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R K Blackman

Publications and source records attributed to R K Blackman.

16 recordsLinked to original sources
PubMed1999

Wingless, decapentaplegic and EGF receptor signaling pathways interact to specify dorso-ventral pattern in the adult abdomen of Drosophila.

Adult abdominal segments of Drosophila are subdivided along the dorso-ventral axis into a dorsal tergite, a ventral sternite and ventro-lateral pleural cuticle. We report that this pattern is largely specified during the pupal stage by Wingless (Wg), Decapentaplegic (Dpp) and Drosophila EGF Receptor (DER) signaling. Expression of wg and dpp is activated at the posterior edge of the anterior compartment by Hedgehog signaling. Within this region, wg and dpp are expressed in domains that are mutually exclusive along the dorso-ventral axis: wg is expressed in the sternite and medio-lateral tergite, whereas dpp expression is confined to the pleura and the dorsal midline. Neither gene is expressed in the lateral tergite. Shirras and Couso (1996, Dev. Biol. 175, 24-36) have shown that tergite and sternite cell fates are specified by Wg signaling. We find that DER acts synergistically with Wg to promote tergite and sternite identities, and that Wg and DER activities are opposed by Dpp signaling, which promotes pleural identity. Wg and Dpp interact antagonistically at two levels. First, their expression is confined to complementary domains by mutual transcriptional repression. Second, Wg and Dpp compete directly with one another by exerting opposite effects on cell fate. DER signaling does not affect the expression of wg or dpp, indicating that it interacts with Wg and Dpp at the level of cell fate determination. Within the tergite, the requirements for Wg and DER function are roughly complementary: Wg is required mainly in the medial region, whereas DER is most important laterally. Finally, we show that Dpp signaling at the dorsal midline controls dorso-ventral patterning within the tergite by promoting pigmentation in the medial region.

Abdomen↗
PubMed1999

Cubitus interruptus is necessary but not sufficient for direct activation of a wing-specific decapentaplegic enhancer.

In Drosophila, the imaginal discs are the primordia for adult appendages. Their proper formation is dependent upon the activation of the decapentaplegic (dpp) gene in a stripe of cells just anterior to the compartment boundary. In imaginal discs, the dpp gene has been shown to be activated by Hedgehog signal transduction. However, an initial analysis of its enhancer region suggests that its regulation is complex and depends upon additional factors. In order to understand how multiple factors regulate dpp expression, we chose to focus on a single dpp enhancer element, the dpp heldout enhancer, from the 3' cis regulatory disc region of the dpp locus. In this report, we present a molecular analysis of this 358 bp wing- and haltere-specific dpp enhancer, which demonstrates a direct transcriptional requirement for the Cubitus interruptus (Ci) protein. The results suggest that, in addition to regulation by Ci, expression of the dpp heldout enhancer is spatially determined by Drosophila TCF (dTCF) and the Vestigial/Scalloped selector system and that temporal control is provided by dpp autoregulation. Consistent with the unexpectedly complex regulation of the dpp heldout enhancer, analysis of a Ci consensus site reporter construct suggests that Ci, a mediator of Hedgehog transcriptional activation, can only transactivate in concert with other factors.

Animals↗
PubMed1996

Promoter specificity mediates the independent regulation of neighboring genes.

Although enhancers can exert their influence over great distances, their effect is generally limited to a single gene. To discern the mechanism by which this constraint can he mediated, we have studied three neighboring Drosophila genes: decapentaplegic (dpp), SLY1 homologous (Slh) and out at first (oaf). Several dpp enhancers are positioned close to Slh and oaf, and yet these genes are unaffected by the dpp elements. However, when a transposon is located within the oaf gene, the dpp enhancers activate the more distant transposon promoters while still ignoring the closer Slh and oaf start sites. To test whether this promoter specificity accounts for the regulatory autonomy normally found for the three genes, we used in vivo gene targeting to replace the oaf promoter with a dpp-compatible one in an otherwise normal chromosome. Strikingly, this chimeric gene is now activated by the dpp enhancers. Thus, the properties of the promoters themselves are sufficient to mediate the autonomous regulation of genes in this region.

Animals↗
PubMed1996

The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development.

