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.

Epithelial morphogenesis: filopodia at work.

Spreading and fusion of epithelial sheets are conserved morphogenetic mechanisms that help shape embryos and tissues. Recent findings suggest that the formation of dynamic filopodia at the leading front of the epithelia plays a critical role in regulating cell movement and recognition during these processes.

The EGF receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning.

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. Here we show that the proneural proteins trigger, in addition, positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (EGFR) and the Ras/Raf pathway. These interactions, which we denominate 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the EGFR/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess EGFR signalling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the EGFR and Notch pathways act antagonistically in notum macrochaetae determination.

JNK and decapentaplegic signaling control adhesiveness and cytoskeleton dynamics during thorax closure in Drosophila.

One of the fundamental events in metamorphosis in insects is the replacement of larval tissues by imaginal tissues. Shortly after pupariation the imaginal discs evaginate to assume their positions at the surface of the prepupal animal. This is a very precise process that is only beginning to be understood. In Drosophila, during embryonic dorsal closure, the epithelial cells push the amnioserosa cells, which contract and eventually invaginate in the body cavity. In contrast, we find that during pupariation the imaginal cells crawl over the passive larval tissue following a very accurate temporal and spatial pattern. Spreading is driven by filopodia and actin bridges that, protruding from the leading edge, mediate the stretching of the imaginal epithelia. Although interfering with JNK (Jun N-terminal kinase) and dpp (decapentaplegic) produces similar phenotypic effects suppressing closure, their effects at the cellular level are different. The loss of JNK activity alters the adhesion properties of larval cells and leads to the detachment of the imaginal and larval tissues. The absence of dpp signaling affects the actin cytoskeleton, blocks the emission of filopodia, and promotes the collapse of the leading edge of the imaginal tissues. Interestingly, these effects are very similar to those observed after interfering with JNK and dpp signaling during embryonic dorsal closure.

p38 MAPK signalling cascades: ancient roles and new functions.

p38 MAPKs are a conserved subfamily of MAPKs involved in the response to stress found in eukaryotic cells from yeast to mammals. The recent isolation of genes coding for members of this signalling cascade in Drosophila has provided us with the genetic tools to study their various biological roles and their regulatory interactions with other signalling pathways. This cascade participates in the immune response, a function that is remarkably conserved between flies and humans. Additionally, it appears to exert other fundamental roles during development, in cell fate specification in imaginal discs, and in cell polarity during oogenesis. These functions involve genetic and biochemical interactions with other signalling cascades, the decapentaplegic/TGFbeta, the wingless/Wnt and the torpedo/Ras-ERK pathways. In the near future, we can expect a flurry of information that will allow us to draw a comprehensive picture of the roles of signalling networks mediated by p38s during development.

DER signaling restricts the boundaries of the wing field during Drosophila development.

Arthropod and vertebrate limbs develop from secondary embryonic fields. In insects, the wing imaginal disk is subdivided early in development into the wing and notum subfields. The activity of the Wingless protein is fundamental for this subdivision and seems to be the first element of the hierarchy of regulatory genes promoting wing formation. Drosophila epidermal growth factor receptor (DER) signaling has many functions in fly development. Here we show that antagonizing DER signaling during the second larval instar leads to notum to wing transformations and wing mirror-image duplications. DER signaling is necessary for confining the wing subregion in the developing wing disk and for the specification of posterior identity. To do so, DER signaling acts by restricting the expression of Wingless to the dorsal-posterior quadrant of wing discs, suppressing wing-organizing activities, and by cooperating in the maintenance of Engrailed expression in posterior compartment cells.

A temporal switch in DER signaling controls the specification and differentiation of veins and interveins in the Drosophila wing.

The Drosophila EGF receptor (DER) is required for the specification of diverse cell fates throughout development. We have examined how the activation of DER controls the development of vein and intervein cells in the Drosophila wing. The data presented here indicate that two distinct events are involved in the determination and differentiation of wing cells. (1) The establishment of a positive feedback amplification loop, which drives DER signaling in larval stages. At this time, rhomboid (rho), in combination with vein, initiates and amplifies the activity of DER in vein cells. (2) The late downregulation of DER activity. At this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Together, these temporal and spatial changes in the activity of DER constitute an autoregulatory network that controls the definition of vein and intervein cell types.

Neuronal differentiation of PC12 cells induced by engrailed homeodomain is DNA-binding specific and independent of MAP kinases.

