Drosophila puckered regulates Fos/Jun levels during follicle cell morphogenesis.

puckered (puc) encodes a VH1-like phosphatase that down-regulates Jun kinase (JNK) activity during dorsal closure of the Drosophila embryo. We report a role for puc in follicle cell morphogenesis during oogenesis. puc mRNA accumulates preferentially in the centripetally migrating follicle cells and cells of the elongating dorsal appendages. Proper levels of Puc activity in the follicle cells are critical for the production of a normal egg: either reduced or increased Puc activity result in incomplete nurse cell dumping and aberrant dorsal appendages. Phenotypes associated with puc mutant follicle cells include altered DE-cadherin expression in the follicle cells and a failure of nurse cell dumping to coordinate with dorsal appendage elongation, leading to the formation of cup-shaped egg chambers. The JNK pathway target A251-lacZ showed cell-type-specific differences in its regulation by puc and by the small GTPase DRac1. puc mutant cells displayed region-specific ectopic expression of the A251-lacZ enhancer trap whereas overexpression of a transgene encoding Puc was sufficient to suppress lacZ expression in a cell autonomous fashion. Strikingly, decreased or increased puc function leads to a corresponding increase or decrease, respectively, of Fos and Jun protein levels. Taken together, these data indicate that puc modulates gene expression responses by antagonizing a Rho GTPase signal transduction pathway that stabilizes the AP-1 transcription factor. Consistent with this, overexpression of a dominant negative DRac1 resulted in lower levels of Fos/Jun.

The zinc finger protein schnurri acts as a Smad partner in mediating the transcriptional response to decapentaplegic.

In Drosophila, a BMP-related ligand Decapentaplegic (Dpp) is essential for cell fate specification during embryogenesis and in imaginal disc development. Dpp signaling culminates in the phosphorylation and nuclear translocation of Mothers against dpp (Mad), a receptor-specific Smad that can bind DNA and regulate the transcription of Dpp-responsive genes. Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study we show that Shn acts as a DNA-binding Mad cofactor in the nuclear response to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore we find that Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the regulation of endogenous Dpp target genes we developed a cell culture assay and show that Shn and Mad act synergistically to induce transcription. Our results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor.

Integration of epithelial patterning and morphogenesis in Drosophila ovarian follicle cells.

Drosophila oogenesis involves the coordinated development of germ cells and an overlying follicular epithelium. The follicle cells provide a genetically tractable system to investigate the cell biology of patterning and morphogenesis. Follicle cells initially form a cuboidal epithelium surrounding a syncytium of nurse cells and oocyte. Epithelial structure is maintained as these cells reorganize to create the three dimensional architecture of the eggshell. Both long-range and short-range cell-cell communications pattern the domains of follicle cells that will create specific eggshell structures. After terminal differentiation to deposit the eggshell proteins, the follicle cells die. This review summarizes recent progress in understanding the cell-cell communication that orchestrates follicle cell patterning and migrations. DE-cadherin-mediated adhesion is important at several steps in egg chamber formation and follicle cell migration. Notch signaling is critical during each successive round of patterning and migration. Integration of bone morphogenetic protein (BMP) and epidermal growth factor (EGF) signals patterns the elaborate structures of the dorsal-anterior eggshell.

Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary.

The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Here we show that opposing DPP and EGF signals set the boundary for an ovarian follicle cell fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein MAD; ectopic expression of BUNCHED suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. We previously demonstrated that the bunched expression pattern requires the EGF receptor ligand GURKEN. Here we show that activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. We propose that the juxtaposition of cells with high and low BUNCHED activity organizes a sharp boundary for the operculum fate.

Dynamic expression of TSC-22 at sites of epithelial-mesenchymal interactions during mouse development.

The leucine zipper transcription factor TSC-22 (TGF-beta1 Stimulated Clone-22) was first isolated from a mouse osteoblast cell line as an immediate-early target gene of TGF-beta1. However, work with other cell lines, as well as with a Drosophila homolog, bunched, suggests that it is an effector gene of various growth factors and potentially involved in the integration of multiple extracellular signals. Throughout mouse embryogenesis TSC-22 is expressed in a dynamic pattern. Although early TSC-22 expression is ubiquitous in 6.5 day embryos, as development proceeds TSC-22 expression is upregulated at sites of epithelial-mesenchymal interactions such as the limb bud, tooth primordiurn, hair follicle, kidney, lung, and pancreas. TSC-22 is also expressed in many neural crest-derived tissues including the mesenchyme of the branchial arches, the cranial, dorsal root, and sympathetic ganglia, as well as the facial cartilage and bone. Other areas of expression are the otic and optic vesicles, the heart, and cartilage and bone forming regions throughout the embryo.

