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.

Sp5 and Sp8 recruit ß-catenin and Tcf1-Lef1 to select enhancers to activate Wnt target gene transcription.

The ancient, highly conserved, Wnt signaling pathway regulates cell fate in all metazoans. We have previously shown that combined null mutations of the specificity protein (Sp) 1/Klf-like zinc-finger transcription factors Sp5 and Sp8 (i.e., Sp5/8) result in an embryonic phenotype identical to that observed when core components of the Wnt/ß-catenin pathway are mutated; however, their role in Wnt signal transduction is unknown. Here, we show in mouse embryos and differentiating embryonic stem cells that Sp5/8 are gene-specific transcriptional coactivators in the Wnt/ß-catenin pathway. Sp5/8 bind directly to GC boxes in Wnt target gene enhancers and to adjacent, or distally positioned, chromatin-bound T-cell factor (Tcf) 1/lymphoid enhancer factor (Lef) 1 to facilitate recruitment of ß-catenin to target gene enhancers. Because Sp5 is itself directly activated by Wnt signals, we propose that Sp5 is a Wnt/ß-catenin pathway-specific transcript on factor that functions in a feed-forward loop to robustly activate select Wnt target genes.

Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation.

In the development of the vertebrate body plan, Wnt3a is thought to promote the formation of paraxial mesodermal progenitors (PMPs) of the trunk region while suppressing neural specification. Recent lineage-tracing experiments have demonstrated that these trunk neural progenitors and PMPs derive from a common multipotent progenitor called the neuromesodermal progenitor (NMP). NMPs are known to reside in the anterior primitive streak (PS) region; however, the extent to which NMPs populate the PS and contribute to the vertebrate body plan, and the precise role that Wnt3a plays in regulating NMP self-renewal and differentiation are unclear. To address this, we used cell-specific markers (Sox2 and T) and tamoxifen-induced Cre recombinase-based lineage tracing to locate putative NMPs in vivo. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/ß-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2(+)T(+) multipotent NMPs and form the bulk of neural progenitors and PMPs of the posterior trunk region. Finally, we find that Wnt3a/ß-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors.

Mesogenin 1 is a master regulator of paraxial presomitic mesoderm differentiation.

Neuromesodermal (NM) stem cells generate neural and paraxial presomitic mesoderm (PSM) cells, which are the respective progenitors of the spinal cord and musculoskeleton of the trunk and tail. The Wnt-regulated basic helix-loop-helix (bHLH) transcription factor mesogenin 1 (Msgn1) has been implicated as a cooperative regulator working in concert with T-box genes to control PSM formation in zebrafish, although the mechanism is unknown. We show here that, in mice, Msgn1 alone controls PSM differentiation by directly activating the transcriptional programs that define PSM identity, epithelial-mesenchymal transition, motility and segmentation. Forced expression of Msgn1 in NM stem cells in vivo reduced the contribution of their progeny to the neural tube, and dramatically expanded the unsegmented mesenchymal PSM while blocking somitogenesis and notochord differentiation. Expression of Msgn1 was sufficient to partially rescue PSM differentiation in Wnt3a(-/-) embryos, demonstrating that Msgn1 functions downstream of Wnt3a as the master regulator of PSM differentiation. Our data provide new insights into how cell fate decisions are imposed by the expression of a single transcriptional regulator.

Reciprocal myocardial-endocardial interactions pattern the delay in atrioventricular junction conduction.

Efficient blood flow depends on two developmental processes that occur within the atrioventricular junction (AVJ) of the heart: conduction delay, which entrains sequential chamber contraction; and valve formation, which prevents retrograde fluid movement. Defects in either result in severe congenital heart disease; however, little is known about the interplay between these two crucial developmental processes. Here, we show that AVJ conduction delay is locally assigned by the morphogenetic events that initiate valve formation. Our data demonstrate that physical separation from endocardial-derived factors prevents AVJ myocardium from becoming fast conducting. Mechanistically, this physical separation is induced by myocardial-derived factors that support cardiac jelly deposition at the onset of valve formation. These data offer a novel paradigm for conduction patterning, whereby reciprocal myocardial-endocardial interactions coordinate the processes of valve formation with establishment of conduction delay. This, in turn, synchronizes the electrophysiological and structural events necessary for the optimization of blood flow through the developing heart.

Isolation and culture of mouse proepicardium using serum-free conditions.

The proepicardium (PE) is an embryonic tissue that gives rise to multipotent vascular progenitors. Most notably the PE gives rise to the epicardium, cardiac fibroblasts, myocardium, and coronary vessels including both vascular smooth muscle and vascular endothelium. Much attention has been given to epicardial-derived cells that show the capacity to differentiate into a wide variety of vascular progenitors including cardiomyocytes. However, it is the PE itself that possesses the greatest potential as a source of multipotent vascular progenitors. We show here a simple method to manually isolate mouse PE at the ninth day of mouse embryonic development and culture highly pure PE tissue in serum-free conditions. This PE culture method allows for the ex vivo analysis of specific growth factors on PE and epicardial development with greater efficiency and precision than existing epicardial culture methods.

