Drosophila Notch Research Articles

Functional analysis of the TAN-1 gene, a human homolog of Drosophila notch.

The TAN-1 gene was originally discovered at the breakpoint of a recurrent (7;9)(q34;q34.3) chromosomal translocation found in a subset of human T-lymphoblastic leukemias (Reynolds et al. 1987; Smith et al. 1988; Ellisen et al. 1991). This translocation joins roughly the 3′ half of TAN-1 head-to-head with the 3′ portion of the β T-cell-receptor gene (TCRB) beginning at the 5′ boundary of one or the other J segment. Intact TAN-1 is normally transcribed into an 8.2-kb transcript that is present in many tissues, most abundantly in developing thymus and spleen (Ellisen et al. 1991). This tissue distribution and the apparent involvement of an altered version of the gene in T-cell cancers have suggested that TAN-1 normally has some special function in lymphocytes or their precursors.

Further evidence for function of the Drosophila Notch protein as a transmembrane receptor.

N locus mutations associated with unusual mutant phenotypes were found to alter the structure of the encoded protein. Two mutations, NCo and N60g11, eliminate much of the cytoplasmic domain. NCo can act as a null allele or as a competitive inhibitor of N+ function, whereas N60g11 produces dominant gain of function in some cell types. This difference in function can be attributed to retention of cdc10/SWI6 repeats in the Notch60g11 protein. The results suggest a role for these repeats in intracellular signaling and are consistent with action of Notch as a receptor. nd3 and l(1)NB alter extracellular epidermal growth factor-like and lin-12/Notch elements, respectively. nd3 eliminates a conserved cysteine residue, so the mutation may result in complete loss of function for a single Notch epidermal growth factor element. N60g11 and l(1)NB produce related gain-of-function phenotypes. It is proposed that l(1)NB produces an extracellular modification of the protein that stimulates aberrant intracellular signaling by the Notch cytoplasmic domain.

An activated Notch receptor blocks cell-fate commitment in the developing Drosophila eye.

The Notch locus of Drosophila melanogaster encodes a 2,703-amino-acid transmembrane protein required for a variety of developmental processes, including neurogenesis, oogenesis and ommatidial assembly. The Notch protein contains a large extracellular domain of 36 epidermal growth factor-like repeats as well as three Notch/Lin-12 repeats and an intracellular domain with 6 Cdc10/ankyrin repeats, motifs that are highly conserved in several vertebrate Notch homologues. Truncation of the extracellular domain of the Drosophila Notch protein produces an activated receptor, as judged by its ability to cause phenotypes similar to gain-of-function alleles of duplications of the Notch locus. Equivalent truncations of vertebrate Notch-related proteins have been associated with malignant neoplasma and other developmental abnormalities. We present here an analysis of activated Notch function at single-cell resolution in the Drosophila compound eye. We find that overexpression of full-length Notch in defined cell types has no apparent effects but that overexpression of activated Notch in the same cells transiently blocks their proper cell-fate commitment, causing them either to adopt incorrect cell fates or to differentiate incompletely. Moreover, an activated Notch protein lacking the transmembrane domain is translocated to the nucleus, raising the possibility that Notch may participate directly in nuclear events.

Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor

The Notch gene of Drosophila plays an important role in cell fate specification throughout development. To investigate the functions of specific structural domains of the Notch protein in vivo, a series of deletion mutants have been ectopically expressed under the hsp70 heat shock promoter. Two classes of dominant phenotypes are observed, one suggestive of Notch loss-of-function mutations and the other of Notch gain-of-function mutations. Dominant activated phenotypes result from overexpression of a protein lacking most extracellular sequences, while dominant negative phenotypes result from overexpression of a protein lacking most intracellular sequences. These results support the notion that Notch functions as a receptor whose extracellular domain mediates ligand binding, resulting in the transmission of developmental signals by the cytoplasmic domain. Finally, the phenotypes observed suggest that the cdc10/ankyrin repeat region within the intracellular domain plays an essential role in the postulated signal transduction events.

Genetic and molecular characterization of a Notch mutation in its Delta- and Serrate-binding domain in Drosophila.