TGF-beta family signal transduction in Drosophila development: from Mad to Smads.

The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of soluble extracellular proteins that are potent regulators of development in both vertebrates and invertebrates. Drosophila TGF-beta family members include three proteins with homology to vertebrate bone morphogenetic proteins (BMPs): Decapentaplegic (Dpp), Screw, and Glass bottom boat-60A. Genetic studies of Dpp signaling led to the identification of Smad proteins as central mediators of signal transduction by TGF-beta family members. Work in mammalian tissue culture has elucidated a biochemical model for signal transduction, in which activation of receptor serine-threonine kinase activity leads to phosphorylation of specific Smad proteins and translocation of heteromeric Smad protein complexes to the nucleus. Once in the nucleus Smad proteins interact with other DNA binding proteins to regulate transcription of specific target genes. Dissection of Dpp-response elements from genes expressed during embryonic mesoderm patterning and midgut morphogenesis provides important insights into the contributions of Smad proteins and tissue-specific transcription factors to spatial regulation of gene expression. Genetic studies in Drosophila are now expanding to include multiple BMP ligands and receptors and have uncovered activities not explained by the current signal transduction model. Identification of more ligand sequences and demonstration of a functional Drosophila activin-like signal transduction pathway suggest that all TGF-beta signal transduction pathways are present in flies.

Medea is a Drosophila Smad4 homolog that is differentially required to potentiate DPP responses.

Mothers against dpp (Mad) mediates Decapentaplegic (DPP) signaling throughout Drosophila development. Here we demonstrate that Medea encodes a MAD-related protein that functions in DPP signaling. MEDEA is most similar to mammalian Smad4 and forms heteromeric complexes with MAD. Like dpp, Medea is essential for embryonic dorsal/ventral patterning. However, Mad is essential in the germline for oogenesis whereas Medea is dispensable. In the wing primordium, loss of Medea most severely affects regions receiving low DPP signal. MEDEA is localized in the cytoplasm, is not regulated by phosphorylation, and requires physical association with MAD for nuclear translocation. Furthermore, inactivating MEDEA mutations prevent nuclear translocation either by preventing interaction with MAD or by trapping MAD/MEDEA complexes in the cytosol. Thus MAD-mediated nuclear translocation is essential for MEDEA function. Together these data show that, while MAD is essential for mediating all DPP signals, heteromeric MAD/MEDEA complexes function to modify or enhance DPP responses. We propose that this provides a general model for Smad4/MEDEA function in signaling by the TGF-beta family.

The Drosophila bunched gene is a homologue of the growth factor stimulated mammalian TSC-22 sequence and is required during oogenesis.

A Drosophila melanogaster sequence homologous to the mammalian growth factor-stimulated TSC-22 gene was isolated in an enhancer trap screen for genes expressed in anterodorsal follicle cells during oogenesis. This sequence includes a 225 aa residue open reading frame that encompasses a leucine zipper motif immediately preceded by a highly conserved region (TSC box), similarly located but distinct from the basic domain of bZIP proteins. The gene encoding this sequence, bunched (bun), has been independently isolated and characterized with respect to its role in peripheral nervous system development and eye development (Treisman, J.E., Lai, Z.-C. and Rubin, G.M. (1995) Shortsighted acts in the decapentaplegic pathway in the Drosophila eye development and has homology to a mouse TGF-beta-responsive gene. Development 121, 2835-2845). In agreement with the expression of the enhancer detector insertion, in situ hybridization reveals that bun transcripts localize to the anterior dorsal follicle cells at stages 10-12 of oogenesis. Changes in bun enhancer trap expression in genetic backgrounds that disrupt the grk/Egfr signaling pathway suggest that bun is regulated by growth factor patterning of dorsal anterior follicle cell fates. Clonal analysis shows that bun is required for the proper elaboration of dorsal cell fates leading to the formation of the dorsal appendages.

Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a growth factor that belongs to the transforming growth factor-beta (TGF-beta) superfamily and that plays a central role in multiple cell-cell signaling events throughout development. Through genetic screens we are seeking to identify other functions that act upstream, downstream or in concert with dpp to mediate its signaling role. We report here the genetic characterization and cloning of Mothers against dpp (Mad), a gene identified in two such screens. Mad loss-of-function mutations interact with dpp alleles to enhance embryonic dorsal-ventral patterning defects, as well as adult appendage defects, suggesting a role for Mad in mediating some aspect of dpp function. In support of this, homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk development and embryonic dorsal-ventral patterning that are very reminiscent of dpp mutant phenotypes. We cloned the Mad region and identified the Mad transcription unit through germline transformation rescue. We sequenced a Mad cDNA and identified three Mad point mutations that alter the coding information. The predicted MAD polypeptide lacks known protein motifs, but has strong sequence similarity to three polypeptides predicted from genomic sequence from the nematode Caenorhabditis elegans. Hence, MAD is a member of a novel, highly conserved protein family.

Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila.

Pathways for regulation of signaling by transforming growth factor-beta family members are poorly understood at present. The best genetically characterized member of this family is encoded by the Drosophila gene decapentaplegic (dpp), which is required for multiple events during fly development. We describe here the results of screens for genes required to maximize dpp signaling during embryonic dorsal-ventral patterning. Screens for genetic interactions in the zygote have identified an allele of tolloid, as well as two novel alleles of screw, a gene recently shown to encode another bone morphogenetic protein-like polypeptide. Both genes are required for patterning the dorsalmost tissues of the embryo. Screens for dpp interactions with maternally expressed genes have identified loss of function mutations in Mothers against dpp and Medea. These mutations are homozygous pupal lethal, engendering gut defects and severely reduced imaginal disks, reminiscent of dpp mutant phenotypes arising during other dpp-dependent developmental events. Genetic interaction phenotypes are consistent with reduction of dpp activity in the early embryo and in the imaginal disks. We propose that the novel screw mutations identified here titrate out some component(s) of the dpp signaling pathway. We propose that Mad and Medea encode rate-limiting components integral to dpp pathways throughout development.

The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary?

Imaginal disks, the primordia of the adult appendages in Drosophila, are divided into anterior and posterior compartments. However, the developmental role of such compartments remains unclear. The expression of decapentaplegic (dpp), a pattern formation gene required for imaginal disk development, has the intriguing property of being expressed in a line at or near the boundary between these compartments. Here, we compare the distribution of dpp-driven reporter gene expression to the pattern of expression of the engrailed (en) gene, known to be required for the maintenance of the compartment boundary. Using confocal microscopy to obtain single cell resolution, we have determined that the majority of the en+ imaginal disk cells expressing the dpp-driven reporter genes about those cells expressing en, while a small percentage of dpp reporter gene expressing cells also express en. In posterior regions of en mutant disks, where compartmentalization is abnormal, we observe ectopic expression of the dpp-driven reporter genes. We conclude that the pattern of dpp expression in imaginal disks is delimited in part through the direct or indirect repression by engrailed. Our results lead us to question the widely held assumption that the anterior edge of en expression demarcates the A/P compartment boundary.

An extensive 3' cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-beta family in Drosophila.

The decapentaplegic (dpp) gene in Drosophila melanogaster encodes a TGF-beta-like signalling molecule that is expressed in a complex and changing pattern during development. One of dpp's contributions is to proximal-distal outgrowth of the adult appendages, structures derived from the larval imaginal disks. Appendage specific mutations of dpp fall in a 20 kb interval 3' to the known dpp transcripts. Here, we directly test the hypothesis that these mutations define an extended 3' cis-regulatory region. By analysis of germ-line transformants expressing a reporter gene, we show that sequences from this portion of the gene, termed the dppdisk region, are capable of directing expression comparable to that defined by RNA in situ hybridization. We localize two intervals of the dppdisk region that appear to account for much of the dpp spatial pattern in imaginal disks and discuss the positions of these important elements in terms of the genetics of dpp. Finally, we provide evidence to suggest that one of our constructs expresses beta-galactosidase in the early imaginal disk primordia in the embryo, at approximately the time when they are set aside from surrounding larval epidermal tissues. Thus, dpp may be involved directly in the determination of the imaginal disks.

