Generation and PCR screening of bacteriophage gamma sublibraries enriched for rare clones (the "sublibrary method").
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Animals↗
Biomedical subjects
M Lardelli
Publications and source records attributed to M Lardelli.
Expression of the Notch 3 intracellular domain in mouse central nervous system progenitor cells is lethal and leads to disturbed neural tube development.
Notch-like receptors are found in organisms ranging from nematodes to mammals. In Drosophila, Notch plays a key role in cell fate decisions in the early nervous system. In this report we analyse the effects of excess Notch 3 activity in central nervous system (CNS) progenitor cells. A mutated Notch gene encoding the intracellular domain of mouse Notch 3 transcribed from the nestin promoter was expressed in CNS progenitor cells in transgenic mice. This mutation resulted in a phenotypic series of neural tube defects in embryonic day 10.5-12.5 embryos and proved lethal to embryos beyond this age. In the milder phenotype the neural tube displayed a zig-zag morphology and the CNS was slightly enlarged. More severely affected embryos showed a lack of closure of the anterior neural pore, resulting in the externalization of neural tissue and the complete collapse of the third and fourth ventricles. The expanded ventricular zone of the neuroepithelium, a correspondingly enlarged area of nestin expression, and an increase in the number of proliferating cells in the neural tube suggested that these phenotypes resulted from an expanded CNS progenitor cell population. These data provide support in vivo for the notion that Notch activity plays a role in mammalian CNS development and may be required to guide CNS progenitor cells in their choice between continued proliferation or neuronal differentiation.
Animals↗
Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system.
Nestin is a recently discovered intermediate filament (IF) gene. Nestin expression has been extensively used as a marker for central nervous system (CNS) progenitor cells in different contexts, based on observations indicating a correlation between nestin expression and this cell type in vivo. To evaluate this correlation in more detail nestin mRNA expression in developing and adult mouse CNS was analysed by in situ hybridization. We find that nestin is expressed from embryonic day (E) 7.75 and that expression is detected in many proliferating CNS regions: at E10.5 nestin is expressed in cells of both the rostral and caudal neural tube, including the radial glial cells; at E15.5 and postnatal day (P) 0 expression is observed largely in the developing cerebellum and in the ventricular and subventricular areas of the developing telencephalon. Furthermore, the transition from a proliferating to a post-mitotic cell state is accompanied by a rapid decrease in nestin mRNA for motor neurons in the ventral spinal cord and for neurons in the marginal layer of developing telencephalon. In contrast to these data we observe two proliferating areas, the olfactory epithelium and the precursor cells of the hippocampal granule neurons, which do not express nestin at detectable levels. Thus, nestin mRNA expression correlates with many, but not all, regions of proliferating CNS progenitor cells. In addition to its temporal and spatial regulation nestin expression also appears to be regulated at the level of subcellular mRNA localization: in columnar neuroepithelial and radial glial cells nestin mRNA is predominantly localized to the pial endfeet.
Animals↗
Complementary and combinatorial patterns of Notch gene family expression during early mouse development.
The Drosophila Notch gene encodes a transmembrane receptor involved in the regulation of cell fate. It exerts its effect by lateral specification, inductive signaling and is also important for cell adhesion and axonal pathfinding. In this report we analyse the expression of the three mammalian Notch homologues during early mouse development by in situ hybridization. The Notch 1, 2 and 3 genes show dynamic and complex expression patterns, in particular during gastrulation and somitogenesis and in early nervous system formation. During gastrulation, the Notch genes are expressed in non-overlapping, successive patterns. Notch 3 is widely expressed in both ectoderm and mesoderm. Notch 2 is then expressed in the node, notochord and neural groove while Notch 1 becomes highly expressed in presomitic mesoderm. As somitogenesis begins, Notch 2 expression is activated in newly forming somites while Notch 3 is activated in mature somites. Various neural crest cell populations and ectodermal placode cells can be defined by expression of specific combinations of Notch genes. All three Notch genes are expressed within cells of the dorsal neural tube at E9.5, although neural crest cells that have begun migrating all show distinct patterns of Notch expression. Finally, Notch 1 expression is observed not only in placodes, but also in cells migrating from placodes to the site of the ganglia anlagen. This expression pattern may be analogous to Notch expression in the peripheral nervous system of Drosophila, suggesting that mammalian Notch genes may also be involved in axonal pathfinding.
Animals↗
Expression of Notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate.
