Yu-Ping Yang

Fgf8 is required for anterior heart field development

In the mouse embryo, the splanchnic mesodermal cells of the anterior heart field (AHF) migrate from the pharynx to contribute to the early myocardium of the outflow tract (OT) and right ventricle (RV). Recent studies have attempted to distinguish the AHF from other precardiac populations, and to determine the genetic and molecular mechanisms that regulate its development. Here, we have used an Fgf8lacZ allele to demonstrate that Fgf8 is expressed within the developing AHF. In addition, we use both a hypomorphic Fgf8 allele (Fgf8neo) and Cre-mediated gene ablation to show that Fgf8 is essential for the survival and proliferation of the AHF. Nkx2.5Cre is expressed in the AHF, primary heart tube and pharyngeal endoderm, while TnT-Cre is expressed only within the specified heart tube myocardium. Deletion of Fgf8 by Nkx2.5Cre results in a significant loss of the Nkx2.5Cre lineage and severe OT and RV truncations by E9.5, while the remaining heart chambers (left ventricle and atria) are grossly normal. These defects result from significant decreases in cell proliferation and aberrant cell death in both the pharyngeal endoderm and splanchnic mesoderm. By contrast, ablation of Fgf8 in the TnT-Cre domain does not result in OT or RV defects, providing strong evidence that Fgf8 expression is crucial in the pharyngeal endoderm and/or overlying splanchnic mesoderm of the AHF at a stage prior to heart tube elongation. Analysis of downstream signaling components, such as phosphorylated-Erk and Pea3, identifies the AHF splanchnic mesoderm itself as a target for Fgf8 signaling.

Context-specific α-to-β-cell reprogramming by forced Pdx1 expression

Using single transcription factors to reprogram cells could produce important insights into the epigenetic mechanisms that direct normal differentiation, or counter inappropriate plasticity, or even provide new ways of manipulating normal ontogeny in vitro to control lineage diversification and differentiation. We enforced Pdx1 expression from the Neurogenin-3-expressing endocrine commitment point onward and found during the embryonic period a minor increased β-cell allocation with accompanying reduced α-cell numbers. More surprisingly, almost all remaining Pdx1-containing glucagon/Arx-producing cells underwent a fairly rapid conversion at postnatal stages, through glucagon-insulin double positivity, to a state indistinguishable from normal β cells, resulting in complete α-cell absence. This α-to-β conversion was not caused by activating Pdx1 in the later glucagon-expressing state. Our findings reveal that Pdx1 can work single-handedly as a potent context-dependent autonomous reprogramming agent, and suggest a postnatal differentiation evaluation stage involved in normal endocrine maturation.

The Bone Morphogenetic Protein Antagonist Noggin Regulates Mammalian Cardiac Morphogenesis

Bone morphogenetic proteins (BMPs) play many roles in mammalian cardiac development. Here we address the functions of Noggin, a dedicated BMP antagonist, in the developing mouse heart. In early cardiac tissues, the Noggin gene is mainly expressed in the myocardial cells of the outflow tract, atrioventricular canal, and future right ventricle. The major heart phenotypes of Noggin mutant embryos are thicker myocardium and larger endocardial cushions. Both defects result from increased cell number. Cell proliferation is increased and cell cycle exit is decreased in the myocardium. Although we find evidence of increased BMP signal transduction in the myocardium and endocardium, we show that the cardiac defects of Noggin mutants are rescued by halving the gene dosage of Bmp4. In culture, BMP increases the epithelial-to-mesenchymal transformation (EMT) of endocardial explant cells. Increased EMT likely accounts for the enlarged atrioventricular cushion. In the outflow tract cushion, we observed an increased contribution of cardiac neural crest cells to the mutant cushion mesenchyme, although many cells of the cushion were not derived from neural crest. Thus the enlarged outflow tract cushion of Noggin mutants likely arises by increased contributions both of endocardial cells that have undergone EMT as well as cells that have migrated from the neural crest. These data indicate that antagonism of BMP signaling by Noggin plays a critical role in ensuring proper levels of cell proliferation and EMT during cardiac morphogenesis in the mouse.

