formation during embryogenesis. The development of the colon starts with the formation and extension of the hindgut. We show that Wnt3a is expressed in the caudal embryo in a dorsal-ventral (DV) gradient across all three germ layers, including the hindgut. Using genetic and lineage-tracing approaches, we describe novel dorsal and ventral hindgut domains, and show that ventrolateral hindgut cells populate the majority of the colonic epithelium. A Wnt3a-ß-catenin-Sp5/8 pathway, which is active in the dorsal hindgut endoderm, is required for hindgut extension and colon formation. Interestingly, the absence of Wnt activity in the ventral hindgut is crucial for proper hindgut morphogenesis, as ectopic stabilization of ß-catenin in the ventral hindgut via gain- or loss-of-function mutations in Ctnnb1 or Apc, respectively, leads to severe colonic hyperplasia. Thus, the DV Wnt gradient is required to coordinate growth between dorsal and ventral hindgut domains to regulate the extension of the hindgut that leads to colon formation.

A new gain-of-function mouse line to study the role of Wnt3a in development and disease.

Wnt/ß-catenin signals are important regulators of embryonic and adult stem cell self-renewal and differentiation and play causative roles in tumorigenesis. Purified recombinant Wnt3a protein, or Wnt3a-conditioned culture medium, has been widely used to study canonical Wnt signaling in vitro or ex vivo. To study the role of Wnt3a in embryogenesis and cancer models, we developed a Cre recombinase activatable Rosa26(Wnt3a) allele, in which a Wnt3a cDNA was inserted into the Rosa26 locus to allow for conditional, spatiotemporally defined expression of Wnt3a ligand for gain-of-function (GOF) studies in mice. To validate this reagent, we ectopically overexpressed Wnt3a in early embryonic progenitors using the T-Cre transgene. This resulted in up-regulated expression of a ß-catenin/Tcf-Lef reporter and of the universal Wnt/ß-catenin pathway target genes, Axin2 and Sp5. Importantly, T-Cre; Rosa26(Wnt3a) mutants have expanded presomitic mesoderm (PSM) and compromised somitogenesis and closely resemble previously studied T-Cre; Ctnnb1(ex3) (ß-catenin(GOF) ) mutants. These data indicate that the exogenously expressed Wnt3a stimulates the Wnt/ß-catenin signaling pathway, as expected. The Rosa26(Wnt3a) mouse line should prove to be an invaluable tool to study the function of Wnt3a in vivo.

Zfp703 Is a Wnt/ß-Catenin Feedback Suppressor Targeting the ß-Catenin/Tcf1 Complex.

The Wnt/ß-catenin signaling pathway controls embryonic development and adult stem cell maintenance through the regulation of transcription. Failure to downregulate Wnt signaling can result in embryonic malformations and cancer, highlighting the important role of negative regulators of the pathway. The Wnt pathway activates several negative feedback targets, including axin2 and Dkk1, that function at different levels of the signaling cascade; however, none have been identified that directly target active ß-catenin/Tcf1 transcriptional complexes. We show that Zfp703 is a Wnt target gene that inhibits Wnt/ß-catenin activity in Wnt reporter assays and in Wnt-dependent mesoderm differentiation in embryonic stem cells. Zfp703 binds directly to Tcf1 to inhibit ß-catenin/Tcf1 complex formation and does so independently of the Groucho/Tle transcriptional corepressor. We propose that Zfp703 is a novel feedback suppressor of Wnt/ß-catenin signaling that functions by inhibiting the association of ß-catenin with Tcf1 on Wnt response elements in target gene enhancers.

An anteroposterior wave of vascular inhibitor downregulation signals aortae fusion along the embryonic midline axis.