Notch 1, Notch 2, and Notch 3 are three highly conserved mammalian homologues of the Drosophila Notch gene, which encodes a transmembrane protein important for various cell fate decisions during development. Little is yet known about regulation of mammalian Notch gene expression, and this issue has been addressed in the developing rodent tooth during normal morphogenesis and after experimental manipulation. Notch 1, 2, and 3 genes show distinct cell-type specific expression patterns. Most notably, Notch expression is absent in epithelial cells in close contact with mesenchyme, which may be important for acquisition of the ameloblast fate. This reveals a previously unknown prepatterning of dental epithelium at early stages, and suggests that mesenchyme negatively regulates Notch expression in epithelium. This hypothesis has been tested in homo- and heterotypic explant experiments in vitro. The data show that Notch expression is downregulated in dental epithelial cells juxtaposed to mesenchyme, indicating that dental epithelium needs a mesenchyme-derived signal in order to maintain the downregulation of Notch. Finally, Notch expression in dental mesenchyme is upregulated in a region surrounding beads soaked in retinoic acid (50-100 micrograms/ml) but not in fibroblast growth factor-2 (100-250 micrograms/ml). The response to retinoic acid was seen in explants of 11-12-d old mouse embryos but not in older embryos. These data suggest that Notch genes may be involved in mediating some of the biological effects of retinoic acid during normal development and after teratogenic exposure.
Ameloblasts↗
Notch-related genes in animal development.
The Drosophila melanogaster gene Notch is central to many cell differentiation events during development. It encodes a large transmembrane signal receptor protein that acts in a poorly understood mechanism of communication affecting the choice of alternative differentiation fates by cells in close proximity. Genes with homology to Notch have been isolated from the nematode Caenorhabditis elegans and a number laboratories, including our own, have isolated multiple vertebrate Notch homologs. In this article we briefly outline the current state of research on Notch and our contribution to it. First, we examine the structure of Notch-related proteins. We then examine the requirements for Notch activity in the development of different organisms and how genetic and transgenic studies are helping us to understand the mechanism(s) by which these proteins function. We present models for the action of Notch receptors during signal transduction and for the interaction of multiple vertebrate Notch receptors. Finally, we discuss current ideas about the role played by Notch in differentiation and cell-cell communication.
Amino Acid Sequence↗
The human NOTCH1, 2, and 3 genes are located at chromosome positions 9q34, 1p13-p11, and 19p13.2-p13.1 in regions of neoplasia-associated translocation.
In Drosophila the Notch gene controls differentiation to various cell fates in many tissues. Three mammalian Notch homologs have recently been identified: Notch 1, 2, and 3. All three homologs are very highly conserved relative to the Drosophila Notch gene, which suggests that they are important for cell differentiation in mammals. This notion is supported by the previous finding of a truncated, translocated form of the human NOTCH1 gene (formerly TAN1) in three cases of leukemia. Given this genetic link between NOTCH1 and tumor formation, it is of interest to establish the chromosomal positions of the other two homologs. We report the identification of cosmid clones for the human NOTCH1, 2, and 3 genes. These clones were used as probes in fluorescence in situ hybridization to human metaphase chromosomes, and the results, combined with data from somatic cell hybrid panels, show that the NOTCH2 and 3 genes are located at positions 1p13-p11 and 19p13.2-p13.1, respectively, which are regions of neoplasia-associated translocation.
Amino Acid Sequence↗
Thyroid abnormalities and hepatocellular carcinoma in mice transgenic for v-erbA.
The v-erbA oncogene consists of an avian retroviral gag gene fused to a mutated thyroid hormone receptor. To define better its role as an oncogene in mammals and its ability to function as a dominant negative transcription factor, transgenic mice expressing v-erbA ubiquitously were generated. The effects of v-erbA are pleiotropic, tissue-specific and dose dependent. Mice have breeding disorders, abnormal behavior, reduced adipose tissue, hypothyroidism with inappropriate TSH response, and enlarged seminal vesicles. This provides an animal model consistent with the proposal that v-ErbA functions as a dominant negative receptor by transcriptional interference or squelching of normal receptors or associated proteins. Finally, male animals develop hepatocellular carcinoma, demonstrating that v-erbA can promote neoplasia in mammals.
Adipose Tissue↗
The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium.
