Control of endodermal endocrine development by Hes-1.

Development of endocrine cells in the endoderm involves Atonal and Achaete/Scute-related basic helix-loop-helix (bHLH) proteins. These proteins also serve as neuronal determination and differentiation factors, and are antagonized by the Notch pathway partly acting through Hairy and Enhancer-of-split (HES)-type proteins. Here we show that mice deficient in Hes1 (encoding Hes-1) display severe pancreatic hypoplasia caused by depletion of pancreatic epithelial precursors due to accelerated differentiation of post-mitotic endocrine cells expressing glucagon. Moreover, upregulation of several bHLH components is associated with precocious and excessive differentiation of multiple endocrine cell types in the developing stomach and gut, showing that Hes-1 operates as a general negative regulator of endodermal endocrine differentiation.

Genomic imprinting of Mash2, a mouse gene required for trophoblast development.

The mouse gene Mash2 encodes a transcription factor required for development of trophoblast progenitors. Mash2-homozygous mutant embryos die at 10 days postcoitum from placental failure. Here we show that Mash2 is genomically imprinted. First, Mash2+/- embryos inheriting a wild-type allele from their father die at the same stage as -/- embryos, with a similar placental phenotype. Second, the Mash2 paternal allele is initially expressed by groups of trophoblast cells at 6.5 and 7.5 days post-coitum, but appears almost completely repressed by 8.5 days post-coitum. Finally, we have genetically and physically mapped Mash2 to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor-2 and H19.

Cells expressing Ia antigens in the avian thymus.

The various cell types expressing Ia antigens in the chick and quail thymus have been studied by means of monoclonal antibodies (mAb) prepared by using chick and quail thymic adherent cells (macrophages and dendritic cells) as immunogens. Three reagents were selected by the following criteria: (a) they react with a surface determinant carried by thymic adherent cells and bursal lymphocytes, (b) they can be used to immunoprecipitate from spleen cell membrane extracts molecular entities of an apparent molecular weight close to 55,000, which can be fractionated into monomers at approximately 30,000 mol wt in dissociating conditions. Among these three reagents, two are strictly species specific, i.e., they recognize either chick (TaPl) or quail (TaCl) Ia determinants, whereas the third, TaC2, recognizes both chick and quail Ia molecules. Chimeric thymuses in which the epithelioconnective stroma is derived from the quail thymic primordium and the whole hemopoietic cell population (lymphocytes and accessory cells) are of chick origin were constructed as previously described by our group (20). The different mAb were applied on normal (quail and chick) and chimeric thymuses. It appears that the thymus is divided into two compartments in terms of the nature of cells expressing Ia: the cortex, in which class II antigens are exclusively expressed by endodermal epithelial cells, and the medulla, where the majority of nonlymphoid cells are Ia-positive cells of hemopoietic origin. A few epithelial cells only are present in the thymic medulla. They are closely intricated with the sessile Ia-positive cells, whose precursors penetrate the thymus along with the lymphocyte progenitors and which are renewed in the course of thymic development. In contrast, the epithelial reticulum, expressing Ia both in the cortex and medulla, contributes a stable thymic component. During early thymic ontogeny, the hemopoietic cells expressed detectable levels of Ia antigen before the epithelial cell network.

Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord.

We have examined how genetic pathways that specify neuronal identity and regulate neurogenesis interface in the vertebrate neural tube. Here, we demonstrate that expression of the proneural gene Neurogenin2 (Ngn2) in the ventral spinal cord results from the modular activity of three enhancers active in distinct progenitor domains, suggesting that Ngn2 expression is controlled by dorsoventral patterning signals. Consistent with this hypothesis, Ngn2 enhancer activity is dependent on the function of Pax6, a homeodomain factor involved in specifying the identity of ventral spinal cord progenitors. Moreover, we show that Ngn2 is required for the correct expression of Pax6 and several homeodomain proteins expressed in defined neuronal populations. Thus, neuronal differentiation involves crossregulatory interactions between a bHLH-driven program of neurogenesis and genetic pathways specifying progenitor and neuronal identity in the spinal cord.

Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors.

We have addressed the role of the proneural bHLH genes Neurogenin2 (Ngn2) and Mash1 in the selection of neuronal and glial fates by neural stem cells. We show that mice mutant for both genes present severe defects in development of the cerebral cortex, including a reduction of neurogenesis and a premature and excessive generation of astrocytic precursors. An analysis of wild-type and mutant cortical progenitors in culture showed that a large fraction of Ngn2; Mash1 double-mutant progenitors failed to adopt a neuronal fate, instead remaining pluripotent or entering an astrocytic differentiation pathway. Together, these results demonstrate that proneural genes are involved in lineage restriction of cortical progenitors, promoting the acquisition of the neuronal fate and inhibiting the astrocytic fate.

