Molecular Analysis of Scabrous Mutant Alleles from Drosophila Melanogaster Indicates a Secreted Protein with Two Functional Domains

Mutations at the scabrous locus (sca) affect cell-cell signaling during neural developent. Twenty-one mutant alleles of scabrous have been analyzed. Many synthesize no sca protein. In others, a defective protein is arrested intracellularly. Two mutants in which protein is not arrested must affect sca protein function outside the cell. Both affect the fibrinogen related domain (FReD), a 200-amino acid segment conserved in fibrinogen, tenascins, and other proteins. In fibrinogen, this region is involved in protein interactions and is altered in human mutations affecting blood clotting. In sca(UM2), an invariant Asp residue is replaced by Asn. In sca(MSKF), an insertion of the hobo transposable element truncates the sca protein at the start of the FReD. The sca(MSKF) allele has dominant negative properties, indicating that the truncated amino-terminal portion interferes with the function of some other gene product. These mutations show that the conserved FReD is essential for wild-type sca function, but suggest that the amino-terminal domain also interacts with other proteins. Genetic interactions identify the neurogenic genes Notch and Delta as potential interacting proteins, but other neural mutations were without effect. Models for the role of a two-domain protein in neural development are discussed.     

The action of the Notch locus in Drosophila melanogaster

Summary It is shown that the Notch⁸ deficiency in Drosophila melanogaster affects a number of enzyme activities localized in the mitochondria, such as NADH oxidase (activity of the complete respiratory chain), NADH dehydrogenase (the first step in the respiratory chain before transfer to ubiquinone), Succinate dehydrogenase and -Glycerophosphate dehydrogenase. The experiments reported here do not exclude the possibility of involvement of other genes in the deficiency. The effect of duplications of the Notch locus on NADH oxidase and NADH dehydrogenase suggest that this locus determines the enzyme activities.The dosage effects of the Notch locus on activity suggest that this locus contains the structural genes for these enzymes.     

Mutations in the heatshock cognate 70 protein (hsc4) modulate Notch signaling

In our effort to dissect the Notch signaling mechanism we have conducted a screen for mutations that reduce Notch signaling activity. We recovered nine complementation groups as modifiers of the hypomorphic Notch allele notchoid. Apart from the known Notch signaling modulators Notch, Delta and mastermind we isolated alleles in vestigial, wingless, scalloped and clipped, genes known to affect wing morphogenesis. In addition, we identified mutations in Bag, the gene encoding clathrin heavy chain and a dominant mutation of the cytosolic 70 kDa heatshock cognate protein encoded by the hsc4 gene, as Notch signaling modifier. We focused our attention on the latter mutation because it displays dramatic genetic interactions with mutations of the Notch receptor as well as several additional Notch signaling pathway elements. We discuss how hsc4, a gene thought to be involved in subcellular trafficking, may affect the number of functional Notch receptors on the cell surface.     

scabrous modifies epithelial cell adhesion and extends the range of lateral signalling during development of the spaced bristle pattern in Drosophila.

The role of scabrous (sca) in the evenly spaced bristle pattern of Drosophila is explored. Loss-of-function of sca results in development of an excess of bristles. Segregation of alternately spaced bristle precursors and epidermal cells from a group of equipotential cells relies on lateral inhibition mediated by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit the neural fate of neighbouring cells, causing them to adopt the epidermal fate. We show that Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signalling has been increased. scabrous acts nonautonomously, and we present evidence that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signalling are discussed. We also show that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and speculate on a possible role for these structures during signalling.     

一号冰川地区四种藓类植物的解剖学研究

运用石蜡切片技术和扫描电镜方法,对一号冰川地区生长的4种藓类植物茎、叶的内部结构及叶表皮角质层褶皱、疣和中肋等进行观察,结果表明：红扭口藓(Barbula asperifolia Mitt.)茎横切面呈多棱形;叶背、腹面角质层厚,粗疣不分叉,但顶端朝细胞凹陷处倾斜;叶背面细胞壁凹陷深,象张开的气孔,粗疣也藏在其中,中肋突出明显。丛叶扭藓(Tortella humilis (Hedw.) Jenn.)茎横切面呈椭圆形;叶背、腹面中上部均密被鹿角状粗疣,这些分叉的粗疣,从凹陷的细胞壁处成束突起,顶端向下弯曲成钩状,在叶的下部疣状突起则逐渐减少至无,中肋较宽。异叶提灯藓(Mnium heterophyllum (Hook.)Schwagr.)茎横切面呈五棱形;叶细胞一层,呈不规则多边形,细胞壁凹陷使叶片呈网状;中肋红色。大灰藓(Hypnum plumaeforme Wils.)茎的横切面呈圆形;叶背、腹面均密被疣状突起,且差异不大,粗疣顶部均倒向孔口呈遮盖状;中肋短而弱。     

