Understanding the molecular mechanisms of stem cell maintenance is crucial for the ultimate goal of manipulating stem cells for the treatment of disease. Foxd3 is required early in mouse embryogenesis; Foxd3 -/-embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and blastocyst-derived stem cell lines cannot be established. Here, we report that Foxd3 is required for maintenance of the multipotent mammalian neural crest. Using tissue-specific deletion of Foxd3 in the neural crest, we show that Foxd3 flox/-; Wnt1-Cre mice die perinatally with a catastrophic loss of neural crest-derived structures. Cranial neural crest tissues are either missing or severely reduced in size, the peripheral nervous system consists of reduced dorsal root ganglia and cranial nerves, and the entire gastrointestinal tract is devoid of neural crest derivatives. These results demonstrate a global role for this transcriptional repressor in all aspects of neural crest maintenance along the anterior-posterior axis, and establish an unprecedented molecular link between multiple divergent progenitor lineages of the mammalian embryo.
Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of Insulin-expressing cells and Insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of Insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of Insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic Insulin granules and the presence of abnormal granules in Insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation.
A structure-activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known BMP inhibitors dorsomorphin, 1, LDN193189, 2, and DMH1, 3, led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation.
SUMMARY Canonical Wnt signaling pathway, mediated by the transcription factor β-catenin, plays critical roles in embryonic development, and represents an important therapeutic target. In a zebrafish-based in vivo screen for small molecules that specifically perturb embryonic dorsoventral patterning, we discovered a novel compound, named windorphen, which selectively blocks the Wnt signal required for ventral development. Windorphen exhibits remarkable specificity toward β-catenin-1 function, indicating that the two β-catenin isoforms found in zebrafish are not functionally redundant. We show that windorphen is a selective inhibitor of p300 histone acetyl transferase, a co-activator that associates with β-catenin. Lastly, windorphen robustly and selectively kills cancer cells that harbor Wnt-activating mutations, supporting the therapeutic potential of this novel Wnt inhibitor class.
A complete molecular understanding of -cell mass expansion will be useful for the improvement of therapies to treat diabetic patients. During normal periods of metabolic challenges, such as pregnancy, -cells proliferate, or self-renew, to meet the new physiological demands. The transcription factor Forkhead box D3 (Foxd3) is required for maintenance and self-renewal of several diverse progenitor cell lineages, and Foxd3 is expressed in the pancreatic primordium beginning at 10.5 d postcoitum, becoming localized predominantly to -cells after birth. Here, we show that mice carrying a pancreas-specific deletion of Foxd3 have impaired glucose tolerance, decreased -cell mass, decreased -cell proliferation, and decreased -cell size during pregnancy. In addition, several genes known to regulate proliferation, Foxm1, Skp2, Ezh2, Akt2, and Cdkn1a, are misregulated in islets isolated from these Foxd3 mutant mice. Together, these data place A pproximately 7% of pregnant women are affected by gestational diabetes mellitus (GDM), a disease resulting from the inability of the resident -cell population to produce sufficient insulin during pregnancy (1). GDM increases the risk of complications to both mother and newborn child; the mother is more likely to develop type 2 diabetes later in life, and the child is more likely to be born with birth defects, macrosomia, and an increased risk of developing type 2 diabetes (2-6). During normal murine pregnancy, -cells proliferate, or self-renew, thereby expanding the total -cell mass to meet the mother's increasing demand for insulin (1,(7)(8)(9)(10)(11)(12). This mechanism of -cell mass expansion during human pregnancy remains controversial. Analogous measurements are not ethically feasible in humans. However, morphological analyses of human pancreata indicate that -cell mass is increased during pregnancy (13,14). Recent work analyzing pancreata from 38 cadaveric donors (18 pregnant, 20 controls) suggested that -cells do not proliferate during human pregnancy. Instead, an increased number of smaller islets and insulin-positive cells was observed within the ductal epithelium (15). However, this work is not without caveats. It is well known that murine -cells proliferate within a defined time window, and it is likely that the 18 pregnant human donors examined were not within an analogous gestational age (16,17). This study also included donors with inflammatory disease that may have adversely affected -cell mass expansion (18). It is important to note that the pregnancy-associated hormones prolactin, placental lactogen (PL), and human growth hormone all stimulate -cell proliferation in islets isolated from mice, rats, and humans, suggesting that cell proliferation is a conserved mechanism of Abbreviations: BrdU, 5-Bromo-2-deoxyuridine; Cdkn, cyclin-dependent kinase inhibitor; ES, embryonic stem; Ezh2, enhancer of zeste homolog 2; Foxa2, Forkhead box A2; Foxd3, Forkhead box D3; Foxm1, forkhead box M1; GDM, gestational diabetes mellitus; IPGTT, ip glucose tolerance testing;...
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