Aaron M. Zorn

Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro

Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal ‘organoids’ consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.

The Genome of the Western Clawed Frog Xenopus tropicalis

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633, see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes. Assembly, annotation, and analysis of the frog genome compares gene content and synteny with the human and chicken genomes. The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Vertebrate Endoderm Development and Organ Formation

The endoderm germ layer contributes to the respiratory and gastrointestinal tracts and to all of their associated organs. Over the past decade, studies in vertebrate model organisms, including frog, fish, chick, and mouse, have greatly enhanced our understanding of the molecular basis of endoderm organ development. We review this progress with a focus on early stages of endoderm organogenesis including endoderm formation, gut tube morphogenesis and patterning, and organ specification. Lastly, we discuss how developmental mechanisms that regulate endoderm organogenesis are used to direct differentiation of embryonic stem cells into specific adult cell types, which function to alleviate disease symptoms in animal models.

Repression of Wnt/β-catenin signaling in the anterior endoderm is essential for liver and pancreas development

The liver and pancreas are specified from the foregut endoderm through an interaction with the adjacent mesoderm. However, the earlier molecular mechanisms that establish the foregut precursors are largely unknown. In this study, we have identified a molecular pathway linking gastrula-stage endoderm patterning to organ specification. We show that in gastrula and early-somite stage Xenopus embryos, Wnt/β-catenin activity must be repressed in the anterior endoderm to maintain foregut identity and to allow liver and pancreas development. By contrast, high β-catenin activity in the posterior endoderm inhibits foregut fate while promoting intestinal development. Experimentally repressing β-catenin activity in the posterior endoderm was sufficient to induce ectopic organ buds that express early liver and pancreas markers. β-catenin acts in part by inhibiting expression of the homeobox gene hhex, which is one of the earliest foregut markers and is essential for liver and pancreas development. Promoter analysis indicates that β-catenin represses hhex transcription indirectly via the homeodomain repressor Vent2. Later in development,β -catenin activity has the opposite effect and enhances liver development. These results illustrate that turning Wnt signaling off and on in the correct temporal sequence is essential for organ formation, a finding that might directly impact efforts to differentiate liver and pancreas tissue from stem cells.

Sox17 and β-catenin cooperate to regulate the transcription of endodermal genes

Recent studies have led to a model of the molecular pathway that specifies the endoderm during vertebrate gastrulation. The HMG box transcription factor Sox17 is a key component of this pathway and is essential for endoderm formation; however, the molecular events controlled by Sox17 are largely unknown. We have identified several direct transcriptional targets of Sox17, including Foxa1 and Foxa2. We show that β-catenin, a component of Wnt signaling pathway, physically interacts with Sox17 and potentiates its transcriptional activation of target genes. We identify a motif in the C terminus of Sox17, which is conserved in all the SoxF subfamily of Sox proteins, and this motif is required for the ability of Sox17 to both transactivate target genes and bind β-catenin. Nuclear β-catenin is present in endoderm cells of the gastrula, and depletion of β-catenin from embryos results in a repression of Sox17 target genes. These data suggest that in a mechanism analogous to Tcf/Lef interacting with β-catenin, Sox17 and β-catenin interact to transcribe endodermal target genes.

Mutations in Cardiac T-Box Factor Gene TBX20 Are Associated with Diverse Cardiac Pathologies, Including Defects of Septation and Valvulogenesis and Cardiomyopathy

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. We report here analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carried strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bound specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacted with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, led to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. We propose that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development.

Sox17 and Sox4 Differentially Regulate β-Catenin/T-Cell Factor Activity and Proliferation of Colon Carcinoma Cells

ABSTRACT The canonical Wnt pathway is necessary for gut epithelial cell proliferation, and aberrant activation of this pathway causes intestinal neoplasia. We report a novel mechanism by which the Sox family of transcription factors regulate the canonical Wnt signaling pathway. We found that some Sox proteins antagonize while others enhance β-catenin/T-cell factor (TCF) activity. Sox17, which is expressed in the normal gut epithelium but exhibits reduced expression in intestinal neoplasia, is antagonistic to Wnt signaling. When overexpressed in SW480 colon carcinoma cells, Sox17 represses β-catenin/TCF activity in a dose-dependent manner and inhibits proliferation. Sox17 and Sox4 are expressed in mutually exclusive domains in normal and neoplastic gut tissues, and gain- and loss-of-function studies demonstrate that Sox4 enhances β-catenin/TCF activity and the proliferation of SW480 cells. In addition to binding β-catenin, both Sox17 and Sox4 physically interact with TCF/lymphoid enhancer factor (LEF) family members via their respective high-mobility-group box domains. Results from gain- and loss-of-function experiments suggest that the interaction of Sox proteins with β-catenin and TCF/LEF proteins regulates the stability of β-catenin and TCF/LEF. In particular, Sox17 promotes the degradation of both β-catenin and TCF proteins via a noncanonical, glycogen synthase kinase 3β-independent mechanism that can be blocked by proteasome inhibitors. In contrast, Sox4 may function to stabilize β-catenin protein. These findings indicate that Sox proteins can act as both antagonists and agonists of β-catenin/TCF activity, and this mechanism may regulate Wnt signaling responses in many developmental and disease contexts.

Wnt signalling: antagonistic Dickkopfs.

Dickkopf proteins are secreted antagonists of the Wnt cell signalling molecules, which have a novel mode of action. Dickkopf1 binds to the LRP5/6 Wnt co-receptor and prevents the formation of active Wnt--Frizzled--LRP5/6 receptor complexes, thus blocking the canonical Wnt--beta-catenin pathway.

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ‘fossil’ transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17–18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.