The scabrous protein can act as an extracellular antagonist of notch signaling in the Drosophila wing

Notch (N) is a receptor for signals that inhibit neural precursor specification [1-6]. As N and its ligand Delta (DI) are expressed homogeneously, other molecules may be differentially expressed or active to permit neural precursor cells to arise intermingled with nonneural cells [7,8]. During Drosophila wing development, the glycosyltransferase encoded by the gene fringe (fng) promotes N signaling in response to DI, but inhibits N signaling in response to Serrate (Ser), which encodes a ligand that is structurally similar to DI. Dorsal expression of Fng protein localizes N signaling to the dorsoventral (DV) wing margin [9-11]. The secreted protein Scabrous (Sca) is a candidate for modulation of N in neural cells. Mutations at the scabrous (sca) locus alter the locations where precursor cells form in the peripheral nervous system [12,13]. Unlike fringe, sca mutations act cell non-autonomously [12]. Here, we report that targeted misexpression of Sca during wing development inhibited N signaling, blocking expression of all N target genes. Sca reduced N activation in response to DI more than in response to Ser. Ligand-independent signaling by overexpression of N protein, or by expression of activated truncated N molecules, was not inhibited by Sca. Our results indicate that Sca can act on N to reduce its availability for paracrine and autocrine interactions with DI and Ser, and can act as an antagonist of N signaling.     

The Notch gene, adhesion, and developmental fate in the Drosophila embryo

The Notch locus in Drosophila encodes a transmembrane protein required for the determination of cell fate in ectodermal cells. When these cells are faced with a choice of two possible fates, Notch enables some of them to advance from the fate that represents a "default" state. The decision appears to involve both an interaction between cells and the presence of the Notch product on the surface of those advancing from the default state. The timing of gene action suggests that Notch plays a role in the stabilization of the choice of cell fate and that the Notch-mediated interaction occurs between presumptive epidermal cells. Several properties of the Notch product are characteristic of an adhesion molecule, and thus cell adhesion may play a role in the determination of cell fate.     

Modifications of the Notch Function by Abruptex Mutations in Drosophila Melanogaster

The function of the Notch gene is required in cell interactions defining alternative cell fates in several developmental processes. The Notch gene encodes a transmembrane protein with 36 epidermal growth factor (EGF)-like repeats in its extracellular domain. This protein functions as a receptor that interacts with other transmembrane proteins, such as Serrate and Delta, which also have EGF repeats in their extracellular domain. The Abruptex mutations of the Notch locus are associated with amino acid substitutions in the EGF repeats 24-29 of the Notch protein. We have studied, in genetic combinations, the modifications of Notch function caused by Abruptex mutations. These mutations lead to phenotypes which are opposite to those caused by Notch deletions. The Abruptex phenotypes are modified by the presence of mutations in other loci, in particular in the genes Serrate and Delta as well as Hairless, and groucho. The results suggest that all Abruptex mutations cause stronger than normal Notch activation by the Delta protein. Some Abruptex alleles also display an insufficiency of N function. Abruptex alleles which produce stronger enhancement of Notch activation also display stronger Notch insufficiency. This insufficiency could be due to reduced ability of Abruptex proteins to interact with Notch ligands and/or to form functional Notch dimers.     

Local function of the Notch gene for embryonic ectodermal pathway choice in Drosophila

Mutations at the Notch locus affect the fate of cells in the neurogenic region of the Drosophila embryo so that epidermal precursors become neuroblasts. We have analyzed the cellular requirements for wild-type Notch gene function by means of genetic mosaics, using a cuticle marker to distinguish hypodermal cell genotype. Cells that were genotypically Notch never gave rise to hypoderm within the neurogenic region of mosaic embryos. Mosaic dividing lines within the neurogenic region juxtapose N+ hypoderm with regions of neural hypertrophy. This autonomous action of Notch in hypodermal cells is consistent with a local function of the protein during neurogenesis. Comparison of clone distribution in Notch mosaics and controls suggests that islands of wild-type hypodermal cells fail to differentiate cuticle.     

Drosophila Notch Receptor Activity Suppresses Hairless Function during Adult External Sensory Organ Development

The neurogenic Notch locus of Drosophila encodes a receptor necessary for cell fate decisions within equivalence groups, such as proneural clusters. Specification of alternate fates within clusters results from inhibitory communication among cells having comparable neural fate potential. Genetically, Hairless (H) acts as an antagonist of most neurogenic genes and may insulate neural precursor cells from inhibition. H function is required for commitment to the bristle sensory organ precursor (SOP) cell fate and for daughter cell fates. Using Notch gain-of-function alleles and conditional expression of an activated Notch transgene, we show that enhanced signaling produces H-like loss-of-function phenotypes by suppressing bristle SOP cell specification or by causing an H-like transformation of sensillum daughter cell fates. Furthermore, adults carrying Notch gain of function and H alleles exhibit synergistic enhancement of mutant phenotypes. Over-expression of an H(+) transgene product suppressed virtually all phenotypes generated by Notch gain-of-function genotypes. Phenotypes resulting from over-expression of the H(+) transgene were blocked by the Notch gain-of-function products, indicating a balance between Notch and H activity. The results suggest that H insulates SOP cells from inhibition and indicate that H activity is suppressed by Notch signaling.     