The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons.

neurogenin2 encodes a neural-specific basic helix-loop-helix (bHLH) transcription factor related to the Drosophila proneural factor atonal. We show here that the murine ngn2 gene is essential for development of the epibranchial placode-derived cranial sensory ganglia. An ngn2 null mutation blocks the delamination of neuronal precursors from the placodes, the first morphological sign of differentiation in these lineages. Mutant placodal cells fail to express downstream bHLH differentiation factors and the Notch ligand Delta-like 1. These data suggest that ngn2 functions like the Drosophila proneural genes in the determination of neuronal fate in distal cranial ganglia. Interestingly, the homeobox gene Phox2a is activated independently of ngn2 in epibranchial placodes, suggesting that neuronal fate and neuronal subtype identity may be specified independently in cranial sensory ganglia.

Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis.

Mammalian hairy and Enhancer of split homolog 1 (HES1), a basic helix-loop-helix factor gene, is expressed in retinal progenitor cells, and its expression decreases as differentiation proceeds. Retinal progenitor cells infected with HES1-transducing retrovirus did not differentiate into mature retinal cells, suggesting that persistent expression of HES1 blocks retinal development. In contrast, in the retina of HES1-null mutant mice, differentiation was accelerated, and rod and horizontal cells appeared prematurely and formed abnormal rosette-like structures. Lens and cornea development was also severely disturbed. Furthermore, in the mutant retina, bipolar cells extensively died, and finally disappeared. These studies provide evidence that HES1 regulates differentiation of retinal neurons and is essential for eye morphogenesis.

The critical role of wavelength in the UV-activated grafting of 1-alkene onto silicon and silicon nitride SixN4 surfaces.

The wavelength used during photochemical grafting of alkene onto silicon related surfaces influences molecular surface coverage. Ultraviolet light leads to apparent highly dense layers on UV absorbing materials due to the side reaction between alkenes resulting in strongly physisorbed dimers whereas higher wavelengths lead to dense and well-controlled layers.

The major histocompatibility complex in the chicken.

The chicken B complex is the first non-mammalian MHC characterized at the molecular level. It differs from the human HLA and murine H-2 complexes in the small size of the class I (B-F) and class II (B-L) genes and their close proximity. This proximity accounts for the absence of recombination between B-F and B-L genes and leaves no space for class III genes. Moreover the B-F and B-L genes are tightly linked to unrelated genes absent from mammalian MHCs, such as the polymorphic B-G genes and a member of the G protein beta subunit family. This linkage could form the basis for resistance to viral-induced tumors associated with some B complex haplotypes.

Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle.

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.

Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3.

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class of cut homeodomain transcription factors. During mouse development, HNF-6 is expressed in the epithelial cells that are precursors of the exocrine and endocrine pancreatic cells. We have investigated the role of HNF-6 in pancreas differentiation by inactivating its gene in the mouse. In hnf6(-/-) embryos, the exocrine pancreas appeared to be normal but endocrine cell differentiation was impaired. The expression of neurogenin 3 (Ngn-3), a transcription factor that is essential for determination of endocrine cell precursors, was almost abolished. Consistent with this, we demonstrated that HNF-6 binds to and stimulates the ngn3 gene promoter. At birth, only a few endocrine cells were found and the islets of Langerhans were missing. Later, the number of endocrine cells increased and islets appeared. However, the architecture of the islets was perturbed, and their beta cells were deficient in glucose transporter 2 expression. Adult hnf6(-/-) mice were diabetic. Taken together, our data demonstrate that HNF-6 controls pancreatic endocrine differentiation at the precursor stage and identify HNF-6 as the first positive regulator of the proendocrine gene ngn3 in the pancreas. They also suggest that HNF-6 is a candidate gene for diabetes mellitus in humans.

[p27Kip1 independently promotes neuronal differentiation and migration in the cerebral cortex]. / p27Kip1 independently promotes neuronal differentiation and migration in the cerebral cortex.