TGF-beta-related signaling pathways play diverse roles during vertebrate and invertebrate development. A common mechanism for regulating the activity of TGF-beta family members is inhibition by extracellular antagonists. Recently, the Drosophila short gastrulation (sog) gene was shown to encode a predicted diffusible factor which antagonizes signaling mediated by the TGF-beta-like Decapentaplegic (Dpp) pathway in the early blastoderm embryo. sog and dpp, which are among the earliest zygotic genes to be activated, are expressed in complementary dorsal-ventral domains. The opposing actions of sog and dpp in the early embryo have been highly conserved during evolution as their vertebrate counterparts, chordin and BMP-4, function homologously to define neural versus non-neural ectoderm in Xenopus. Here we exploit the genetically sensitive adult wing vein pattern to investigate the generality of the antagonistic relationship between sog and dpp. We show that dpp is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating Dpp feedback loop. These data support the view that Sog is a dedicated Dpp antagonist.

Animals↗
PubMed1996

The TGF-beta signaling pathway is essential for Drosophila oogenesis.

We examine roles of signaling by secreted ligands of the TGF-beta family during Drosophila oogenesis. One family member, the DPP ligand encoded by the decapentaplegic (dpp) gene, is required for patterning of anterior eggshell structures. This requirement presumably reflects the expression pattern of dpp in an anterior subset of somatic follicle cells: the centripetally migrating and the nurse cell-associated follicle cells. Similar requirements are also revealed by mutations in the saxophone (sax)-encoded receptor, consistent with the idea that DPP signaling is, at least in part, mediated by the SAX receptor. A loss of germline sax function results in a block in oogenesis associated with egg chamber degeneration and a failure of the transfer of nurse cell contents to the oocyte, indicating that TGF-beta signaling is required for these events. Some phenotypes of sax mutations during oogenesis suggest that SAX responds to at least one other TGF-beta ligand as well in the posterior follicle cells.

Actins↗
PubMed1995

Regulatory autonomy and molecular characterization of the Drosophila out at first gene.

Our previous work has shown that the expression of the Drosophila decapentaplegic (dpp) gene in imaginal disks is controlled by a 30 kb array of enhancers located 3' of the dpp coding region. Here, we describe the cloning and characterization of out at first (oaf), a gene located near this enhancer region. Transcription of oaf results in three classes of alternatively polyadenylated RNAs whose expression is developmentally regulated. All oaf transcripts contain two adjacent open reading frames separated by a single UGA stop codon. Suppression of the UGA codon during translation, as seen previously in Drosophila, could lead to the production of different proteins from the same RNA. During oogenesis, oaf RNA is expressed in nurse cells of all ages and maternally contributed to the egg. During embryonic development, zygotic transcription of the gene occurs in small clusters of cells in most or all segments at the time of germband extension and subsequently in a segmentally repeated pattern in the developing central nervous system. The gene is also expressed in the embryonic, larval and adult gonads of both sexes. We also characterize an enhancer trap line with its transposon inserted within the oaf gene and use it to generate six recessive oaf mutations. All six cause death near the beginning of the first larval instar, with two characterized lines showing nervous system defects. Last, we discuss our data in light of the observation that the enhancers controlling dpp expression in the imaginal disks have no effect on the relatively nearby oaf gene.

Amino Acid Sequence↗
PubMed1995

The tolkin gene is a tolloid/BMP-1 homologue that is essential for Drosophila development.

The Drosophila decapentaplegic (dpp) gene, a member of the transforming growth factor beta superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interactions between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp.

Amino Acid Sequence↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1990

Molecular organization of the decapentaplegic gene in Drosophila melanogaster.

The decapentaplegic (dpp) locus of Drosophila melanogaster is a greater than 55 kb genetic unit required for proper pattern formation during the embryonic and imaginal development of the organism. We have proposed that these morphogenetic functions result from the action of a secreted transforming growth factor-beta (TGF-beta)-related protein product encoded by dpp. In this paper we localize 60 mutations on the molecular map of dpp. The positions of these mutations cluster according to phenotypic class, identifying the locations of specific dpp functions. By Northern and cDNA analysis, we characterize five overlapping dpp transcripts. On the basis of the locations of the overlaps relative to a previously sequenced cDNA, it is likely that these transcripts all encode similar or identical polypeptides. We propose that the bulk of dpp DNA consists of extensive arrays of cis-regulatory information. The large (greater than 25-kb) 3' cis-regulatory region represents a novel feature of dpp gene organization

Alleles↗
PubMed1989

Identification of a fully-functional hobo transposable element and its use for germ-line transformation of Drosophila.