Twist is an essential regulator of the skeletogenic gene regulatory network in the sea urchin embryo

Recent work on the sea urchin endomesoderm gene regulatory network (GRN) offers many opportunities to study the specification and differentiation of each cell type during early development at a mechanistic level. The mesoderm lineages consist of two cell populations, primary and secondary mesenchyme cells (PMCs and SMCs). The micromere-PMC GRN governs the development of the larval skeleton, which is the exclusive fate of PMCs, and SMCs diverge into four lineages, each with its own GRN state. Here we identify a sea urchin ortholog of the Twist transcription factor, and show that it plays an essential role in the PMC GRN and later is involved in SMC formation. Perturbations of Twist either by morpholino knockdown or by overexpression result in defects in progressive phases of PMC development, including specification, ingression/EMT, differentiation and skeletogenesis. Evidence is presented that Twist expression is required for the maintenance of the PMC specification state, and a reciprocal regulation between Alx1 and Twist offers stability for the subsequent processes, such as PMC differentiation and skeletogenesis. These data illustrate the significance of regulatory state maintenance and continuous progression during cell specification, and the dynamics of the sequential events that depend on those earlier regulatory states.

Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9(+) bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3(TA.LO) cell population, defined as Neurog3 transcriptionally active and Sox9(+) and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9(+) Neurog3(TA.LO) progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3(HI) cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9(+) Neurog3(TA.LO) progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9(+) Neurog3(TA.LO) endocrine-biased progenitors feed production of Neurog3(HI) endocrine-committed cells during pancreas organogenesis.

Roles of Bone Morphogenetic Protein signaling and its antagonism in holoprosencephaly

Holoprosencephaly (HPE) is the most common malformation of the forebrain, resulting from a failure to completely septate the left and right hemispheres at the rostral end of the neural tube. Because of the tissue interactions that drive head development, these forebrain defects are typically accompanied by midline deficiencies of craniofacial structures. Early events in setting up tissue precursors of the head, as well as later interactions between these tissues, are critical for normal head formation. Defects in either process can result in HPE. Signaling by bone morphogenetic proteins (BMPs), a family of secreted cytokines, generally plays negative roles in early stages of head formation, and thus must be attenuated in multiple contexts to ensure proper forebrain and craniofacial development. Chordin and Noggin are endogenous, extracellular antagonists of BMP signaling that promote the normal organization of the forebrain and face. Mouse mutants with reduced levels of both factors display mutant phenotypes remarkably analogous to the range of malformations seen in human HPE sequence. Chordin and Noggin function in part by antagonizing the inhibitory effects of BMP signaling on the Sonic hedgehog and Nodal pathways, genetic lesions in each being associated with human HPE. Study of Chordin;Noggin mutant mice is helping us to understand the molecular, cellular, and genetic pathogenesis of HPE and associated malformations.

BMP antagonism protects Nodal signaling in the gastrula to promote the tissue interactions underlying mammalian forebrain and craniofacial patterning

Holoprosencephaly (HPE) is the most common forebrain and craniofacial malformation syndrome in humans. The genetics of HPE suggest that it often stems from a synergistic interaction of mutations in independent loci. In mice, several combinations of mutations in Nodal signaling pathway components can give rise to HPE, but it is not clear whether modest deficits of Nodal signaling along with lesions in other pathways might also cause such defects. We find that HPE results from simultaneous reduction of Nodal signaling and an organizer BMP (bone morphogenetic protein) antagonist, either Chordin or Noggin. These defects result from reduced production of tissues that promote forebrain and craniofacial development. Nodal promotes the expression of genes in the anterior primitive streak that are important for the development of these tissues, whereas BMP inhibits their expression. Pharmacological and transgenic manipulation of these signaling pathways suggests that BMP and Nodal antagonize each other prior to intracellular signal transduction. Biochemical experiments in vitro indicate that secreted Bmp2 and Nodal can form extracellular complexes, potentially interfering with receptor activation. Our results reveal that the patterning of forebrain and medial craniofacial elements requires a fine balance between BMP and Nodal signaling during primitive streak development, and provide a potential mechanistic basis for a new multigenic model of HPE.

Chemicals turn human embryonic stem cells towards beta cells

Small-molecule library screening identifies simple imitators of the cellular signaling events that normally guide formation of the pancreas and its insulin-secreting beta cells, further enabling detailed analysis in vitro, or eventual diabetes therapies via large-scale differentiation of human stem cells.

The mammal-specific Pdx1 Area II enhancer has multiple essential functions in early endocrine cell specification and postnatal β-cell maturation

ABSTRACT The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5′ conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (−2139 to −1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo. Pdx1ΔAREAII/− mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon+ α relative to insulin+ β cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions. Summary: A crucial enhancer element in the Pdx1 promoter, termed Area II, is required for endocrine progenitor specification, α/β-cell lineage diversification and β-cell maturation in mice.

Clustering of integrin α5 at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells

Integrin α5 clustering at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells via deposition and polymerization of fibronectin and recruitment of paxillin to cluster sites, followed by tight junction formation.