The generation of glutamatergic neurons by stem and progenitor cells is a complex process involving the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation and cell migration. The mechanisms that integrate these different events into a coherent program are not well understood. Here we show that the cyclin-dependent kinase inhibitor p27Kip1 plays an important role in neurogenesis in the mouse cerebral cortex, by promoting the differentiation and radial migration of cortical projection neurons. Importantly, p27Kip1 promotes neuronal differentiation and neuronal migration via two distinct mechanisms, which are themselves independent of the cell cycle regulatory function of p27Kip1. p27Kip1 inactivation by gene targeting or RNA interference results in neuronal differentiation and radial migration defects, demonstrating that p27Kip1 regulates cell migration in vivo. The differentiation defect, but not the migration defect, is rescued by overexpression of the proneural gene Neurogenin 2. p27Kip1 acts by stabilizing Neurogenin 2 protein, an activity carried by the N-terminal half of the protein. The migration defect resulting from p27Kp1 inactivation is rescued by blocking RhoA signalling, an activity that resides in the c-terminal half of p27Kip1. Thus, p27Kip1 plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.

Gene targeting and development of the nervous system.

Gene targeting provides a means of directly assaying the function of specific genes during mouse nervous system development. Generation of targeted mutant mice has provided the first evidence of developmental roles for genes whose function was suggested based on their expression, but for which appropriate assay systems were lacking. In other cases, where gene function was known, targeted mutations have revealed in which cell population, and at what developmental stage, particular genes are first indispensable. The existing targeted mutants suggest that an early mechanism of pattern formation in mammals involves regional control of proliferation or survival of neural precursors, and that later general functions, such as the control of differentiation of precursors, may be performed by different genes in distinct neural lineages. As many genes display complex temporal and spatial patterns of expression, analysis of the full range of functions of such genes will require the generation of a series of alleles, including stage- and tissue-specific mutations.

Hes1 and Hes5 activities are required for the normal development of the hair cells in the mammalian inner ear.

The mammalian inner ear contains two sensory organs, the cochlea and vestibule. Their sensory neuroepithelia are characterized by a mosaic of hair cells and supporting cells. Cochlear hair cells differentiate in four rows: a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs). Recent studies have shown that Math1, a mammalian homolog of Drosophila atonal is a positive regulator of hair cell differentiation. The basic helix-loop-helix (bHLH) genes Hes1 and Hes5 (mammalian hairy and Enhancer-of-split homologs) can influence cell fate determination by acting as negative regulators to inhibit the action of bHLH-positive regulators. We show by using reverse transcription-PCR analysis that Hes1, Hes5, and Math1 are expressed in the developing mouse cochleae. In situ hybridization revealed a widespread expression of Hes1 in the greater epithelial ridge (GER) and in lesser epithelial ridge (LER) regions. Hes5 is predominantly expressed in the LER, in supporting cells, and in a narrow band of cells within the GER. Examination of cochleae from Hes1(-/-) mice showed a significant increase in the number of IHCs, whereas cochleae from Hes5(-/-) mice showed a significant increase in the number of OHCs. In the vestibular system, targeted deletion of Hes1 and to a lesser extent Hes5 lead to formation of supernumerary hair cells in the saccule and utricle. The supernumerary hair cells in the mutant mice showed an upregulation of Math1. These data indicate that Hes1 and Hes5 participate together for the control of inner ear hair cell production, likely through the negative regulation of Math1.

A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.

We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.

Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system.

The Drosophila Achaete-Scute Complex genes encode transcriptional regulators belonging to the basic-helix-loop-helix family which control early steps of development of the central and peripheral nervous systems. We have isolated two mouse homologues of Achaete-Scute Complex genes, Mash-1 and Mash-2, by using the conservation of the basic-helix-loop-helix domain in this family. In this article, we analyse the expression of Mash-1 from its onset during neurulation to adult stages by RNA in situ hybridization on whole mounts and sections. As was observed for the rat Mash-1 protein, mouse Mash-1 RNA expression is restricted to cells of the developing central and peripheral nervous systems. We have observed three successive phases in the distribution of Mash-1 transcripts in the developing central nervous system. Initially, between embryonic day 8.5 and 10.5, Mash-1 transcripts are found in restricted domains in the neuroepithelium of the midbrain and ventral forebrain, as well as in the spinal cord. Between embryonic day 10.5 and 12.5, Mash-1 expression pattern changes from a restricted to a widespread one. Mash-1 transcripts are then found at variable levels in the ventricular zone in all regions of the brain. From embryonic day 12.5 to post-natal stages, Mash-1 is also expressed in cells outside of the ventricular zone throughout the brain. In addition, Mash-1 is expressed during development of the olfactory epithelium and neural retina. Overall, its expression pattern suggest that Mash-1 plays a role at early stages of development of specific neural lineages in most regions of the central nervous system and of several lineages in the peripheral nervous system. We have also compared the expression of Mash-1 and mouse Notch because their Drosophila homologues have been shown to interact genetically. The two genes show very similar expression patterns, both spatially and temporally, in the early developing brain and in the retina, suggesting that both genes may participate in the development of the same neural lineages.