Wing formation in Drosophila melanogaster requires decapentaplegic gene function along the anterior-posterior compartment boundary.

Previous analyses of the decapentaplegic (dpp) gene in Drosophila melanogaster have suggested that its product, a polypeptide of the transforming growth factor-beta family of secreted factors, acts at the level of intercellular communication to control several events in spatial pattern formation. In this report, we use clonal analysis to demonstrate a localized requirement for wild-type dpp expression along the anterior-posterior (A/P) compartment boundary of the developing wing primordium. Clonal analysis reveals that normal wing blade development is solely dependent on dpp+ function in those anterior compartment cells that border the anterior-posterior (A/P) compartment boundary of the wing imaginal disk. Conversely, the wing blade will not develop if these boundary cells lack dpp activity. The localized requirement for dpp coincides with the spatial distribution of dpp transcripts, which accumulate in a stripe of cells at or near the known A/P compartment boundary of the wing imaginal disk. Thus, only a small subset of the cells that normally comprise the wing must express dpp to permit development of the entire structure. We propose that this localized expression of dpp is essential to proximal-distal appendage development. We discuss the possibility that dpp expression serves as a landmark for establishing and/or maintaining positional information in imaginal disks.

Systematic alterations in the anticodon arm make tRNA(Glu)-Suoc a more efficient suppressor.

Using site-specific mutagenesis, we constructed five more efficient variants of tRNA(Glu)-Suoc, an extremely inefficient ochre suppressor. Each variant has an extended anticodon, or region of the anticodon arm, which is more similar to that found in normal tRNAs which translate codons Uxx. Suppressor efficiency invariably increases with similarity of the extended anticodon to that of a normal Uxx-translating tRNA. Altered nucleotides in both helix and loop strongly affect efficiency, with no position dependence and no significant interaction between substitutions. The variant with all substitutions is 230-fold more efficient (in one context) than the parental tRNA(Glu)-Suoc. Two other unexpected variants seem to be 'context mutants', having altered response to message context.

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli.

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.

Site-specific mutagenesis of Escherichia coli gltT yields a weak, glutamic acid-inserting ochre suppressor.

Of all the Escherichia coli tRNA genes that can give rise to an amber or an ochre suppressor by a single-nucleotide mutation, only the tRNAGlu genes have not been observed to do so. A study of the relationship between the sequences of tRNAs and the codons they translate predicts that the ochre suppressor derived from tRNAGlu would function very poorly on the ribosome. We have used site-specific mutagenesis to create the gene for such a tRNA in order to test this prediction. We cloned the tRNAGlu-Suoc gene into a high copy number plasmid, under control of the lacUV5 promoter. The mutant tRNA suppresses both amber and ochre nonsense mutations. As predicted, it is less efficient than other suppressors expressed under similar conditions.

Defined set of cloned termination suppressors: in vivo activity of isogenetic UAG, UAA, and UGA suppressor tRNAs.

We have cloned an isogenetic set of UAG, UAA, and UGA suppressors. These include the Su7 -UAG, Su7 -UAA, and Su7 -UGA suppressors derived from base substitutions in the anticodon of Escherichia coli tRNATrp and also Su9 , a UGA suppressor derived from a base substitution in the D-arm of the same tRNA. These genes are cloned on high-copy-number plasmids under lac promoter control. The construction of the Su7 -UAG plasmid and the wild-type trpT plasmid have been previously described ( Yarus , et al., Proc. Natl. Acad. Sci. U.S.A. 77:5092-5097, 1980). Su7 -UAA ( trpT177 ) is a weak suppressor which recognizes both UAA and UAG nonsense codons and probably inserts glutamine. Su7 -UGA ( trpT176 ) is a strong UGA suppressor which may insert tryptophan. Su9 ( trpT178 ) is a moderately strong UGA suppressor which also recognizes UGG (Trp) codons, and it inserts tryptophan. The construction of these plasmids is detailed within. Data on the DNA sequences of these trpT alleles and on amino acid specificity of the suppressors are presented. The efficiency of the cloned suppressors at certain nonsense mutations has been measured and is discussed with respect to the context of these codons.