Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors.

Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling ß cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2) as a specific cell surface marker for isolating pancreatic endoderm cells (PECs) from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK) inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived ß cells.

ß-Catenin Regulates Primitive Streak Induction through Collaborative Interactions with SMAD2/SMAD3 and OCT4.

Canonical Wnt and Nodal signaling are both required for induction of the primitive streak (PS), which guides organization of the early embryo. The Wnt effector ß-catenin is thought to function in these early lineage specification decisions via transcriptional activation of Nodal signaling. Here, we demonstrate a broader role for ß-catenin in PS formation by analyzing its genome-wide binding in a human embryonic stem cell model of PS induction. ß-catenin occupies regulatory regions in numerous PS and neural crest genes, and direct interactions between ß-catenin and the Nodal effectors SMAD2/SMAD3 are required at these regions for PS gene activation. Furthermore, OCT4 binding in proximity to these sites is likewise required for PS induction, suggesting a collaborative interaction between ß-catenin and OCT4. Induction of neural crest genes by ß-catenin is repressed by SMAD2/SMAD3, ensuring proper lineage specification. This study provides mechanistic insight into how Wnt signaling controls early cell lineage decisions.

Neogenin regulates Sonic Hedgehog pathway activity during digit patterning.

BACKGROUND: Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. RESULTS: We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. CONCLUSIONS: Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning.

Murine models of holoprosencephaly.

Holoprosencephaly (HPE), the most common developmental defect of the forebrain and midface, is caused by a failure to delineate the midline in these structures. Both genetic and environmental etiologies exist for HPE, and clinical presentation is highly variable. HPE occurs in sporadic and inherited forms, and even HPE in pedigrees is characterized by incomplete penetrance and variable expressivity. Heterozygous mutations in eight different genes have been identified in human HPE, and disruption of Sonic hedgehog expression and/or signaling in the rostroventral region of the embryo is a major common effect of these mutations. An understanding of the mechanisms whereby genetic defects and teratogenic exposures become manifest as developmental anomalies of varying severity requires experimental models that accurately reproduce the spectrum of defects seen in human HPE. The mouse has emerged as such a model, because of its ease of genetic manipulation and similarity to humans in development of the forebrain and face. HPE is generally observed in mice homozygous for mutations in orthologs of human HPE genes though, unlike humans, rarely in mice with heterozygous mutations. Moreover, reverse genetics in the mouse has provided a wealth of new candidate human HPE genes. Construction of hypomorphic alleles, interbreeding to produce double mutants, and analysis of these mutations on different genetic backgrounds has generated multiple models of HPE and begun to provide insight into the conundrum of the HPE spectrum. Here, we review forebrain development with an emphasis on the pathways known to be defective in HPE and describe the strengths and weaknesses of various murine models of HPE.

Dynamic positive feedback phosphorylation of mixed lineage kinase 3 by JNK reversibly regulates its distribution to Triton-soluble domains.

MLK3 (mixed lineage kinase 3) is a widely expressed, mammalian serine/threonine protein kinase that activates multiple MAPK pathways. Previously our laboratory used in vivo labeling/mass spectrometry to identify phosphorylation sites of activated MLK3. Seven of 11 identified sites correspond to the consensus motif for phosphorylation by proline-directed kinases. Based on these results, we hypothesized that JNK, or another proline-directed kinase, phosphorylates MLK3 as part of a feedback loop. Herein we provide evidence that MLK3 can be phosphorylated by JNK in vitro and in vivo. Blockade of JNK results in dephosphorylation of MLK3. The hypophosphorylated form of MLK3 is inactive and redistributes to a Triton-insoluble fraction. Recovery from JNK inhibition restores MLK3 solubility and activity, indicating that the redistribution process is reversible. This work describes a novel mode of regulation of MLK3, by which JNK-mediated feedback phosphorylation of MLK3 regulates its activation and deactivation states by cycling between Triton-soluble and Triton-insoluble forms.

Cdc42 induces activation loop phosphorylation and membrane targeting of mixed lineage kinase 3.

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation-dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.