Neuronal differentiation may be induced by different mechanisms. In PC12 cells, differentiation can be achieved after stimulation by nerve growth factor through the sustained activation and nuclear translocation of MAPKs. A peptide covering the homeodomain of Drosophila Antennapedia translocates through the cell membrane in primary neurons in culture and reaches their nuclei. This process accelerates neurite elongation. We have examined whether the capacity for neuronal induction is a general characteristic of homeodomains, and whether differentiation proceeds through the same pathway as that induced by growth factors or represents a distinct cellular response. We show here that Engrailed homeodomain is internalized by UR61 cells, a PC12 cell derivative, and that it promotes and sustains neurite outgrowth. This event appears to proceed independently of MAPKs activation, suggesting that either parallel signal transduction pathways are under the control of homeoproteins or that they act downstream of MAPKs. The Fushi tarazu homeodomain also causes neurite outgrowth in UR61 cells and the neurotrophic activities of Engrailed and Fushi tarazu homeodomains correlate with their DNA binding specificities. However, neurite outgrowth is not promoted by Bicoid homeodomain, which recognizes a different DNA sequence. Therefore, the neurotrophic activity of the homeodomains depends not only on DNA-binding ability but also on the specificity of this binding.

Regulatory control of signal transduction during morphogenesis in Drosophila.

Morphogenesis shapes pattern and size during development. The initiation and propagation of morphogenetic processes is led by the integrated activation of signaling cascades. Much is known about regulatory control of signaling cascades in cell culture systems. However, how this regulatory elements act when cells need to behave coordinately is still unknown territory. The morphogenetic process of dorsal closure proceeds through changes in cell shape and polarity under the control of JNK signaling. Amongst other regulatory elements, Puckered, a Drosophila MAPK phosphatase, is involved in a negative feedback loop that controls JNK signaling activity. puckered is expressed in many other tissues, could influence other developmental events and might regulate different signaling cascades. The negative regulatory control of signal transduction pathways could be a general mechanism regulating differentiation and morphogenesis.

Genetic interactions and cell behaviour in blistered mutants during proliferation and differentiation of the Drosophila wing.

In this work, we analyse the blistered function in wing vein development by studying genetic mosaics of mutant cells, genetic interactions with other genes affecting vein development and blistered expression in several mutant backgrounds. blistered encodes for a nuclear protein homologous to the mammalian Serum Response Factor and is expressed in presumptive intervein cells of third larval instar and pupal wing discs. Clones of blistered mutant cells proliferate normally but tend to grow along veins and always differentiate as vein tissue. These observations indicate that vein-determined wing cells show a particular behaviour that is responsible for their allocation to vein regions. We observe strong genetic interactions between blistered, veinlet and genes of the Ras signaling cascade. During disc proliferation, blistered expression is under the control of the Ras signal transduction pathway, but its expression is independent of veinlet. During the pupal period, blistered and veinlet expression become interdependent and mutually exclusive. These results link the activity of the Ras pathway to the process of early determination of intervein cells, by the transcriptional control of the blistered nuclear factor.

puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila.

The activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities. The latter is mediated by a subset of phosphatases with dual specificity (VH-1 family). Here, we describe a new member of this family encoded by the puckered gene of Drosophila. Mutations in this gene lead to cytoskeletal defects that result in a failure in dorsal closure related to those associated with mutations in basket, the Drosophila JNK homolog. We show that puckered mutations result in the hyperactivation of DJNK, and that overexpression of puc mimics basket mutant phenotypes. We also show that puckered expression is itself a consequence of the activity of the JNK pathway and that during dorsal closure, JNK signaling has a dual role: to activate an effector, encoded by decapentaplegic, and an element of negative feedback regulation encoded by puckered.

Regulation of cell differentiation by the Drosophila Jun kinase cascade.

Recent studies have defined a new Drosophila Jun amino-terminal kinase (DJNK) pathway. The first role that has been uncovered for this pathway is the control of cell differentiation and morphogenesis during the process of dorsal closure. This phosphorylation cascade has been conserved during evolution and several reports suggest that it becomes activated in response to the action of small GTPases. DJNK signalling impinges on transcription factors as DJun and Anterior open and eventually controls the expression of target genes such as decapentaplegic and puckered. Important progress has been made in the study of the coordination of DJNK and Decapentaplegic signalling during dorsal closure and their role in the control of cell shape changes--and possibly cell polarity--by promoting cytoskeletal changes.

Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila.

In bilateral animals, the left and right sides of the body usually present asymmetric structures, the genetic bases of whose generation are still largely unknown [CIBA Foundation (1991) Biological Asymmetry and Handedness, CIBA Foundation Symposium 162 (Wiley, New York), pp. 1-327]. In Drosophila melanogaster, mutations in the rotated abdomen (rt) locus cause a clockwise helical rotation of the body. Even null alleles are viable but exhibit defects in embryonic muscle development, rotation of the whole larval body, and helical staggering of cuticular patterns in abdominal segments of the adult. rotated abdomen is expressed in the embryonic mesoderm and midgut but not in the ectoderm; it encodes a putative integral membrane glycoprotein (homologous to key yeast mannosyltransferases). Mesodermal cells defective in O-glycosylation lead to an impaired larval muscular system. We propose that the staggering of the adult abdominal segments would be a consequence of the relaxation of intrinsic rotational torque of muscle architecture, preventing the colateral alignment of the segmental histoblast cells during their proliferation at metamorphosis.

Phosphorylation of the Drosophila engrailed protein at a site outside its homeodomain enhances DNA binding.

The engrailed gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled engrailed protein. The primary embryonic protein kinase modifying engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II-dependent phosphorylation of an N-truncated derivative of engrailed protein purified from bacteria increased its DNA binding 2-4-fold.

DR-78, a novel Drosophila melanogaster genomic DNA fragment highly homologous to the DNA-binding domain of thyroid hormone-retinoic acid-vitamin D receptor subfamily.

Degenerate oligodeoxyribonucleotides were designed for both ends of the DNA-binding domain of members of the nuclear receptor superfamily. PCR amplified Drosophila melanogaster DNA was purified and cloned (DR plasmids). Genomic lambda DASH clones were identified at high stringency with an amplified DR-78 plasmid DNA and isolated. The partial sequence shows a very probable open reading frame which would encode a peptide highly homologous to members of the thyroid hormone-retinoic acid-vitamin D receptor subfamily. The fragment corresponds to a single copy gene and was mapped at position 78D of chromosome three by in situ hybridization.

S1 nuclease-sensitive sites in the bithoraxoid region of the Drosophila Ultrabithorax gene.

The Ultrabithorax gene is required to specify the third thoracic and first abdominal segments of Drosophila melanogaster. Mutations in the bithoraxoid region, a 40 kb DNA stretch upstream of the Ultrabithorax promoter, affect cis-regulatory elements of the Ultrabithorax gene. We now have identified specific sites in the bithoraxoid region that exhibit S1 nuclease sensitivity in vitro. These sites are not scattered along the DNA but are grouped instead in specific domains. Some of these S1-sensitive sites correlate with known breakpoint or insertional mutations. Others correspond to putative binding sites for transcription factors. The results suggest that unusual secondary structure might be important in chromosomal translocation within regulatory sequences of the Ultrabithorax product or its transcriptional regulation.

Analysis of genetic variability of populations of herpes simplex viruses.

An analysis of genetic variability of herpes simplex virus type 1 and type 2 populations of the Madrid (Spain) area has been carried out by digestion of viral DNA with restriction endonucleases. The index of nucleotide diversity indicated that herpes simplex virus type 1 has a slightly, although statistically significant, higher degree of heterogeneity than type 2. A phylogenetic tree for each type of virus has been constructed. The evolutionary pattern followed by both types of viruses ('star-like' topology) suggest that all the isolates analyzed evolved from a unique origin for each type of virus.

Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions.

The crystal structure of a complex containing the engrailed homeodomain and a duplex DNA site has been determined at 2.8 A resolution and refined to a crystallographic R factor of 24.4%. In this complex, two separate regions of the 61 amino acid polypeptide contact a TAAT subsite. An N-terminal arm fits into the minor groove, and the side chains of Arg-3 and Arg-5 make contacts near the 5' end of this "core consensus" binding site. An alpha helix fits into the major groove, and the side chains of IIe-47 and Asn-51 contact base pairs near the 3' end of the TAAT site. This "recognition helix" is part of a structurally conserved helix-turn-helix unit, but these helices are longer than the corresponding helices in the lambda repressor, and the relationship between the helix-turn-helix unit and the DNA is significantly different.

Expression of engrailed proteins in arthropods, annelids, and chordates.

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.