In Drosophila, the Notch gene is pivotal for cell fate decisions at many stages of development and, in particular, during the formation of the nervous system. Absence of Notch results in the generation of excessive numbers of neural cells at the expense of epidermal cells. Two previously identified mammalian Notch homologous encode all the principal features of the Drosophila gene, e.g. 36 EGF-repeats and 3 Notch/lin-12 repeats extracellularly and 6 intracellular cdc10/SWI6 repeats. We report here the characterisation of a third mammalian homologue, mouse Notch 3, which shares the same remarkable conservation relative to the Drosophila gene as the two previously identified homologues, but with three important distinctions. First, Notch 3 specifically lacks the equivalent of EGF-repeat 21; second, it lacks an EGF-repeat-sized region comprising parts of EGF-repeats 2 and 3; and third, it encodes a considerably shorter intracellular domain. The Notch 3 gene is expressed at high levels in proliferating neuroepithelium and expression is downregulated at later stages. The expression patterns of the Notch 1, 2 and 3 genes are quite distinct during central nervous system (CNS) development, and all possible combinations of expression, i.e. none, one, two, or all three genes, are seen, suggesting a combinatorial code of Notch function in mammals. Considering the predominantly early expression in CNS and its distinct structural features, the Notch 3 gene is likely to contribute significantly to vertebrate Notch function during CNS development.
Amino Acid Sequence↗
Generating bacteriophage lambda sublibraries enriched for rare clones.
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Animals↗
Motch A and motch B--two mouse Notch homologues coexpressed in a wide variety of tissues.
Notch is one of the neurogenic genes of Drosophila controlling the decision between ectodermal and neural fate for cells in the early embryo. We have used a polymerase chain reaction (PCR)-based strategy to identify cDNA clones representing two mouse homologues to the Drosophila Notch gene. One of the genes, Motch A, is the mouse orthologue to the previously cloned Notch genes in Xenopus, rat, and man. The other gene, Motch B, is more distantly related to the characterized vertebrate Notch genes, but of equal homology to the Drosophila Notch as is Motch A, and therefore represents a distinct branch of a vertebrate Notch gene family. The Motch A and Motch B branches probably arose by gene duplication early in vertebrate evolution. Both Motch A and Motch B retain the same principal structure, encode mRNAs of approximately 10 kilobases, are expressed during mouse embryogenesis, and have largely overlapping expression patterns in adult tissues. We discuss possible consequences of expressing two closely related Notch homologues in the same set of tissues in terms of cell-cell signaling and differentiation control.
Amino Acid Sequence↗
Drosophila hairy pair-rule gene regulates embryonic patterning outside its apparent stripe domains.
The hairy (h) segmentation gene of Drosophila regulates segmental patterning of the early embryo, and is expressed in a set of anteroposterior stripes during the blastoderm stage. We have used a set of h gene deletions to study the h promoter and the developmental requirements for individual h stripes. The results confirm upstream regulation of h striping but indicate that expression in the anterodorsal head domain depends on sequences downstream of the two transcription initiation sites. Surprisingly, the two anterior-most h domains appear to be dispensable for head development and embryonic viability. One partial promoter deletion expresses ectopic h, leading to misexpression of other segmentation genes and embryonic pattern defects. We demonstrate that h affects patterning outside its apparent stripe domains, supporting a model in which primary pair-rule genes act as concentration-dependent transcriptional regulators, i.e. as local morphogens.
Animals↗
Synthesis in Escherichia coli of the major glycoprotein of human rotavirus: analysis of the antigenic regions.
Various regions of the gene encoding the major neutralization antigen, VP7, of human rotavirus have been expressed in Escherichia coli, as N-terminal fusions to beta-galactosidase under the control of the lac promoter. We have determined that the fusion products of two clones containing regions AB (aa 69-158) and ABC (aa 69-319) were antigenic, reacting with antibodies raised against whole virus. When guinea pigs were immunized with fusion protein purified by monoclonal antibody affinity columns, no neutralizing or virus-binding antibodies were detected, but antibodies binding to denatured VP7 were present.
Antigens, Viral↗
The Drosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc.
The Drosophila segmentation gene, hairy (h), acts to regulate embryonic segmentation and bristle pattern. We present the DNA sequence of the h gene and of h cDNAs, thereby deducing the organization of the h transcripts. The h gene encodes a 337 amino acid protein that acts in both embryonic segmentation and adult bristle patterning. The h protein includes a domain that shows extensive similarity to a domain of the proto-oncogene N-myc that may be involved in DNA binding and/or protein dimerization. We discuss mechanisms of h action as a transcriptional regulator.
Amino Acid Sequence↗