Developmental expression and cardiac transcriptional regulation of Myh7b, a third myosin heavy chain in the vertebrate heart.

The mammalian heart expresses two myosin heavy chain (MYH) genes (Myh6 and Myh7), which are major components of the thick filaments of the sarcomere. We have determined that a third MYH, MYH7B, is also expressed in the myocardium. Developmental analysis shows Myh7b expression in cardiac and skeletal muscle of Xenopus, chick and mouse embryos, and in smooth muscle tissues during later stages of mouse embryogenesis. Myh7b is also expressed in the adult human heart. The promoter region of the Myh7b gene shows remarkable similarity between diverse species, suggesting that transcriptional control mechanisms have been conserved. Using luciferase reporter analysis in rat cardiomyocytes, it can be shown that MEF2, GATA, and E-box regulatory elements are essential for efficient expression of the Myh7b gene. In addition two conserved elements that do not correspond to consensus binding sites for known transcription factors are also essential for full transcriptional activity of the Myh7b reporter. Finally, the Myh7b gene shows a transcriptional response similar to Myh6 in response to cardiac hypertrophy.

Early arterial differentiation and patterning in the avian embryo model.

Of the many models to study vascular biology the avian embryo remains an informative and powerful model system that has provided important insights into endothelial cell recruitment, assembly and remodeling during development of the circulatory system. This review highlights several discoveries in the avian system that show how arterial patterning is regulated using the model of dorsal aortae development along the embryo midline during gastrulation and neurulation. These discoveries were made possible through spatially and temporally controlled gain-of-function experiments that provided direct evidence that BMP signaling plays a pivotal role in vascular recruitment, patterning and remodeling and that Notch-signaling recruits vascular precursor cells to the dorsal aortae. Importantly, BMP ligands are broadly expressed throughout embryos but BMP signaling activation region is spatially defined by precisely regulated expression of BMP antagonists. These discoveries provide insight into how signaling, both positive and negative, regulate vascular patterning. This review also illustrates similarities of early arterial patterning along the embryonic midline in amniotes both avian and mammalians including human, evolutionarily specialized from non-amniotes such as fish and frog.

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.

BMP signals promote proepicardial protrusion necessary for recruitment of coronary vessel and epicardial progenitors to the heart.

The coronary vessels and epicardium arise from an extracardiac rudiment called the proepicardium. Failed fusion of the proepicardium to the heart results in severe coronary and heart defects. However, it is unknown how the proepicardium protrudes toward and attaches to the looping heart tube. Here, we show that ectopic expression of BMP ligands in the embryonic myocardium can cause proepicardial cells to target aberrant regions of the heart. Additionally, misexpression of a BMP antagonist, Noggin, suppresses proepicardium protrusion and contact with the heart. Finally, proepicardium explant preferentially expands toward a cocultured heart segment. This preference can be mimicked by BMP2/4 and suppressed by Noggin. These results support a model in which myocardium-derived BMP signals regulate the entry of coronary progenitors to the specific site of the heart by directing their morphogenetic movement.

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation.

Knowledge of the molecular mechanisms regulating cell ingression, epithelial-mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.

Expression of the fast twitch troponin complex, fTnT, fTnI and fTnC, in vascular smooth muscle.

It is generally believed that proteins of the troponin complex are not expressed in smooth muscle. We have directly assayed for expression of troponin transcripts in mouse vascular smooth muscle and found that troponin sequences normally associated with fast twitch skeletal muscle (fTnT, fTnI, fTnC) were present at significant levels in the thoracic aorta. In situ hybridization experiments demonstrated that fTnT, fTnI and fTnC transcripts were expressed in the smooth muscle layer of mouse blood vessels of all sizes. Protein blot analysis using rat tissue showed that at least two members of the troponin complex, Troponin T and Troponin I, were translated in vascular smooth muscle of the aorta. Finally, immuno-fluorescence microscopy of rat aortic smooth muscle revealed that TnT and TnI are localized in a unique pattern, coincident with the distribution of tropomyosin. It seems likely therefore, that a complete troponin complex is expressed in vascular smooth muscle and is associated with the contractile machinery of the cell. These observations raise the possibility that troponins play a role in regulation of smooth muscle function.

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.

Developmental expression and comparative genomic analysis of Xenopus cardiac myosin heavy chain genes.

