Nkx6.1 and nkx6.2 regulate α- and β-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells

In mice, the Nkx6 genes are crucial to α- and β-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in β-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of α-cells but has no effect on β-, δ-, or ε-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of β-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both β- and α-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for α-cell than for β-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control α- and β-cell differentiation.     

Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development

Mice carrying loss-of-function mutations in certain Notch pathway genes display increased and accelerated pancreatic endocrine development, leading to depletion of precursor cells followed by pancreatic hypoplasia. Here, we have investigated the effect of expressing a constitutively active form of the Notch1 receptor (Notch1(ICD)) in the developing pancreas using the pdx1 promoter. At e10.5 to e12.5, we observe a disorganized pancreatic epithelium with reduced numbers of endocrine cells, confirming a repressive activity of Notch1 upon the early differentiation program. Subsequent branching morphogenesis is impaired and the pancreatic epithelium forms cyst-like structures with ductal phenotype containing a few endocrine cells but completely devoid of acinar cells. The endocrine cells that do form show abnormal expression of cell type-specific markers. Our observations show that sustained Notch1 signaling not only significantly represses endocrine development, but also fully prevents pancreatic exocrine development, suggesting that a possible role of Notch1 is to maintain the undifferentiated state of common pancreatic precursor cells.     

Activation of Tolloid‐like 1 gene expression by the cardiac specific homeobox gene Nkx2–5

Mammalian Tolloid‐like 1 (Tll‐1) is a pleiotropic metalloprotease that is expressed by a small subset of cells within the precardiac mesoderm and is necessary for proper heart development. Following heart tube formation Tll‐1 is expressed by the endocardium and regions of myocardium overlying the region of the muscular interventricular septum. Mutations in Tll‐1 lead to embryonic lethality due to cardiac defects. We demonstrate that the Tll‐1 promoter contains Nkx2–5 binding sites and that the Tll‐1 promoter is activated by and directly binds Nkx2–5. Tll‐1 expression is ablated by a dominant negative Nkx2–5 or by mutation of the Nkx2–5 binding sites within the Tll‐1 promoter. In vivo, Tll‐1 expression is decreased in the hearts of Nkx2–5 knockout embryos when compared with hemizygous and wild‐type embryos. These results show that Nkx2–5 is a direct activator of Tll‐1 expression and provide insight into the mechanism of the defects found in both the Tll‐1 and Nkx2–5 knockout mice.     

Differential expression of insulin genes 1 and 2 in MIN6 cells and pseudoislets

There is some evidence that the two rodent insulin genes are differentially regulated in mice, although there is no satisfactory consensus on the relative levels and patterns of expression for the two genes. Using the mouse insulinoma cell line MIN6, we have demonstrated by quantitative RT-PCR, differential patterns of expression for the two genes. In mouse islets and early passage MIN6 cells, expression of ins 1 and ins 2 were found to be approximately equal, but levels of ins 1 mRNA diminished rapidly with continued passage. Furthermore, the ins 1 gene was found to be up-regulated in response to glucose stimulation and as a result of increased cell-cell contact, but no effect on the ins 2 gene was observed. Since the MIN6 cell line is frequently used as a beta-cell model for gene expression studies, consideration should be given to both insulin genes.     

Appropriate expression of imprinted genes on mouse chromosome 12 extends development of bi-maternal embryos to term

Recently, we reported that the restored regulation of imprinted gene expression from two regions -H19 differentially methylated region (H19-DMR) and intergenic germline-derived DMR (IG-DMR) - is sufficient for accomplishing full-term development in mice. In the present study, we determined the developmental ability of the bi-maternal embryos (BMEs) containing the non-growing oocyte genome with the IG-DMR deletion (ng(Deltach12)) and fully-grown (fg) oocyte genome. Foetuses derived from ng(Deltach12)/fg BMEs were alive at E19.5 but could not survive further. Comparison with BMEs derived from Igf2+/- ng/fg genomes suggests that bi-allelic H19 expression might be involved in foetal development.     