Mash2 is expressed in oogenesis and preimplantation development but is not required for blastocyst formation.

The basic helix-loop-helix transcription factor, Mash2, has been shown to be necessary for the development of the spongiotrophoblast of the mature chorioallantoic placenta of the mouse. Here we show that Mash2 is transcribed during oogenesis and expressed throughout preimplantation development, only becoming restricted to the diploid trophoblast around the time of implantation. This expression raised the possibility that Mash2 has earlier functions in the trophoblast lineage that were not detectable in mutant embryos because of the persistence of oogenetically derived protein. This was tested by generating viable Mash2-/- females by tetraploid rescue of the extraembryonic defect. Mutant embryos derived from such females showed no enhanced phenotype over embryos produced from heterozygous females, demonstrating unequivocally that neither maternal nor zygotic Mash2 is required for early trophoblast development. If Mash2 functions in other aspects of trophoblast development, it must act cooperatively with other factors.

Detailed transcript map of a 810-kb region at 11p14 involving identification of 10 novel human 3' exons.

A limited number of genes, including the human brain-derived neutrotrophic factor (BDNF) gene, have been identified in the human chromosome 11p14 region. Since this area is involved in a genetic disorder (WAGR syndrome) and because of interest in studying the regulation of the human BDNF gene, we have established a detailed transcript map of a 810-kb region clone in a yeast artificial chromosome (YAC), corresponding to a portion of this genomic locus. A set of nested deletion mutants has been generated to map genes at a mean resolution of 75kb. Four genic markers from available mapping databases have been mapped on the YAC. Ten potential novel human exons have been isolated by a 3' terminal exon trapping procedure directly applied to purified YAC DNA. Most of these exons display polyadenylation signals and they all yield positive signals in RT-PCR experiments, confirming their status of transcribed sequences. The BDNF gene is now co-localised with three other genes on a 120 kb DNA fragment.

Molecular analysis of chicken immune response genes.

We have recently isolated immune response genes of the major histocompatibility B complex of the chicken (the B-L beta genes) by cross-hybridization in low stringency with an HLA class II beta chain probe. After reviewing the main results obtained, we present a detailed analysis of the region flanking the first gene characterized, B-L beta III. By Southern blot analysis with exon-specific probes, we demonstrate the presence of another related B-L beta gene 10 kb on the 3' side of B-L beta III, the B-L beta V gene. Moreover, retrospective analysis of the phage clones initially isolated with the HLA-DQ beta probe, using a chicken class I probe that we isolated by chromosome walking from the B-L beta genes, indicates that the B-L beta III gene is closely linked on its 5' side to a class I gene, B-FVI.

Influence of the timing of administration of 300 mg ranitidine on 24-hour gastric pH in patients acute duodenal ulcer.

The 24-hour intragastric pH of 12 patients with an acute duodenal ulcer was recorded with the aim of comparing the effects of two different times of administration of 300 mg ranitidine: post evening meal, or bedtime. This double-blind crossover trial involved 3 centres. Twenty-four-hour gastric pH was measured under standard conditions (meals, time schedule) at the middle of each 14-day treatment period. The analysis was performed on the percentage of times spent at pH levels below 1.5, 2, 3 and 4 for different periods and for the total 24 hours. During the whole day and night combined, as well as during the afternoon (12.00 hours-19.00 hours), there was no difference between the 2 regimens regardless of the pH profile studied. During the morning (07.30 hours-12.00 hours), the time spent below pH 1.5 and 2 was less when the drug was taken at bedtime (P less than 0.05). In contrast, during the whole night (19.00 hours-07.30 hours) the percentage of time spent below pH 1.5, 2 and 3 was significantly less when the drug was taken at post evening meal (P less than 0.05). These results show that in patients with acute duodenal ulcer, 300 mg ranitidine administered at the end of the evening meal provides better control of nocturnal acidity than administration at bedtime and hence is suggested for optimization of therapeutic efficacy.