Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.

Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.

Molecular cloning and expression pattern of the Fkbp25 gene during cerebral cortical neurogenesis.

Neocortical neurons are generated predominantly from the cells that proliferate in the ventricular zone of the telencephalon. In order to understand the nature of these expanding cortical neuronal progenitor cells, we selected by differential display some transcripts that were enriched in the telencephalon as compared to the more caudal regions (diencephalon/mesencephalon). This systematic screening revealed one of the differentially expressed transcripts, namely the Fkbp25 mRNA that encodes a member of the FK506 binding proteins (FKBPs). Northern blot analysis showed that the expression of the single 1.4kb Fkbp25 transcript reached a maximum level on embryonic day 11.5 at the start of cortical neurogenesis in the mouse and was followed by a weak basal expression in the adult brain. In the embryo, Fkbp25 gene was strongly expressed in the telencephalon ventricular zone but also in areas active in myogenesis (walls of the ventricle and the atrium) and chondrogenesis (the cartilage of the rib and the hindlimb). An increase in the transcript levels of the Fkbp25 gene was also observed during the two successive proliferation waves of the cerebellum development. Immunostaining on primary cultures of embryonic day 10.5 telencephalon stem cells showed that the Fkbp25 protein was present in the cytoplasm and nuclei of cells cultured for 6h but exclusively in the nuclei of the Tuj-1 immunoreactive neurons obtained after 3 days of culture (The sequence data reported here have been submitted to GenBank under accession no. AF135595.).

Nestin expression in pancreatic exocrine cell lineages.

Expression of nestin has been suggested to be a characteristic of pancreatic islet stem cells. To determine whether nestin is indeed expressed in such putative cells during embryonic development, or in the adult pancreas after injury, we performed a cell lineage analysis using two independent lines of transgenic mice encoding Cre recombinase under the control of rat nestin cis-regulatory sequences, each crossed with loxP-bearing R26R mice. F1 animals produced the reporter molecule beta-galactosidase only upon Cre-mediated recombination, thus solely in cells using (or having used) the transgenic nestin promoter. In early pancreatic primordia, beta-galactosidase was observed in mesenchymal and epithelial cells. At later developmental stages or in adults, vast clusters of acinar cells and few ductal cells were labeled, in addition to fibroblasts and vascular cells, but no endocrine cells were tagged by beta-galactosidase. This correlated with the transient expression, observed with an anti-nestin antibody, of endogenous nestin in about 5% of epithelial cells during development (whether in cord-forming arrangements or in nascent acini), and in vascular and mesenchymal structures. After partial pancreatectomy, there was a transient increase of the number of anti-nestin-labeled endothelial cells, but again, no endocrine cells bore beta-galactosidase. Together, these findings show that nestin is expressed in the pancreatic exocrine cell lineage, and suggest that consistent nestin expression is not a major feature of islet endocrine progenitor cells.

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells.

Spinal muscular atrophy (SMA) is a recessive disorder involving the loss of motor neurons from the spinal cord. Homozygous absence of the survival of motor neuron 1 gene (SMN1) is the main cause of SMA, but disease severity depends primarily on the number of SMN2 gene copies. SMN protein levels are high in normal spinal cord and much lower in the spinal cord of SMA patients, suggesting neuron-specific regulation for this ubiquitously expressed gene. We isolated genomic DNA from individuals with SMN1 or SMN2 deletions and sequenced 4.6 kb of the 5' upstream regions of the these. We found that these upstream regions, one of which is telomeric and the other centromeric, were identical. We investigated the early regulation of SMN expression by transiently transfecting mouse embryonic spinal cord and fibroblast primary cultures with three transgenes containing 1.8, 3.2 and 4.6, respectively, of the SMN promoter driving beta-galactosidase gene expression. The 4.6 kb construct gave reporter gene expression levels five times higher in neurons than in fibroblasts, due to the combined effects of a general enhancer and a non-neuronal cell silencer. The differential expression observed in neurons and fibroblasts suggests that the SMN genes play a neuron-specific role during development. An understanding of the mechanisms regulating SMN promoter activity may provide new avenues for the treatment of SMA.

Murine peripherin gene sequences direct Cre recombinase expression to peripheral neurons in transgenic mice.

Spatially and temporally regulated somatic mutations can be achieved by using the Cre/loxP recombination system of bacteriophage P1. To develop a cell type-specific system of gene targeting in the peripheral nervous system, we generated the transgenic mouse lines expressing Cre recombinase under the control of the mouse peripherin gene promoter. The activity of the Cre recombinase during embryonic development was examined by mating the peripherin-Cre transgenic mice to the knock-in Cre-mediated recombination reporter strain, R26R. Analysis of F1 embryos from this cross showed specific excision of loxP-flanked sequences in the dorsal root ganglia, trigeminal ganglia, and olfactory epithelium, in a pattern very similar to the expression of the endogenous mouse peripherin gene, and the previously reported peripherin-lacZ transgenic mice. Thus, the peripherin-Cre mouse described here will provide a valuable tool for Cre-loxP-mediated conditional expression in the peripheral nervous system.

An amylase/Cre transgene marks the whole endoderm but the primordia of liver and ventral pancreas.

Mice bearing a Cre-encoding transgene driven by a compound [SV40 small t antigen/mousealpha-amylase-2] promoter expressed the recombinase at early developmental stages broadly in the embryonic endoderm before the pancreas and lungs begin to outgrow, but not in other germ layers, as determined indirectly by beta-galactosidase and YFP reporter activity, indicating that the transgene is in fact an endodermic marker. Interestingly, the liver and ventral pancreas were excluded from this expression pattern, denoting that the chimerical alpha-amylase-2 promoter was not active in the anterior leading edge of the endoderm (the presumptive region from which liver and ventral pancreas form). These transgenics thus confirm, among other findings, that dorsal and ventral pancreatic primordia have different intrinsic transcriptional capabilities. In conclusion, we have generated a new transgenic mouse that should be useful to target endoderm at early stages, without affecting the liver or ventral pancreas before embryonic day E12.5.

