Elisabeth Knust

Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of drosophila

The crumbs protein of Drosophila is an integral membrane protein, with 30 EGF-like and 4 laminin A G domain-like repeats in its extracellular segment, which is expressed on the apical plasma membrane of all ectodermally derived epithelia. Here, we present evidence to show that the insertion of crumbs into the plasma membrane is necessary and sufficient to confer apical character on a membrane domain. Overexpression of crumbs results in an enormous expansion of the apical plasma membrane and the concomitant reduction of the basolateral domain. This is followed by the redistribution of beta Heavy-spectrin, a component of the membrane cytoskeleton, and by the ectopic deposition of cuticle and other apical components into these areas. Strikingly, overexpression of the membrane-bound cytoplasmic portion of crumbs alone is sufficient to produce this dominant phenotype. Our results suggest that crumbs plays a key role in specifying the apical plasma membrane domain of ectodermal epithelial cells of Drosophila.

crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia

We describe the molecular characterization of the Drosophila gene crumbs, which encodes an integral membrane protein with 30 EGF-like repeats in the extracellular part and exhibits a striking expression pattern. The protein is exclusively localized on the apical membranes of epithelial cells and concentrated at the borders between cells. Mutations in crumbs lead to severe disruptions in the organization of ectodermally derived epithelia and in some cases to cell death in these tissues. The structure and the expression pattern of the protein and the phenotype of mutations indicate a function of crumbs during the development of epithelia, possibly for the establishment and/or maintenance of cell polarity.

The neurogenic gene Delta of Drosophila melanogaster is expressed in neurogenic territories and encodes a putative transmembrane protein with EGF-like repeats

The decision of an ectodermal cell to take on a neural or an epidermal fate depends on its interactions with the neighbouring cells. In Drosophila melanogaster, the available evidence suggests that a regulatory signal necessary for epidermal commitment is built by the products of the so‐called neurogenic genes. We have cloned 180 kb of genomic DNA surrounding the neurogenic gene Delta (Dl). Restriction fragment‐length polymorphisms were mapped to a region of 25 kb. These 25 kb of DNA are assumed to contain essential parts, or all, of the Dl gene. Northern blots detect two developmentally regulated transcripts, of 5.4 and 4.6 kb, which are associated with the region where the mutants map. Serveral cDNA clones were recovered from embryonic cDNA libraries by homology to the 25 kb of genomic DNA. The complete sequence of a cDNA clone containing an insert of 4.73 kb was determined. The conceptual translation of the longest open reading frame yields a protein of 880 amino acids. This protein displays characteristics of a membrane protein, with intercellular, transmembrane and extracellular domains. The extracellular domain contains a tandem array of nine EGF‐like repeats. In in situ hybridizations to tissue sections, transcripts homologous to Dl are detected in all territories with neurogenic abilities, e.g. the neurogenic ectoderm and the primordia of the sensory organs. Initially all cells of these neurogenic territories express Dl, but later on transcription of Dl becomes restricted to the cells that have adopted the neural fate. The topological specificity in the transcription of Dl corresponds to the one expected for a regulatory signal that mediates epidermal commitment.

Closely related transcripts encoded by the neurogenic gene complex enhancer of split of Drosophila melanogaster.

Genetic evidence suggests that E(spl), one of the neurogenic loci of Drosophila, is a gene complex comprising an as yet incompletely established number of transcription units. In order to correlate the various transcription units with E(spl) functions, wild‐type flies were transformed with genomic DNA encoding the transcription unit m8 from the mutant E(spl)D, which was known to be altered in embryos carrying this mutant allele. Transformants show the same dominant enhancement of the spl phenotype as E(spl)D itself. Since m8 has a virtually identical pattern of expression as m4, m5 and m7, we have determined the sequence of these four transcripts. The deduced protein products of m5, m7 and m8 exhibit extensive sequence homology with each other. All three encode a sequence similar to one of the conserved domains of representatives of the vertebrate myc gene family which is also present in the deduced protein sequences of the Drosophila achaete‐scute gene complex. Sequence analysis of the m8 transcription unit in the E(spl)D mutation revealed several DNA lesions. One of the lesions is a deletion in the region upstream of the transcription start site. Another lesion is a deletion in the coding region that leads to a shorter protein which, in addition, differs in its carboxy‐terminal end from the wild‐type protein by the presence of nine amino acids.

Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila

BACKGROUNDnThe appendages of insects, like the limbs of vertebrates, grow out of the body wall after the establishment of a proximo-distal axis among a group of primordial cells. In Drosophila, the wing develops in the limbless larva from one of the imaginal discs of the thorax, which give rise to the adult epidermis. The earliest identified requirement in wing development is for the induction of vestigial (vg) gene expression at the interface between ventral cells and dorsal cells of the wing disc. It has been proposed that this event requires two reciprocal signals--one from the dorsal to the ventral cells and the other from the ventral to the dorsal cells--which trigger vg expression at the presumptive wing margin and hence initiate the development of the wing tissue.nnnRESULTSnWe have identified four genes--Serrate (Ser), wingless (wg), Notch and Suppressor of Hairless (Su(H))--whose activity is required during the second and early third larval instars for the expression of vg. Analysis of the functions and patterns of expression of these genes at the time of the inductive event indicates that the Ser protein acts as a dorsal signal, and the Wg protein as a ventral signal for the induction of vg expression. Furthermore, the expression of both Ser and Wg is sufficient to trigger ectopic wing development in the wing disc and leg discs. The product of the Notch gene, which encodes a receptor, is also required for this event and we suggest that its role is to integrate the inputs of Ser and Wg.nnnCONCLUSIONSnWe show that the induction of vg, which initiates wing development in Drosophila, requires the combined activities of Ser, wg and Notch. Based on the patterns of expression and requirements for Ser and wg in this process, we propose that Ser is a dorsal signal and that Wg is a ventral signal, and that their combination at the dorso-ventral interface activates the Notch receptor and leads to vg expression.

bHLH proteins encoded by theEnhancer of split complex ofDrosophila negatively interfere with transcriptional activation mediated by proneural genes

TheEnhancer of split complex [E(SPL)-C] ofDrosophila participates in the control of cell fate choice by uncommitted neuroectodermal cells in the embryo. It encodes seven proteins that belong to the basic helix-loop-helix (bHLH) family, six of which are expressed in very similar patterns in the neuroectoderm. Here we describe experiments aimed at unravelling the molecular basis of their function. We found that two products of the complex, HLH-M5 andEnhancer of split, are capable of binding as homo-and heterodimers to a sequence in the promoters of theEnhancer of split andachaete genes, called the N-box, which differs slightly from the consensus binding site (the E-box) for other bHLH proteins. In transient expression assays in cell culture, both proteins were found to attenuate the transcriptional activation mediated by the proneural bHLH proteinslethal of scute anddaughterless at theEnhancer of split promoter.

Molecular analysis of the neurogenic locus Enhancer of split of Drosophila melanogaster

Enhancer of split [E(spl)], one of the neurogenic loci of Drosophila, is located in bands 96F8‐13. One hundred and fifty kilobases of genomic DNA, spanning the E(spl) locus, were cloned by chromosomal walking. DNA heterogeneities associated with eleven E(spl) mutations, including three Pr alleles, were mapped to a region of 36 kb, and an additional one outside of this region. One of these mutations is a deletion of ˜34 kb that causes severe neural hyperplasia of homozygous embryos with complete penetrance. Mutations associated with DNA polymorphisms mapping within smaller regions do not lead to a fully penetrant neurogenic phenotype. The 36‐kb region encodes 11 major transcripts, which exhibit distinct temporal and/or spatial patterns of expression. The expression of one of these transcripts is modified in two different mutants. In addition, one of the mutants [E(spl)D] shows another transcript, which is not present in the wild‐type and co‐exists with the remaining transcripts. We suggest that more than one of the 11 transcripts are necessary for a normal function of the E(spl) locus. The spatial distribution of four of these RNAs, which exhibit almost identical patterns of expression, strongly suggests that the encoded proteins are required for the process of segregation of neural and epidermal lineages.

