Danny L. Brower

GAL4 ENHANCER TRAPS EXPRESSED IN THE EMBRYO, LARVAL BRAIN, IMAGINAL DISCS,AND OVARY OF DROSOPHILA

We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS‐lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported. Dev. Dyn. 209:310–322, 1997.

Platelets with wings: the maturation of Drosophila integrin biology

The integrin family of cell surface receptors is strongly conserved in metazoans, making simple invertebrate genetic systems valuable contributors to understanding integrin function. The Drosophila integrins have long served as a paradigm for genetic studies of adhesion proteins during development. Currently, Drosophila experiments are exploring more general aspects of integrin biology. Genetic screens are identifying proteins involved in integrin adhesion complexes and signaling, and structures such as embryonic muscle attachments can be manipulated experimentally to dissect the functions of cytoplasmic components of integrin adhesion sites in whole animals. Drosophila also is beginning to yield some insights into integrin heterodimer structure and function.

Splice Variants of the Drosophila PS2 Integrins Differentially Interact with RGD-containing Fragments of the Extracellular Proteins Tiggrin, Ten-m, and D-Laminin α2

Two new potential ligands of theDrosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin α2 chain encoded by the wing blister locus and Ten-m, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand tiggrin (and the tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the αPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the αPS2 m8 splice variant. The βPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the β splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the βPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure.

A DM domain protein from a coral, Acropora millepora, homologous to proteins important for sex determination

SUMMARY The identification and functional studies of DM domain‐containing proteins Doublesex, MAB‐3, and DMRT1 indicated that flies, nematodes, and humans share at least some of the molecular mechanisms of sex determination. We identified a gene, AmDM1, from the coral Acropora millepora that encodes a homologous DM domain‐containing protein. Molecular analyses show that the AmDM1 primary transcript is processed to generate four different messenger RNAs. Alternative use of two polyadenylation sites produces transcripts that vary only in the 3′ untranslated regions, whereas alternative splicing generates transcripts with and without the region coding for the DM domain. All the transcripts include a second motif, the DMA domain, which is found in a number of other proteins containing a DM domain. Hermaphroditic A. millepora differentiates sexual cells seasonally before the spring spawn, and Northern blot analysis shows that the AmDM1 transcripts are present at higher levels during sexual differentiation. The non‐DM domain‐containing messages are also present at significant levels in late embryos, but DM domain transcripts are extremely rare at this stage. These data suggest that the association of DM domain proteins and sexual determination or differentiation predates the separation of the Cnidaria from the rest of the Metazoa.

Expression of the Sex combs reduced protein in Drosophila larvae

We have generated a monoclonal antibody that binds specifically to the protein product of the homeotic Sex combs reduced (Scr) gene of Drosophila, and have mapped the patterns of Scr expression in late third instar larvae. Virtually the entire prothoracic leg imaginal disc expresses the gene, although the levels of expression vary in different disc regions. This heterogeneity does not reflect the compartmental domains defined by engrailed gene expression. Expression is also observed in the cells of the humeral and labial discs, and there is a small patch of Scr-expressing cells in the antenna disc. The gene is expressed in adepithelial cells of the three thoracic leg discs, but not in the wing or haltere discs. In the central nervous system, Scr expression is confined to a narrow band of cells in the subesophageal region of the ventral ganglion. The results are discussed with respect to the known genetic requirements for Scr+ function.

Functional Properties of Alternatively Spliced Forms of the Drosophila PS2 Integrin α Subunit

The Drosophila αPS2 protein is encoded by two alternatively spliced transcripts. The respective αPS2 proteins differ by the presence of 25 amino acids in the αPS2 (C) protein, not found in the αPS2 (m8) subunit, in a region thought to be important for ligand binding. We examined the functional properties of Drosophila S2 cells transformed with genes expressing either of these proteins, in association with a Pre subunit. Both PS2 integrins support cell spreading on vertebrate vitronectin or, to a lesser extent, on fibronectin. Interestingly, the PS2(C) form promotes spreading more efficiently on vitronectin than does the PS2(m8) form, with an opposite relative efficiency seen for fibronectin. Also, the two forms of PS2 show different requirements for divalent cations in order to mediate efficient cell spreading. These divalent cations are not required to maintain the association of α and β sub-units. Spreading of both cell types is similarly RGD sensitive, and both PS2 integrins appear to associate with th...

