Insulin gene is a target in activin receptor-like kinase 7 signaling pathway in pancreatic beta-cells

Activins regulate pancreatic development, differentiation and insulin secretion. Activin receptor-like kinase 7 (ALK7) has been identified as a receptor for Nodal and Activin AB and B, and is expressed in pancreatic islets and β-cell lines. In this study, human insulin promoter was activated by Smad2, Smad3 and the pancreatic and duodenal homeobox factor-1 (PDX-1) in the ALK7 pathway. A conserved Smad binding element was related to the promoter activation. Phosphorylated Smad2/Smad3 and PDX-1 were bound to insulin gene with Nodal and Activin AB, and the phosphorylated Smad2/Smad3 interacted with PDX-1. These results indicate that one of the direct target genes of Nodal and Activin AB signals is the insulin gene in pancreatic β-cells and that PDX-1 is directly involved in the ALK7-Smad pathway.     

Pre-sertoli specific gene expression profiling reveals differential expression of Ppt1 and Brd3 genes within the mouse genital ridge at the time of sex determination

In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated genital ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female genital ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.     

Induction of pancreatic β-cell-like cells from CD44+/CD105+ human amniotic fluids via epigenetic regulation of the pancreatic and duodenal homeobox factor 1 promoter

Pancreatic and duodenal homeobox factor 1 (PDX-1) maintains β-cell function and differentiation via direct regulation of multiple islet cell genes. However, the molecular mechanisms involved in this process remain unknown. Here, we show that PDX-1 plays an important role in the induction of CD44+/CD105+ human amniotic fluid cells (HuAFCs) into functional pancreatic β-cell-like cells in vitro. CD44+/CD105+ HuAFCs were transfected with either siRNA targeting PDX-1 (siRNA-PDX-1) or mock plasmid (siRNA-MOCK). Following induction, siRNA-MOCK-transfected cells differentiated into β-cell-like cells that expressed multiple islet cell markers and produced insulin and C-peptide in a glucose-regulated manner. However, siRNA-PDX-1-transfected cells did not fully differentiate into β-cell-like cells. Further, we observed epigenetic changes at the PDX-1 gene locus in induced CD44(+)/CD105(+) HuAFCs. Therefore, CD44+/CD105+ HuAFCs could be a source of human pancreatic β-cell-like cells with potential uses in cell replacement therapy for diabetes.     

Control of mouse U1a and U1b snRNA gene expression by differential transcription.

The expression of mouse embryonic U1 snRNA (mU1b) genes is subject to stage- and tissue-specific control, being restricted to early embryos and adult tissues that contain a high proportion of stem cells capable of further differentiation. To determine the mechanism of this control we have sought to distinguish between differential RNA stability and regulation of U1 gene promoter activity in several cell types. We demonstrate here that mU1b RNA can accumulate to high levels in permanently transfected mouse 3T3 and C127 fibroblast cells which normally do not express the endogenous U1b genes, and apparently can do so without significantly interfering with cell growth. Expression of transfected chimeric U1 genes in such cells is much more efficient when their promoters are derived from a constitutively expressed mU1a gene rather than from an mU1b gene. In transgenic mice, introduced U1 transgenes with an mU1b 5' flanking region are subject to normal tissue-specific control, indicating that U1b promoter activity is restricted to tissues that normally express U1b genes. Inactivation of the embryonic genes during normal differentiation is not associated with methylation of upstream CpG-rich sequences; however, in NIH 3T3 fibroblasts, the 5' flanking regions of endogenous mU1b genes are completely methylated, indicating that DNA methylation serves to imprint the inactive state of the mU1b genes in cultured cells. Based on these results, we propose that the developmental control of U1b gene expression is due to differential activity of mU1a and mU1b promoters rather than to differential stability of U1a and U1b RNAs.     

Generation of insulin-producing cells from PDX-1 gene-modified human mesenchymal stem cells

Islet cell replacement is considered as the optimal treatment for type I diabetes. However, the availability of human pancreatic islets for transplantation is limited. Here, we show that human bone marrow-derived mesenchymal stem cells (hMSCs) could be induced to differentiate into functional insulin-producing cells by introduction of the pancreatic duodenal homeobox-1 (PDX-1). Recombinant adenoviral vector was used to deliver PDX-1 gene into hMSCs. After being infected with Ad-PDX-1, hMSCs were successfully induced to differentiate into insulin-secreting cells. The differentiated PDX-1+ hMSCs expressed multiple islet-cell genes including neurogenin3 (Ngn3), insulin, GK, Glut2, and glucagon, produced and released insulin/C-peptide in a weak glucose-regulated manner. After the differentiated PDX-1+ hMSCs were transplanted into STZ-induced diabetic mice, euglycemia can be obtained within 2 weeks and maintained for at least 42 days. These findings validate the hMSCs model system as a potential basis for enrichment of human beta cells or their precursors, and a possible source for cell replacement therapy in diabetes.     