The transposable element hobo can be mobilized to induce a variety of genetic abnormalities within the germ-line of Drosophila melanogaster. Strains containing hobos have 3.0 kb elements and numerous smaller derivatives of the element. By analogy with other transposable element systems, it is likely that only the 3.0 kb elements are capable of inducing hobo mobilization. Here, we report that a cloned 3.0 kb hobo, called HFL1, is able to mediate germ-line transformation and therefore is an autonomous (fully-functional) transposable element. Germ-line transformation was observed when HFL1 and a marked hobo element were co-injected into recipient embryos devoid of endogenous hobos. Integration did not occur in the absence of the 3.0 kb element. A single copy of the marked hobo transposon inserted at each site, and the target sites were widely distributed throughout the genome. Integration occurred at (or very near) the termini of hobo, without internal rearrangement of the hobo or marker gene sequences. The hobo transformation system will allow us to determine the structural and regulatory features of hobo responsible for its mobilization and will provide novel approaches for the molecular and genetic manipulation of the Drosophila genome.

Animals↗
PubMed1987

Mobilization of hobo elements residing within the decapentaplegic gene complex: suggestion of a new hybrid dysgenesis system in Drosophila melanogaster.

We present results demonstrating that the hobo family of transposable elements can promote high rates of chromosomal instability. Using strains with a hobo element inserted within the decapentaplegic gene complex (DPP-C), we have recovered numerous DPP-C mutations involving chromosomal rearrangements and deletions with one endpoint in the vicinity of the pre-existing hobo element. This hypermutability occurred in the germ lines of hybrid progeny from crosses involving strains containing hobo elements to strains lacking them. In some crosses, the offspring had rudimentary gonads, reminiscent of GD sterility. The germline hypermutability and infertility are similar to those produced by P-element-mediated hybrid dysgenesis. Given the many genetic and molecular similarities of the P and hobo systems, we propose that a system analogous to P-M hybrid dysgenesis has been activated in the hobo+ X hobo- crosses.

Alleles↗
PubMed1986

Interspecific nucleotide sequence comparisons used to identify regulatory and structural features of the Drosophila hsp82 gene.

We have sequenced the hsp82 heat shock gene and 5'-flanking DNA of four species of Drosophila in order to identify conserved features of possible regulatory significance and to determine the nature and rate of evolutionary change in various domains of the gene. All conserved sequences identified in the 5' non-transcribed region of the hsp82 gene of melanogaster, simulans, pseudoobscura and virilis lie within 150 base-pairs of the RNA initiation site. These include a 34 base-pair imperfect but highly conserved dyad made up of three overlapping copies of the consensus heat shock regulatory sequence dyad made up of three overlapping copies of the consensus heat shock regulatory sequence C-T-N-G-A-A-N-N-T-T-C-N-A-G. Several other highly conserved features are also seen. In pseudoobscura only, the region from -21 to -133 is almost exactly repeated 698 base-pairs upstream. The upstream repeat retains all of the sequences common to the four species in the -21 to -133 interval except for the T-A-T-A motif. One possibility is that this upstream sequence is involved in dosage compensation of the hsp82 gene, which is pseudoobscura is on the X chromosome. In melanogaster, simulans and virilis there is an oppositely oriented, non-heat shock gene 0.6 to 0.8 kb (kb = 10(3) base-pairs) upstream from the hsp82 transcription initiation site. Comparisons of the hsp82 transcription unit from the distantly related species show little or no sequence conservation in the intron and non-translated exon I sequences. In contrast, the coding regions of these species are 90% homologous at the DNA level and 97 to 99% identical at the amino acid level. Half of the amino acid changes have occurred within a 15-amino acid region that lies within an unusually polar domain of hsp82. From the sequence comparisons, we estimate the rate of silent (synonymous) site substitution in Drosophila as 1 X 10(-8)/nucleotide site per year, similar to that for mammals.

Amino Acid Sequence↗