The Drosophila Notch gene product is a transmembrane protein that functions as a receptor of intercellular signals in several Drosophila developmental processes. Two other transmembrane proteins, encoded by the genes Delta and Serrate, genetically and molecularly behave as Notch ligands. All these proteins share the presence of epidermal growth factor (EGF)-like repeats in their extracellular domain. The Notch protein has 36 EGF-like repeats, 2 of which, numbers 11 and 12, are required for the interaction with the Delta and Serrate ligands. We have isolated and molecularly characterized a Notch mutation in its Delta- and Serrate-binding domain that behaves genetically as both a Notch antimorphic and a loss-of-function mutation. This mutation, NM1, carries a Glu-->Val substitution in the Notch EGF repeat 12. The NM1 allele interacts with other Notch alleles such as Abruptex and split and with mutations in the Notch-ligand genes Delta and Serrate. The basis for the genetic antimorphism of NM1 seems to reside in the titration of Notch wild-type products into NM1/N+ nonfunctional dimers and/or the titration of Delta products into nonfunctional ligand-receptor complexes.

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.

Cloning, Analysis, and Chromosomal Localization of Notch-1, a Mouse Homolog of Drosophila Notch

The Notch gene of Drosophila encodes a large transmembrane protein involved in cell-cell interactions and cell fate decisions in the Drosophila embryo. We report here the isolation of cDNA clones encompassing the full-length coding sequence of Notch-1, a mouse homolog of Drosophila Notch. The predicted amino acid sequence of the Notch-1 protein retains all of the conserved amino acid motifs of Notch and the other vertebrate Notch homologs. The cDNA sequence predicts a 2531-amino-acid protein containing a signal peptide, 36 epidermal growth factor-like repeats, 3 Notch/lin-12 repeats, a transmembrane domain, and 6 cdc10/ankyrin repeats. The Notch-1 gene was localized to the proximal portion of mouse chromosome 2 by mapping with an interspecific backcross panel.

Notch2: a second mammalian Notch gene.

Notch is a cell surface receptor that mediates a wide variety of cellular interactions that specify cell fate during Drosophila development. Recently, homologs of Drosophila Notch have been isolated from Xenopus, human and rat, and the expression patterns of these vertebrate proteins suggest that they may be functionally analogous to their Drosophila counterpart. We have now identified a second rat gene that exhibits substantial nucleic and amino acid sequence identity to Drosophila Notch. This gene, designated Notch2, encodes a protein that contains all the structural motifs characteristic of a Notch protein. Thus, mammals differ from Drosophila in having more than one Notch gene. Northern and in situ hybridisation analyses in the developing and adult rat identify distinct spatial and temporal patterns of expression for Notch1 and Notch2, indicating that these genes are not redundant. These results suggest that the great diversity of cell-fate decisions regulated by Notch in Drosophila may be further expanded in vertebrates by the activation of distinct Notch proteins.

Expression analysis of a Notch homologue in the mouse embryo

The Drosophila Notch gene has been shown to be involved in the determination of fate in a number of different cell types. Similarly, Notch homologues in Caenorhabditis elegans are involved in cell decision-making steps. It is of interest to determine if a mammalian Notch homologue plays a role in cell fate determination. We have isolated cDNA from a mouse Notch gene using low-stringency hybridization with probes derived from the Xenopus Notch gene. Sequence analysis reveals that this gene possesses EGF repeats, Notch/lin-12 repeats, and CDC-10/SWI-6 repeats, characteristic of other Notch homologues. Northern analysis revealed that the transcript size was roughly 10 kb as has been found for the other Notch genes. We have studied the expression pattern of the gene by both conventional and whole mount in situ hybridization. Expression patterns were consistent with mouse Notch having a determinative role in the formation of mesoderm, somites, and the nervous system.

Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins

An oligonucleotide based on the cdc 10 SW16 repeats of the Drosophila Notch protein was used to isolate other Drosophila genes with these repeats. One of these genes is the cactus locus, 1 of 11 genes required maternally for the establishment in embryos of a gradient of nuclear localization of dorsal protein, a rel-like transcription factor. Previous work showed that in cactus mutants more dorsal protein enters the nucleus in dorsal regions, resulting in a ventralized phenotype. It is now shown that the cactus locus produces two proteins that differ at their carboxy termini; both contain six cdc 10 SW16 repeats that are sufficient for binding to dorsal and for inhibiting the ability of dorsal to bind DNA. The site on dorsal to which cactus binds was localized to the rel homology domain, where it overlaps with, or is adjacent to, the nuclear localization signal. In vivo the bulk of the cactus protein associated with dorsal is phosphorylated. This, or the association with dorsal, appears to stabilize the maternal cactus protein.