Is the Notch Locus of DROSOPHILA MELANOGASTER a Tandem Repeat? Correlation of the Genetic Map and Complementation Pattern of Selected Mutations

Correlation of the complementation relationships between the Notch locus alleles fa(g), fa(no), Ax(59d) and nd, with their genetic map order, suggests a tandem repetition within the locus of functionally related sites. This observation is discussed in relation to two hypotheses: (1) that the Notch locus contains a tandem repeat of genetic material; and (2) that the tertiary structure of the Notch locus product has a spiral configuration.     

The Notch locus of Drosophila is required in epidermal cells for epidermal development

The Notch locus of Drosophila plays an important role in cell fate decisions within the neurogenic ectoderm, a role thought to involve interactions at the cell surface. We have assayed the requirement for Notch gene expression in epidermal cells by two kinds of genetic mosaics. First, with gynandromorphs, we removed the wild-type gene long before the critical developmental events to produce large mutant clones. The genotype of cells in large clones was scored by means of an antibody to the Notch protein. Second, using mitotic recombination, we removed the gene at successively later times after completion of the mitotically active early cleavage stages, to produce small clones. These clones were detected by means of a linked mutation of cuticle pattern, armadillo. The results of both experiments demonstrate a requirement for Notch expression by epidermal cells, and thus argue against the model that the Notch product acts as a signal required only in the neuroblast to influence neighboring epidermal cells. The mitotic recombination experiment revealed that Notch product is required by epidermal cells subsequent to neuroblast delamination. This result implies that the Notch gene functions to maintain the determined state of epidermal cells, possibly by mediating cell surface interactions within the epidermis.     

Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes.

How enhancers are able to activate promoters located several kilobases away is unknown. Activation by the wing margin enhancer in the cut gene, located 85 kb from the promoter, requires several genes that participate in the Notch receptor pathway in the wing margin, including scalloped, vestigial, mastermind, Chip, and the Nipped locus. Here we show that Nipped mutations disrupt one or more of four essential complementation groups: l(2)41Ae, l(2)41Af, Nipped-A, and Nipped-B. Heterozygous Nipped mutations modify Notch mutant phenotypes in the wing margin and other tissues, and magnify the effects that mutations in the cis regulatory region of cut have on cut expression. Nipped-A and l(2)41Af mutations further diminish activation by a wing margin enhancer partly impaired by a small deletion. In contrast, Nipped-B mutations do not diminish activation by the impaired enhancer, but increase the inhibitory effect of a gypsy transposon insertion between the enhancer and promoter. Nipped-B mutations also magnify the effect of a gypsy insertion in the Ultrabithorax gene. Gypsy binds the Suppressor of Hairy-wing insulator protein [Su(Hw)] that blocks enhancer-promoter communication. Increased insulation by Su(Hw) in Nipped-B mutants suggests that Nipped-B products structurally facilitate enhancer-promoter communication. Compatible with this idea, Nipped-B protein is homologous to a family of chromosomal adherins with broad roles in sister chromatid cohesion, chromosome condensation, and DNA repair.     

Notch2 expression negatively correlates with glial differentiation in the postnatal mouse brain

Notch family molecules are thought to be negative regulators of neuronal differentiation in early brain development. After expression in the embryonic period, Notch2 continues to be expressed postnatally in the specific regions in the rodent brain. Here, we examined Notch2 expression in the postnatal mouse brain using lacZ knockin animals at the Notch2 locus. Notch2 expression was observed in the developing cerebellum and hippocampus, characteristic regions where neurogenesis persists after birth. Double staining of sections revealed that Notch2 was expressed by Bergmann glia in the cerebellum, radial glia in the hippocampus, and some astrocytes in both regions. Notch2 expression by glial cells was clearly confirmed in dissociated cell cultures. Interestingly, neocortical glia, many of which did not express Notch2 in vivo, did express Notch2 in a dissociated culture condition. The triple staining of dissociated cell cultures revealed that stronger Notch2 expression correlated with the immature type of glial gene expressions: stronger vimentin and weaker glial fibrillary acidic protein expressions. In addition, Notch2 expression correlated with the incorporation of bromodeoxyuridine both in vivo and in vitro. Thus, these findings demonstrate that Notch2 is expressed not only by neuronal cells in the embryonic brain, but also by glial cells in the postnatal brain, and that its expression negatively correlates with glial differentiation, proposing its novel function as a negative regulator of glial differentiation in mammalian brain development.