Dominant dysplasia of the lens in transgenic mice expressing the dbl oncogene

To study the transforming activity of the dbl oncogene and its effect on normal development in vivo, we linked dbl cDNA to promoters with different cell type specificities and used the constructs to generate transgenic mice. The promoters included the mouse alpha A-crystallin promoter, the rat insulin II promoter, and a mouse metallothionein promoter. We also generated transgenic mice carrying a recombinant cosmid clone that contains the entire dbl gene. Mice with the crystallin promoter construct developed cataracts and expressed the dbl protein in their lenses. The architecture of the lenses suggested a block to the normal pattern of differentiation and elongation of the secondary fiber cells. Mice with the insulin II promoter expressed dbl protein in the pancreas but showed no evidence of diabetes and no apparent pancreatic beta-cell defects. Similarly, mice with the metallothionein promoter expressed dbl protein in heart and testes, but showed no pathologic abnormalities in these tissues even after treatment with heavy metals. However, one family of mice carrying the metallothionein promoter construct showed cataracts and a dramatic fibroblastic dysplasia of the lens. One family with the cosmid-dbl gene showed a nearly identical lenticular dysplasia, but with a slower developmental time course. Thus, although the dbl oncogene did not induce neoplasia in any of the mice studied, it is apparently capable of interfering with the ability of the lens epithelial cells to differentiate into lens fiber cells, and of inducing metaplasia of the epithelial cells into fibroblastic cells.     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.     

Characterization of histone H2A.X expression in testis and specific labeling of germ cells at the commitment stage of meiosis with histone H2A.X promoter-enhanced green fluorescent protein transgene

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the -600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.     

Functional,persistent, and extended liver to pancreas transdifferentiation

Pancreatic and duodenal homeobox gene-1 (PDX-1) regulates pancreas development during embryogenesis, whereas in the adult it controls beta-cell function. Here we analyze whether PDX-1 functions as a pancreatic differentiation factor and a bona fide master regulator when ectopically expressed in mature fully differentiated liver in vivo. By ectopic and transient PDX-1 expression in liver in vivo, using the first generation recombinant adenoviruses, we demonstrate that PDX-1 induces in liver a wide repertoire of both exocrine and endocrine pancreatic gene expression. Moreover, PDX-1 induces its own expression (auto-induction), which in turn may explain the long lasting nature of the "liver to pancreas" transdifferentiation. Insulin as well glucagon-producing cells are mainly located in the proximity of hepatic central veins, possibly allowing direct hormone release into the bloodstream, without affecting normal hepatic function. Importantly, we demonstrate that hepatic insulin production triggered by Ad-CMV-PDX-1 recombinant adenovirus administration is functional and prevents streptozotocin-induced hyperglycemia in Balb/c mice even 8 months after the initial treatment. We conclude that PDX-1 plays an important instructive role in pancreas differentiation, not only from primitive gut endoderm but also from mature liver. Transconversion of liver to pancreas may serve as a novel approach for generating endocrine-pancreatic tissue that can replace malfunctioning beta-cells in diabetics.     

Inhibin alpha-iCre mice: Cre deleter lines for the gonads, pituitary, and adrenal

To expand our knowledge of reproductive function, Cre lines to conditionally knockout essential genes in the mouse gonads were generated. Three transgenic lines of inhibin-alpha-iCre mice were designed by fusing the mouse inhibin-alpha promoter with a codon-improved Cre recombinase (iCre). alpha-iCre-line-3 expressed high levels of Cre in Sertoli and Leydig cells of the testis and low levels in other tissues, making line 3 an appropriate deleter line for genes expressed in somatic cells of the testis. In contrast, alpha-iCre-line-1 expressed high levels of Cre in granulosa and theca cells of the ovary and very low levels in other tissues, making line 1 a suitable deleter line for genes expressed in somatic cells of the ovary. A third line, alpha-iCre-line-2, had low levels of Cre in the gonads but high levels in anterior pituitary and adrenal medulla. These lines could be useful to understand reproduction and other processes by establishing conditional knockout mouse models.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.