Pancreatic cell lineage analyses in mice.

Considerable knowledge of the ontogeny of the endocrine pancreas has been gained in recent years, mainly through the use of two complementary genetic approaches in transgenic mice: gene inactivation or overexpression (to assess gene function) and genetic labeling of precursor cells (to determine cell lineages). In recent years, in vivo Cre/loxP-based direct cell tracing experiments show that (i) all pancreatic cells differentiate from pdx1-expressing precursors, (ii) p48 is involved in the exocrine and endocrine pancreatic lineages, (iii) islet endocrine cells derive from ngn3-expressing progenitor cells, and (iv) insulin cells do not derive from glucagon- expressing progenitors. Lineage analyses allow the identification of progenitor cells from which mature cell types differentiate. Once identified, such progenitors can be labeled and isolated, and their differentiation and gene expression profiles studied in vitro. Understanding pancreatic cell lineages is highly relevant for future cell replacement therapies in diabetic patients, helping to define the identity of putative (endodermal) pancreatic stem cells.

Glial cell line derived neurotrophic factor is expressed by epithelia of human renal dysplasia.

PURPOSE: Differentiation of the metanephros is abnormal in cases of renal dysplasia, resulting in abnormal kidney organization. In vitro and in vivo studies indicate that glial cell line derived neurotrophic factor (GDNF) is a major regulator of kidney development and ureteral arborization. Therefore, we investigated the pattern of GDNF gene expression in human dysplastic kidneys. MATERIALS AND METHODS: Specimens of whole tissues of human normal and dysplastic kidneys associated with obstructive uropathy were analyzed for GDNF mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemistry with GDNF antibody and laser capture microdissection plus RT-PCR were done to identify cells producing GDNF. Apoptosis, BCL-2 and Ki67 were also studied. RESULTS: There were few if any GDNF transcripts in normal kidneys, whereas GDNF was over expressed in renal dysplasia specimens. Strong GDNF expression was found in the dysplastic tubules of dysplastic kidneys, whereas peritubular mesenchyma expressed no GDNF protein. Laser capture microdissection/RT-PCR detected GDNF mRNA in epithelial cells isolated from dysplastic tubules but not in cells from the surrounding mesenchyma, which was confirmed by sequence analysis. GDNF expression by epithelial cells was associated with high proliferation, BCL-2 expression and rare apoptosis. CONCLUSIONS: GDNF gene expression is restricted to the tubular epithelium of dysplastic human kidneys. Our results strongly suggest that GDNF not only influences kidney morphogenesis, but is also implicated in abnormal kidney development.

Replacement by a lacZ reporter gene assigns mouse connexin36, 45 and 43 to distinct cell types in pancreatic islets.

Transcripts of three connexin isoforms (Cx36, Cx43 and Cx45) have been reported in rodent pancreatic islets, but the precise distribution of the cognate proteins is still unknown. We determined expression of Cx36 in a cell-autonomous manner using mice with a targeted replacement of the Cx36 coding region by a lacZ reporter gene. For cell-autonomous monitoring of Cx43 expression, we used the Cre/loxP system: Mice carrying the Cx43 coding region flanked by loxP sites (floxed) also carried an embedded lacZ gene that is activated after Cre-mediated recombination in cells with transcriptional activity of the Cx43 gene. Deletion of the Cx43 coding region in beta-cells did not result in the activation of the embedded lacZ reporter gene. Instead, Cx43 expression was found in endothelial cells of the islets of Langerhans in mice with endothelium-specific deletion. Ubiquitous deletion of Cx43 led to a similar endothelial lacZ expression, but again, activity of the reporter gene was not detected in beta-cells. Mice with targeted replacement of the Cx45 coding region by lacZ showed a vascular expression similar to Cx43. The data show that native insulin-producing cells express a connexin isoform (Cx36) which differs from those (Cx43 and Cx45) expressed by vascular islet cells.

Noradrenergic neuronal development is impaired by mutation of the proneural HASH-1 gene in congenital central hypoventilation syndrome (Ondine's curse).

Congenital central hypoventilation syndrome (CCHS, Ondine's curse) is a rare disorder of the chemical control of breathing. It is frequently associated with a broad spectrum of dysautonomic symptoms, suggesting the involvement of genes widely expressed in the autonomic nervous system. In particular, the HASH-1-PHOX2A-PHOX2B developmental cascade was proposed as a candidate pathway because it controls the development of neurons with a definitive or transient noradrenergic phenotype, upstream from the RET receptor tyrosine kinase and tyrosine hydroxylase. We recently showed that PHOX2B is the major CCHS locus, whose mutation accounts for 60% of cases. We also studied the proneural HASH-1 gene and identified a heterozygous nucleotide substitution in three CCHS patients. To analyze the functional consequences of HASH-1 mutations, we developed an in vitro model of noradrenergic differentiation in neuronal progenitors derived from the mouse vagal neural crest, reproducing in vitro the HASH-PHOX-RET pathway. All HASH-1 mutant alleles impaired noradrenergic neuronal development, when overexpressed from adenoviral constructs. Thus, HASH-1 mutations may contribute to the CCHS phenotype in rare cases, consistent with the view that the abnormal chemical control of breathing observed in CCHS patients is due to the impairment of noradrenergic neurons during early steps of brainstem development.