Deletion of the Pitx1 genomic locus affects mandibular tooth morphogenesis and expression of the Barx1 and Tbx1 genes

Pitx1 is a bicoid-related homeodomain factor that exhibits preferential expression in the developing hindlimb, mandible, pituitary gland and teeth. Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of both hindlimb and mandible, suggesting a proliferative defect in these two structures. Here, we studied the expression and regulation of Pitx1 in both mandible and developing teeth and analyzed tooth morphology, cell proliferation, apoptosis and expression of Pitx2, Barx1 and Tbx1 in dental tissues of Pitx1−/− mouse embryos. Pitx1 expression is restricted to the epithelium of the growing tooth anlagen. Tissue recombination and bead implantation experiments demonstrated that bone morphogenetic protein-4 down-regulates Pitx1 expression in both mandibular mesenchyme and dental epithelium. Deletion of the Pitx1 locus results in micrognathia and abnormal morphology of the mandibular molars. Although Pitx2 expression in teeth of Pitx1−/− embryos is not altered, expression of Barx1 decreased in the mesenchyme of the mandibular molars. Furthermore, Pitx1 deletion results in suppression of Tbx1 expression in dental epithelium. Taken together, these results indicate that independent genetic pathways in mandibular and maxillary processes determine tooth development and morphology.     

The effect of feed restriction on expression of hepatic lipogenic genes in broiler chickens and the function of SREBP1

To study the role of sterol regulatory element-binding proteins (SREBP) in lipogenesis and cholesterol synthesis in the chicken, two experiments were carried out. In the first study, seven-week-old broilers (n = 16) were allocated into 2 groups, fasted for 24 h or refed for 5 h after a 24 h fasting. The mRNA concentrations for SREBPs and other lipogenic genes in the liver were determined by quantitative real time PCR. The hepatic mRNA relative abundance of lipogenic genes and genes involved in cholesterol synthesis were significantly greater (p < 0.001) in the refed broilers. Similar results were demonstrated with Northern analysis. The data suggest that in the liver of fasted broilers, genes associated with lipogenesis and cholesterol biosynthesis were inhibited. Indeed, the mRNA concentrations for fatty acid synthase (FAS), malic enzyme, and stearoyl coenzyme A desaturase were almost undetectable after the 24 h fasting. The data also demonstrated that the expression of lipogenic genes coordinate well as a group during the refeeding period. Second, three small interfering RNA (siRNA) oligonucleotides against SREBP1 were designed to be used in transfecting a chicken hepatocarcinoma cell line LMH. One of the three siRNAs effectively reduced SREBP1 mRNA concentration (p < 0.01). The acetyl coenzyme A carboxylase_α (ACC_α) mRNA was also significantly reduced by the SREBP1 siRNA treatment, suggesting that SREBP1 can upregulate the expression of this lipogenic gene. This siRNA, however, did not affect the mRNA for FAS. Taken together, the RNA interference study showed that SREBP1 has the ability to regulate the expression of ACC_α. This study has helped us understand more about the function of SREBP1 and the physiology of the broiler chickens.     

Developmental Expression of the Glycine Transporters GLYT1 and GLYT2 in Mouse Brain

Abstract: Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.     

Inactivation of Patched1 in the Mouse Limb Has Novel Inhibitory Effects on the Chondrogenic Program

The bones of the vertebrate limb form by the process of endochondral ossification, whereby limb mesenchyme condenses to form an intermediate cartilage scaffold that is then replaced by bone. Although Indian hedgehog (IHH) is known to control hypertophic differentiation of chondrocytes during this process, the role of hedgehog signaling in the earlier stages of chondrogenesis is less clear. We have conditionally inactivated the hedgehog receptor Ptc1 in undifferentiated limb mesenchyme of the mouse limb using Prx1-Cre, thus inducing constitutively active ligand-independent hedgehog signaling. In addition to major patterning defects, we observed a marked disruption to the cartilage elements in the limbs of Prx1-Cre:Ptc1^c/c embryos. Using an in vitro micromass culture system we show that this defect lies downstream of mesenchymal cell condensation and likely upstream of chondrocyte differentiation. Despite early increases in levels of chondrogenic genes, soon after mesenchymal condensation the stromal layer of Prx1-Cre:Ptc1^c/c-derived micromass cultures is characterized by a loss of cell integrity, which is associated with increased cell death and a striking decrease in Alcian blue staining cartilage nodules. Furthermore, inhibition of the hedgehog pathway activation using cyclopamine was sufficient to essentially overcome this chondrogenic defect in both micromass and ex vivo explant assays of Prx1-Cre:Ptc1^c/c limbs. These data demonstrate for the first time the inhibitory effect of cell autonomously activated hedgehog signaling on chondrogenesis, and stress the importance of PTC1 in maintaining strict control of signaling levels during this phase of skeletal development.