Allelic interactions at the engrailed locus of Drosophila: engrailed protein expression in imaginal discs☆

Many embryonic lethal engrailed (enlethal) mutations are known to partially complement the cuticular defects of the original engrailed mutation, en1. To explore the nature of this complementation, the adult phenotypes of several different en1/enlethal transheterozygotes were compared with the corresponding patterns of engrailed protein expression in third larval instar imaginal discs (determined by immunofluorescence). Transheterozygotes of en1 and deletions of the locus (enDf) typically show slight complementation in the adult cuticle. The pattern of engrailed protein expression in some en1/enDf wing discs is indistinguishable from en1 homozygotes, but in others the pattern is nearly normal. en1/enDf leg discs appear to express engrailed protein normally. Transheterozygotes of en1 and EMS-induced, cytologically normal enlethal alleles have almost normal adult cuticle phenotypes and also exhibit normal patterns of engrailed protein expression in all of the thoracic imaginal discs. Surprisingly, the intensity of anti-engrailed staining in these discs is elevated relative to that in wild type. en2 is an unusual lethal allele in that it does not complement either the en1 adult cuticle phenotype or the protein expression pattern in imaginal discs. Moreover, the cytologically normal enlethal alleles also complement en2, at least partially. Both wing and leg imaginal discs from en2/enlethal transheterozygotes show abnormal patterns of engrailed protein expression. These results are discussed in the context of an autoregulatory model for engrailed regulation.

Differences in Regulation of Drosophila and Vertebrate Integrin Affinity by Talin

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talins role in regulating Drosophila alphaPS2 betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2 betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2 betaPS with those of human alphaIIb beta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2 betaPS with those of alphaIIb beta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIb beta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talins integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2 betaPS affinity because of structural features inherent in the alphaPS2 betaPS extracellular and/or transmembrane domains.

Identification of a specialized extracellular matrix component in Drosophila imaginal discs.

We have generated a monoclonal antibody that recognizes a major component of a specialized extracellular matrix in Drosophila imaginal discs. In mature larvae, antibody binding is observed almost exclusively on imaginal discs. On the basal surface of the thoracic discs, the antigen is localized to particular regions of the epithelium, and ultrastructural studies indicate that the antigen is found in a fibrous network secreted between the cells and the basal lamina. The localized expression indicates that the matrix is not simply related to disc differentiation, as all regions of the columnar disc epithelium are determined to secrete adult cuticle. A correlation of the antigen distribution with known developmental events leads us to propose that the antigen-containing network provides an extensible matrix for the rapid elongation of the disc epithelium during evagination; consistent with this, the antigen is a component of the matrix between the dorsal and ventral surfaces of the evaginated wing pouch. The antigen is very large (greater than 5 X 10(5) Da), can be labeled metabolically with methionine and sulfate, and is digested by chondroitinase ABC; these biochemical characteristics indicate that the antigen is a proteoglycan.

Drosophila cell adhesion molecules.

Publisher Summary This chapter describes Drosophila cell adhesion molecules. The primary reason for the interest in Drosophila cell adhesion proteins is the ability to undertake sophisticated genetic analyses of function in situ. Analysis in situ is especially important when examining the function during development, where cell culture systems are of limited value. Drosophila has become a major experimental system for understanding the fundamental mechanisms of pattern formation and development. Cells transformed with wild-type or altered genes for adhesion proteins have been instrumental in dissecting the molecular and cell biology of vertebrate adhesion and similar approaches are available to the Drosophila biologist. Typically, these studies utilize the S2 Drosophila cell line that normally grows as round, nonadhering cells. One of the lessons emerging from mushrooming molecular anatomy database is that all metazoans are composed of similar proteins or at least the proteins generated from the combinations of similar domains. Cell adhesion proteins are no exception to this generalization. In some proteins, such as the integrin β subunit, nearly all the functional domains appear to be identical with those of the vertebrate homologs.