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Campbell, G., and S.J. Newfeld (2008) Current topics in organogenesis and gametogenesis. Fly 2: At the 49(th) Annual Drosophila Research Conference from April 3-8, 2008 in San Diego there were eight talks and over ninety posters in the section on Organogenesis and Gametogenesis. These covered a wide range of topics within the two broad categories of organ-specific stem cells (including germ cells) and organ-specific developmental programs. Here we discuss eleven of these presentations describing current research into the formation of the gonad, intestine, trachea, muscle and leg joint. The new insights presented advance our understanding of the molecular events that underlie interactions between stem cells and their niches as well as mechanisms underlying tissue-specific differentiation programs. Wehn, A, and G. Campbell (2006) Genetic interactions among scribbler, Atrophin and groucho in Drosophila uncover links in transcriptional repression. Genetics 173:849-861 In eukaryotes, the ability of DNA binding proteins to act as transcriptional repressors often requires that they recruit accessory proteins, known as corepressors, which provide the activity responsible for silencing transcription. Several of these factors have been identified including the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Here we demonstrate strong genetic interactions between gro and Atro and also with mutations in a third gene, scribbler (sbb), which encodes a nuclear protein of unknown function. We show that mutations in Atro and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs, which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro and Atro/sbb mutant phenotypes suggests they do not function together, but that they may interact with the same transcription factors, including Engrailed and C15, to provide these proteins with maximal repressive activity.
Moser, M., and G. Campbell (2005) Generating and interpreting the Brinker gradient in the Drosophila wing. Dev. Biol. 286:647-658 The transcription factor Brinker (Brk) represses gene expression in the Drosophila wing imaginal disc, where it is expressed in symmetrical lateral-to-medial gradients, a pattern that is established by inverse gradients of the TGF-b, Dpp, which is in turn transduced into graded phosphorylated Mad (pMad, an R-Smad). pMad is part of a complex which directly represses brk. sal and omb are targets of Brk and are, thus, only expressed medially with their domains extending mediolaterally into the region where Brk is graded. omb extends more laterally than sal, indicating that higher levels of Brk are required to repress it. This is supported by our demonstration that higher levels of ectopic Brk are required to completely repress omb than sal. We also show, however, that Mad antagonizes the ability of Brk to repress these genes, indicating that pMad directly activates their expression (as well as repressing brk). Thus, whether a gene is expressed at a particular location may depend not only on how much Brk is present, but also on the level of pMad. We have also investigated the mechanism by which the brk expression gradient is established and show that it is not just a simple readout of the pMad gradient but requires Brk to repress its own expression. In brk mutants, the brk gradient is not established: brk is still off medially and on at high levels laterally, but there is almost no graded expression between these extremes. This Brk negative autoregulation appears to increase the sensitivity of the cells to Dpp/pMad and should also function to stabilize the brk gradient.
Campbell, G. (2005) Regulation of gene expression in the distal region of the Drosophila leg by the Hox11 homolog, C15. Dev. Biol. 278:607-618 The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IVand V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and B are mutually antagonistic resulting in exclusion of B expression from the pretarsus. Although Al is necessary, it is not sufficient to repress B, indicating another factor is required. Here, this factor is identified as the product of the C15 gene, which is another homeodomain protein, a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al and, together, C15 and Al appear to directly repress B. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap.
Winter, S.E., and G. Campbell (2004) Repression of Dpp targets in the Drosophila wing by Brinker. Development 131:6071-6081 Patterning along developing body axes is regulated by gradients of transcription factors, which activate or repress different genes above distinct thresholds. Understanding differential threshold responses requires knowledge of how these factors regulate transcription. In the Drosophila wing, expression of genes such as omb and sal along the anteroposterior axis is restricted by lateral-to-medial gradients of the transcriptional repressor Brinker (Brk). omb is less sensitive to repression by Brk than sal and is consequently expressed more laterally. Contrary to previous suggestions, we show that Brk cannot repress simply by competing with activators, but requires specific repression domains along with its DNA-binding domain. Brk possesses at least three repression domains, but these are not equivalent; one, 3R, is sufficient to repress omb but not sal. Thus, although sal and omb show quantitative differences in their response to Brk, there are qualitative differences in the mechanisms that Brk uses to repress them.
Campbell, G. (2002) Distalization of the Drosophila leg by graded EGF-receptor activity. Nature 418:781-785 Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-b (TGF-b) and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. Here I revise our understanding of limb development in flies and show that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals.