Myosin heavy chains (MHC) are cytoskeletal motor proteins essential to the process of muscle contraction. We have determined the complete sequences of the Xenopus cardiac MHC genes, alpha-MHC and ventricular MHC (vMHC), and have characterized their developmental expression profiles. Whereas alpha-MHC is expressed from the earliest stages of cardiac differentiation, vMHC transcripts are not detected until the heart has undergone chamber formation. Early expression of vMHC appears to mark the cardiac conduction system, but expression expands to include the ventricle and outflow tract myocardium during subsequent development. Sequence comparisons, transgenic expression analysis, and comparative genomic studies indicate that Xenopus alpha-MHC is the true orthologue of the mammalian alpha-MHC gene. On the other hand, we show that the Xenopus vMHC gene is most closely related to chicken ventricular MHC (vMHC1) not the mammalian beta-MHC. Comparative genomic analysis has allowed the detection of a mammalian MHC gene (MyH15) that appears to be the orthologue of vMHC, but evidence suggests that this gene is no longer active.

Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus.

Wnt11 is a secreted protein that signals through the non-canonical planar cell polarity pathway and is a potent modulator of cell behavior and movement. In human, mouse, and chicken, there is a single Wnt11 gene, but in zebrafish and Xenopus, there are two genes related to Wnt11. The originally characterized Xenopus Wnt11 gene is expressed during early embryonic development and has a critical role in regulation of gastrulation movements. We have identified a second Xenopus Wnt11-Related gene (Wnt11-R) that is expressed after gastrulation. Sequence comparison suggests that Xenopus Wnt11-R, not Wnt11, is the ortholog of mammalian and chicken Wnt11. Xenopus Wnt11-R is expressed in neural tissue, dorsal mesenchyme derived from the dermatome region of the somites, the brachial arches, and the muscle layer of the heart, similar to the expression patterns reported for mouse and chicken Wnt11. Xenopus Wnt11-R exhibits biological properties similar to those previously described for Xenopus Wnt11, in particular the ability to activate Jun-N-terminal kinase (JNK) and to induce myocardial marker expression in ventral marginal zone (VMZ) explants. Morpholino inhibition experiments demonstrate, however, that Wnt11-R is not required for cardiac differentiation, but functions in regulation of cardiac morphogenesis. Embryos with reduced Wnt11-R activity exhibit aberrant cell-cell contacts within the myocardial wall and defects in fusion of the nascent heart tube.

Frzb modulates Wnt-9a-mediated beta-catenin signaling during avian atrioventricular cardiac cushion development.

Normal development of the cardiac atrioventricular (AV) endocardial cushions is essential for proper ventricular septation and morphogenesis of the mature mitral and tricuspid valves. In this study, we demonstrate spatially restricted expression of both Wnt-9a (formerly Wnt-14) and the secreted Wnt antagonist Frzb in AV endocardial cushions of the developing chicken heart. Wnt-9a expression is detected only in AV canal endocardial cells, while Frzb expression is detected in both endocardial and transformed mesenchymal cells of the developing AV cardiac cushions. We present evidence that Wnt-9a promotes cell proliferation in the AV canal and overexpression of Wnt-9a in ovo results in enlarged endocardial cushions and AV inlet obstruction. Wnt-9a stimulates beta-catenin-responsive transcription in AV canal cells, duplicates the embryonic axis upon ventral injections in Xenopus embryos and appears to regulate cell proliferation by activating a Wnt/beta-catenin signaling pathway. Additional functional studies reveal that Frzb inhibits Wnt-9a-mediated cell proliferation in cardiac cushions. Together, these data argue that Wnt-9a and Frzb regulate mesenchymal cell proliferation leading to proper AV canal cushion outgrowth and remodeling in the developing avian heart.

Regulation of heart size in Xenopus laevis.

The region with the potential to form the heart has traditionally been called the heart field. This region can be approximated by, but is not identical to, the expression domain of the early cardiac gene Nkx2.5. The region expressing Nkx2.5 does not change in size, although there are major shape changes and a subdivision of the region into non-myogenic and myogenic lineages. Using a variety of embryo manipulations, we have sought to determine whether cellular interactions could change the size of the initial Nkx2.5-expressing region and thus change the size of the heart. We have shown that if the heart is isolated from the dorsal half of the embryo, the volume of tissue expressing myocardial differentiation markers increases, indicating that signals restricting the size of the heart come from the dorsal side. Despite the change in myocardial volume, the non-myogenic heart lineages are still present. The ability of dorsal tissues to restrict the size of the heart is further demonstrated by fusing two Xenopus embryos shortly after gastrulation, generating twinned embryos where the heart of one embryo would develop adjacent to different tissues of the second embryo. The final size of the differentiated heart was markedly reduced if it developed in close proximity to the dorso-anterior surface of the head but not if it developed adjacent to the flank or belly. In all cases, the manipulations that restricted the size of the myocardium also restricted the expression of Nkx2.5 and GATA-4, both key regulatory genes in the cardiogenic pathway. These results provide evidence for a model in which signals from dorso-anterior tissues restrict the size of the heart after gastrulation but before neural fold closure.