EGF homologous sequences encoded in the genome of Drosophila melanogaster, and their relation to neurogenic genes.

The function of the neurogenic genes of Drosophila melanogaster is required for a normal pattern of commitment of neural and epidermal progenitor cells. In the course of searching for a molecular basis for the functional interrelationships that exist between the neurogenic genes, fragments of cloned DNA from the genes master mind (mam), Delta (Dl), Enhancer of split [E(spl)] and Notch (N) were hybridized to each other. Strong cross‐hybridization was observed between a fragment of the Dl gene and a fragment of the N gene encoding a peptide with homology to several proteins of mammals, including the epidermal growth factor (EGF). Sequencing of this Dl fragment revealed an open reading frame encoding four EGF‐like repeats with homology to the repeats found in the N gene. Screening genomic and cDNA libraries under conditions of reduced stringency with Dl and N probes that encode EGF‐like repeats uncovered several cross‐hybridizing clones, suggesting that other Drosophila genes may also encode such peptides. Part of a cross‐hybridizing cDNA clone, derived from a gene located at position 95F on the third chromosome, was sequenced and found to encode five repeats with homology to those encoded by N and Dl. Preliminary evidence on the spatial pattern of transcription indicates that the gene at position 95F is regulated in its expression, as it is transcribed in all ectodermal derivatives, with the exception of the central nervous system. Indirect evidence suggests that this clone may derive from the crumbs (crb) gene, which is likely to be an hitherto unknown neurogenic gene.(ABSTRACT TRUNCATED AT 250 WORDS)

The enhancer of split locus and neurogenesis in Drosophila melanogaster

Enhancer of split (E(spl)) is one of a group of so-called neurogenic genes of Drosophila. We describe two different types of E(spl) alleles, dominant and recessive, which exert opposite effects on both central and peripheral nervous system development. The only extant dominant allele determines a reduction in the number of central neurons and peripheral sensilla; this phenotype is not reduced by a normal complement of wild-type alleles. Since animals carrying a triploidy for the wild-type locus develop similar defects, the dominant allele is probably the result of a gain-of-function mutation. Several recessive alleles, obtained as revertants of the dominant allele, are loss-of-function mutations and determine considerable neural hyperplasia. The present evidence suggests that neural defects of E(spl) mutants are due to defective segregation of neural and epidermal lineages, leading to neural commitment of less or of more cells than in the wild type, depending upon whether the animals carry the dominant or any of the recessive alleles, respectively. Therefore, E(spl) formally behaves as a gene switching between neural and epidermal pathways.

Phenotypic and developmental analysis of mutations at thecrumbs locus, a gene required for the development of epithelia inDrosophila melanogaster

SummaryThe genecrumbs (crb) ofDrosophila melanogaster provides an essential function for the embryonic development of ectodermally derived epithelia. Complete loss of function alleles of thecrb gene are recessive embryonic lethals and lead to a disorganization of the primordia of these epithelia, followed by cell death in some tissues. Incrb mutant embryos, different organs are affected to a different extent. Some tissues die almost completely (as the epidermis, the atrium and the pharynx) while others partially survive and conserve their basic epithelial structure (as the tracheal system, the oesophagus, the proventriculus, the salivary glands, the hindgut and the Malpighian tubules). Degeneration is first visible at stage 11 and continues successively throughout development. There is evidence that the loss of epithelial cell polarity may be the cause for the degeneration of these tissues, suggesting that thecrb gene product is involved in stabilizing the apico-basal polarity of epithelial cells. As previously shown, thecrb protein is specifically expressed on the apical side of embryonic epithelia in a reticular pattern outlining the borders of the cells. Here we demonstrate that thecrb protein shows the same subcellular localization in epithelial cells of imaginal discs and in follicle cells, indicating a similar function ofcrb during the development of embryonic, imaginal and follicle epithelia. Clonal analysis experiments indicate that the genecrb is not cell-autonomous in its expression, suggesting that the gene product may act as a diffusible factor and may serve as a signal in a cell-cell communication process. This signal is thought to be required for the formation and/or maintenance of the cell and tissue structure of the respective epithelia.