Paracrine signals, both positive and negative, regulate the positioning and remodeling of embryonic blood vessels. In the embryos of mammals and birds, the first major remodeling event is the fusion of bilateral dorsal aortae at the midline to form the dorsal aorta. Although the original bilaterality of the dorsal aortae occurs as the result of inhibitory factors (antagonists of BMP signaling) secreted from the midline by the notochord, it is unknown how fusion is later signaled. Here, we report that dorsal aortae fusion is tightly regulated by a change in signaling by the notochord along the anteroposterior axis. During aortae fusion, the notochord ceases to exert its negative influence on vessel formation. This is achieved by a transcriptional downregulation of negative regulators while positive regulators are maintained at pre-fusion levels. In particular, Chordin, the most abundant BMP antagonist expressed in the notochord prior to fusion, undergoes a dramatic downregulation in an anterior to posterior wave. With inhibitory signals diminished and sustained expression of the positive factors SHH and VEGF at the midline, fusion of the dorsal aortae is signaled. These results demonstrate a novel mechanism by which major modifications of the vascular pattern can occur through modulation of vascular inhibitors without changes in the levels of positive vascular regulators.

Census of vertebrate Wnt genes: isolation and developmental expression of Xenopus Wnt2, Wnt3, Wnt9a, Wnt9b, Wnt10a, and Wnt16.

The Wnt family of growth factors regulate many different aspects of embryonic development. Assembly of the complete mouse and human genome sequences, plus expressed sequence tag surveys have established the existence of 19 Wnt genes in mammalian genomes. However, despite the importance of model vertebrates for studies in developmental biology, the complete complement of Wnt genes has not been established for nonmammalian genomes. Using genome sequences for chicken (Gallus gallus), frog (Xenopus tropicalis), and fish (Danio rerio and Tetraodon nigroviridis), we have analyzed gene synteny to identify the orthologues of all 19 human Wnt genes in these species. We find that, in addition to the 19 Wnts observed in humans, chicken contained an additional Wnt gene, Wnt11b, which is orthologous to frog and zebrafish Wnt11 (silberblick). Frog and fish genomes contained orthologues of the 19 mammalian Wnt genes, plus Wnt11b and several duplicated Wnt genes. Specifically, the Xenopus tropicalis genome contained 24 Wnt genes, including additional copies of Wnt7-related genes (Wnt7c) and 3 recent Wnt duplications (Wnt3, Wnt9b, and Wnt11). The Danio rerio genome contained 27 Wnt genes with additional copies of Wnt2, Wnt2b, Wnt4b, Wnt6, Wnt7a, and Wnt8a. The presence of the additional Wnt11 sequence (Wnt11b) in the genomes of all ancestral vertebrates suggests that this gene has been lost during mammalian evolution. Through these studies, we identified the frog orthologues of the previously uncharacterized Wnt2, Wnt3, Wnt9a, Wnt9b, Wnt10a, and Wnt16 genes and their expression has been characterized during early Xenopus development.

Wnt11-R signaling regulates a calcium sensitive EMT event essential for dorsal fin development of Xenopus.

In the frog embryo, a sub-population of trunk neural crest (NC) cells undergoes a dorsal route of migration to contribute to the mesenchyme in the core of the dorsal fin. Here we show that a second population of cells, originally located in the dorsomedial region of the somite, also contributes to the fin mesenchyme. We find that the frog orthologue of Wnt11 (Wnt11-R) is expressed in both the NC and somite cell populations that migrate into the fin matrix. Wnt11-R is expressed prior to migration and persists in the mesenchymal cells after they have distributed throughout the fin. Loss of function studies demonstrate that Wnt11-R activity is required for an epithelial to mesenchymal transformation (EMT) event that precedes migration of cells into the fin matrix. In Wnt11-R depleted embryos, the absence of fin core cells leads to defective dorsal fin development and to collapse of the fin structure. Experiments using small molecule inhibitors indicate that dorsal migration of fin core cells depends on calcium signaling through calcium/calmodulin-dependent kinase II (CaMKII). In Wnt11-R depleted embryos, normal migration of NC cells and dorsal somite cells into the fin and normal fin development can be rescued by stimulation of calcium release. These studies are consistent with a model in which Wnt11-R signaling, via a downstream calcium pathway, regulates fin cell migration and, more generally, indicates a role for non-canonical Wnt signaling in regulation of EMT.