Wang, S., A. Simcox, and G. Campbell (2000) Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development. Genes Dev. 14:2271-2276 Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and distinguish the future wing from the body wall (notum). Here we show that EGFR signaling stimulated by its ligand, Vein, has a fundamental role in regulating two of these cell fate choices: 1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes. 2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene and consequently also controls wing development, which depends upon an interaction between dorsal and ventral cells
Campbell, G.L., and A. Tomlinson (2000) Transcriptional regulation of the Hedgehog effector CI by the zinc-finger gene combgap. Development 127:4095-4103 Members of the Hedgehog (HH) family of secreted signaling molecules specify cell fate during animal development by controlling the activity of members of the Gli family of zinc-finger transcription factors in responding cells. In Drosophila the Gli homolog, cubitus interruptus (CI), is expressed only in the anterior compartment where it represses targets such as the signaling molecule genes decapentaplegic (dpp) and wingless (wg). HH is expressed in the posterior and diffuses into the anterior where it antagonizes CI repression resulting in dpp and wg expression immediately anterior to the compartment border. Reducing CI levels results in misexpression of wg and dpp, while CI misexpression in the posterior disrupts differentiation. Thus, normal disc patterning requires high levels of CI in the anterior and the absence of CI in the posterior. Here we show that mutations in combgap (cg) result in deregulation of CI expression, which is now expressed at much lower levels and ubiquitously, i.e., also in the posterior. Consequently, cg mutants phenocopy ci loss-of-function mutants in the anterior and ci gain-of-function mutants in the posterior. cg encodes a putative DNA-binding protein that regulates both transcriptional activation and repression of the ci gene.
Campbell, G., and A. Tomlinson (1999) Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell 96:553-562 Dpp, a TGF-beta, organizes pattern in the Drosophila wing by acting as a graded morphogen, activating different targets above distinct threshold concentrations. Like other TGF-betas, Dpp appears to induce transcription directly via activation of a SMAD, Mad. However, here we demonstrate that Dpp can also control gene expression indirectly by downregulating the expression of the brinker gene, which encodes a putative transcription factor that functions to repress Dpp targets. The medial-to-lateral Dpp gradient along the anterior-posterior axis is complemented by a lateral-to-medial gradient of Brinker, and the presence of these two opposing gradients may function to allow cells to detect small differences in Dpp concentration and respond by activating different target genes.
Campbell, G.L., and A. Tomlinson (1998) The roles of the homoebox genes aristaless and Distal-less in patterning the legs and wings of Drosophila. Development 125:4483-4493 In the leg and wing imaginal discs of Drosophila, the expression domains of the homeobox genes aristaless (al) and Distal-less (Dll) are defined by the secreted signaling molecules Wingless (Wg) and Decapentaplegic (Dpp). Here, the roles played by al and Dll in patterning the legs and wings have been investigated through loss of function studies. In the developing leg, al is expressed at the presumptive tip and a molecularly defined null allele of al reveals that its only function in patterning the leg appears to be to direct the growth and differentiation of the structures at the tip. In contrast, Dll has previously been shown to be required for the development of all of the leg more distal than the coxa. Dll protein can be detected in a central domain in leg discs throughout most of larval development, and in mature discs this domain corresponds to the distal-most region of the leg, the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. However, earlier in development Dll is required in more proximal regions of the leg suggesting it is expressed at high levels in these cells early in development but not later. This reveals a correlation between a temporal requirement for Dll and position along the proximodistal axis; how this may relate to the generation of the P/D axis is discussed. Dll is required in the distal regions of the leg for the expression of tarsal-specific genes including al and bric-a-brac. Dll mutant cells in the leg sort out from wild-type cells suggesting one function of Dll here is to control adhesive properties of cells. Dll is also required for the normal development of the wing, primarily for the differentiation of the wing margin.
Campbell, G.L., and A. Tomlinson (1995) Initiation of the proximodistal axis in insect legs. Development 121:619-628 Much of the cell-cell communication that controls assignment of cell fates during animal development appears to be mediated by extracellular signaling molecules. The formation of the proximodistal (P/D) axis of the legs of flies is controlled by at least two such molecules, a Wnt and a TGFbeta, encoded by the wingless (wg) and decapentaplegic (dpp) genes, respectively. The P/D axis appears to be initiated from the site where cells expressing wg are in close association with those expressing dpp. Support for this hypothesis comes from two sources: classical grafting experiments in cockroaches and ectopic protein expression in Drosophila.