Mouse mammary tumor gene int-3: a member of the notch gene family transforms mammary epithelial cells

Expression of a 2.3-kb RNA species is induced in mammary tumors as a consequence of insertional mutagenesis at the int-3 locus by the mouse mammary tumor virus. The nucleotide sequence and biological activity of this mammary tumor-specific int-3 RNA species were determined. It contains an open reading frame which encodes a 57-kDa protein. The translated protein possesses six nearly contiguous 32-amino-acid repeats which are related to a similar motif in the Saccharomyces cerevisiae cdc-10-encoded cell cycle protein. In addition, the int-3 cdc-10 repeats are bounded by the PEST amino acid sequence motif which is commonly found in proteins having a rapid turnover and may represent sites for phosphorylation. The int-3 cdc-10 repeat sequences are 50% identical to a portion of the intracellular domain of the neurogenic Drosophila notch gene product. Activation of expression of a recombinant int-3 genomic DNA fragment encoding the 2.3-kb RNA species in HC11 mouse mammary epithelial cells in vitro induces anchorage-independent growth in soft agar.

Molecular bases of early neural development in Xenopus embryos.

Genes that are differentially expressed in the ectoderm as it diverts along the neural and epidermal pathways of differentiation can be used to study the inducing signals underlying induction as well as how ectoderm responds to these signals by forming neural tissue. Although these genes have provided, and will continue to provide, new information about the induction process, they are not likely to provide the whole story. For example, Notch is a gene required for neurogenesis in Drosophila embryos. When the Notch gene product is absent, the embryo forms far too many neuroblasts at the expense of the hypodermal cell layer (Artavanis-Tsakonis 1988, Campos-Ortega 1988). Even though Notch appears to play a role in deciding the fate of neural/hypodermal cells, the Notch gene product is expressed ubiquitously in early embryos in the neurogenic region (Hartley et al 1987, Kidd et al 1989). Thus, one possibility is that Notch function does not necessarily depend on differential expression of the Notch gene product within cells in the neurogenic regions [although an alternative view has been suggested by Greenspan (1990)]. Thus, some of the molecules controlling early neural development may not be expressed differentially when the ectoderm forms the neural plate. Obviously, other approaches will have to be taken to isolate and characterize these molecules. In this light, it is noteworthy that a molecule has been identified in Xenopus that is remarkably similar to Drosophila Notch in both structure and developmental expression (Coffman et al 1990). One hope is that the analysis of this molecule in combination with the molecules that are differentially expressed during neural induction will eventually lead to a molecular understanding of early neural development in vertebrate embryos.

An adult male specific gene in Drosophila containing the repetitive element opa

A cDNA has been isolated for an adult male specific gene in Drosophila that contains the repetitive element opa. We have named this gene Dromsopa for Drosophila male specific opa containing gene. The 0.6 kb mRNA for this gene is only found in the abdominal region of adult male Drosophila and in no other tissue or at other developmental stages. The Dromsopa opa repeat codes for the usual stretch of poly(glutamine) interrupted by histidine residues. The opa repetitive element was originally found in the Drosophila Notch gene (Kidd, S. et al. (1983) Cell 34, 431-433 and Wharton, K.A. (1985) Cell 40, 55-62) and has, more recently, been found in genes under developmental or tissue specific control from yeast to humans. The gene was cloned using a genomic fragment during a chromosomal walk upstream of the AP3 gene located at chromosomal location 79CD on the left arm of the third chromosome (Kelley, M.R. et al. (1989) Mol. Cell. Biol. 9, 965-973). The Dromsopa gene has no other identity with genes currently in the databases, once the opa repeat is excluded. The possibility that the Dromsopa gene is a male specific regulatory factor is under investigation as is its precise location within the abdomen, such as in germ line tissue.

TAN-1, the human homolog of the Drosophila Notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms

Previously we described joining of DNA in the β T cell receptor gene to DNA of an uncharacterized locus in a t(7;9)(q34;q34.3) chromosomal translocation from a case of human T lymphoblastic leukemia (T-ALL). We now show that the locus on chromosome 9 contains a gene highly homologous to the Drosophila gene Notch. Transcripts of the human gene, for which we propose the name TAN-1, and its murine counterpart are present in many normal human fetal and adult mouse tissues, but are most abundant in lymphoid tissues. In t(7;9)(q34;q34.3) translocations from three cases of T-ALL, the breakpoints occur within 100 bp of an intron in TAN-1, resulting in truncation of TAN-1 transcripts. These observations suggest that TAN-1 may be important for normal lymphocyte function and that alteration of TAN-1 may play a role in the pathogenesis of some T cell neoplasms.

Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

A homolog of Drosophila Notch expressed during mammalian development

Drosophila Notch and the related Caenorhabditis elegans proteins lin-12 and glp-1 function as mediators of local cell-cell interactions required for cell-fate decisions during invertebrate development. To investigate the possibility that similar proteins play determinative roles during mammalian development, we isolated cDNA clones encoding rat Notch. The deduced amino acid sequence of this protein contains 36 epidermal growth factor (EGF)-like repeats, and is remarkably similar in both its extracellular and cytoplasmic domains to the sequence of Xenopus Xotch and Drosophila Notch. In the developing central nervous system, in situ hybridisation analyses revealed that Notch transcripts were dramatically restricted to the ventricular proliferative zones of embryonic neuroepithelia. Notch was also strongly expressed during development of non-neural tissues, such as hair follicles and tooth buds, whose correct differentiation requires epithelial-mesenchymal interactions. These data support the hypothesis that Notch plays an essential role in mammalian development and pattern formation that closely parallels its role in the development of invertebrates.

Nerve cells of Drosophila Notch mutant are differentiated inside amphibian brain: a new approach for the analysis of genetic control of nerve cell differentiation

Fragments of the neural primordium of a new Notch mutant of Drosophila melanogaster produced in our laboratory were transplanted into the neural tube of embryos of 4 amphibian species (caudate and ecaudate) immediately after completion of neurulation. The grafts were identified by using a light microscope, scanning electron miscroscope, and in situ hybridization with mobile genetic elements of Drosophila and fluorescent dyes as markers. As has been shown, Drosophila nerve cells survive and differentiate inside the neural tube of amphibian embryos. The grafts increase in size by twentyfold and the cell proliferation zones are retained during the period of six months. Differentiated cells of the graft formed axon-dendritic contacts with recipient cells and penetrated into the organisms' brain structures. The effect of Drosophila transplants proved to be different for caudate and ecaudate amphibians. The presence of the graft accelerated the development of Xenopus laevis and it also affected their behavior. This approach can be very useful for the study of genetic basis of development and behavior.

Mammalian cerebellum expresses a new protein homologous to the cytoplasmic domain of the product from drosophila notch gene

Mammalian cerebellum expresses a new protein homologous to the cytoplasmic domain of the product from drosophila notch gene

Spacing Differentiation in the Developing Drosophila Eye: a Fibrinogen-related Lateral Inhibitor Encoded by scabrous

In the development of multicellular organisms a diversity of cell types differentiate at specific positions. Spacing patterns, in which an array of two or more cell types forms from a uniform field of cells, are a common feature of development. Identical precursor cells may adopt different fates because of competition and inhibition between them. Such a pattern in the developing Drosophila eye is the evenly spaced array of R8 cells, around which other cell types are subsequently recruited. Genetic studies suggest that the scabrous mutation disrupts a signal produced by R8 cells that inhibits other cells from also becoming R8 cells. The scabrous locus was cloned, and it appears to encode a secreted protein partly related to the beta and gamma chains of fibrinogen. It is proposed that the sca locus encodes a lateral inhibitor of R8 differentiation. The roles of the Drosophila EGF-receptor homologue (DER) and Notch genes in this process were also investigated.

Xotch , the Xenopus Homolog of Drosophila Notch

During the development of a vertebrate embryo, cell fate is determined by inductive signals passing between neighboring tissues. Such determinative interactions have been difficult to characterize fully without knowledge of the molecular mechanisms involved. Mutations of Drosophila and the nematode Caenorhabditis elegans have been isolated that define a family of related gene products involved in similar types of cellular inductions. One of these genes, the Notch gene from Drosophila, is involved with cell fate choices in the neurogenic region of the blastoderm, in the developing nervous system, and in the eye-antennal imaginal disc. Complementary DNA clones were isolated from Xenopus embryos with Notch DNA in order to investigate whether cell-cell interactions in vertebrate embryos also depend on Notch-like molecules. This approach identified a Xenopus molecule, Xotch, which is remarkably similar to Drosophila Notch in both structure and developmental expression.