Campbell, G., H. Goring, T. Lin, E. Spana, S. Andersson, C.Q. Doe, and A. Tomlinson (1994) RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development 120:2957-2966 We report the identification of RK2, a glial-specific homeodomain protein. RK2 is localized to the nucleus of virtually all embryonic and imaginal glial cells, with the exception of midline glia. Embryos mutant for the gene encoding RK2 are embryonic lethal but normal for early gliogenesis (birth, initial divisions and migration of glia) and axonogenesis (neuronal pathfinding and fasciculation). However, later in development, there are significantly fewer longitudinal glia that are spatially disorganized; in addition, there is a slight disorganization of axon fascicles and a defective nerve cord condensation. This suggests that RK2 is not required for early glial determination, but rather for aspects of glial differentiation or function that are required for embryonic viability.
Campbell, G.L., T. Weaver, and A. Tomlinson (1993) Axis specification in the developing Drosophila appendage: the roles of wingless, decapentaplegic and the homeobox gene aristaless. Cell 74:1113-1123 The wingless (wg) and decapentaplegic (dpp) genes of Drosophila encode homologs of secreted growth factors and are required for the correct patterning of the appendages. We show that the presumptive tips of both the leg and wing, the distal extreme of the proximodistal axis, are characterized by the close association of cells expressing wg, dpp, and the homeobox gene aristaless (al). Ectopic expression of wg can induce both ectopic al expression and a duplication of the proximodistal axis (the development of supernumerary legs), but only in regions expressing high levels of dpp. Ectopic al expression can induce a duplication of the proximodistal axis in the wing, suggesting that it may be directly involved in axis specification. The proximodistal axis may be specified via a mechanism involving a direct interaction between cells expressing wg, dpp, and possibly al.
Campbell, G.L., and S. Caveney (1989) engrailed gene expression in the abdominal segment of Oncopeltus: gradients and cell states in the insect segment. Development 106:727-737 A monoclonal antibody that recognizes the product of the segmental gene, engrailed (en), of Drosophila has been used to analyse expression of the homologous gene of Oncopeltus. engrailed expression in the abdominal segment of larval Oncopeltus is confined to a narrow band of epidermal cells localized immediately anterior to the segment border. Expression varies in intensity during postembryonic development: no gene product is detectable in newly moulted larvae, but reappears soon after initiation of intermoult activities. One possible function of en in this system is revealed by a series of operations confronting cells from different anteroposterior levels in the segment. New segment borders are generated only when en-expressing cells confront cells from the anteriormost region of the segment. All other combinations result in intercalation of intermediate intrasegmental levels. It is therefore suggested that the most important function of en is the establishment of new, and presumably the maintenance of existing, segment borders.
Campbell, G.L., and P.M.J. Shelton (1987) Cell behaviour during pattern regulation in the insect abdomen (Oncopeltus fasciatus). I. Regeneration of segment borders. Development 101:221-235 The confrontation of cells from the anterior region of an abdominal segment of Oncopeltus with those from the posterior region of the same or the adjacent segment results in the generation of a segment border. The behaviour of epidermal cells during this regulation is described. It consists primarily of cell division and transverse elongation of cells at the site of confrontation. This behaviour can be separated from any associated purely with wound healing because a similar-sized wound to that used to ablate the segment border, performed within the segment, does not result in any cell division or elongation. The results are consistent with the view that there is a discontinuity in positional values at the segment border. The stability of such a discontinuity and the regeneration of segment borders are discussed in terms of there being a special population of cells at the segment border that have the property of isolating other cells with the maximum difference in positional values.
Campbell, G.L. (1987) Cell behaviour during pattern regulation in the insect abdomen (Oncopeltus fasciatus). II. Intrasegmental regulation. Development 101:237-246 Cells from different levels in the anteroposterior axis of an abdominal segment of Oncopeltus were confronted by scraping away the strip of epidermis that separated these levels. The cells migrate over the wound and meet in the centre. The subsequent behaviour of the epidermal cells was followed by preparing whole mounts of integument at various times after confrontation. These operations may lead to cell division and an alteration in cell shape at the confrontation site. The intensity of the induced cell behaviour pattern depends on which levels in the segment are confronted and the evidence suggests that it is directly related to the magnitude of the difference in positional values between confronted cells. The results can be explained by a nonlinear gradient of positional values within the segment with a crowding of values in the posterior region. It is also shown that segment border formation requires the confrontation of cells with a near maximum possible difference in positional values.
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