Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling.
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Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation. (+info)
Cubitus interruptus is necessary but not sufficient for direct activation of a wing-specific decapentaplegic enhancer.
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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. (+info)
Genes required for axon pathfinding and extension in the C. elegans nerve ring.
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Over half of the neurons in Caenorhabditis elegans send axons to the nerve ring, a large neuropil in the head of the animal. Genetic screens in animals that express the green fluorescent protein in a subset of sensory neurons identified eight new sax genes that affect the morphology of nerve ring axons. sax-3/robo mutations disrupt axon guidance in the nerve ring, while sax-5, sax-9 and unc-44 disrupt both axon guidance and axon extension. Axon extension and guidance proceed normally in sax-1, sax-2, sax-6, sax-7 and sax-8 mutants, but these animals exhibit later defects in the maintenance of nerve ring structure. The functions of existing guidance genes in nerve ring development were also examined, revealing that SAX-3/Robo acts in parallel to the VAB-1/Eph receptor and the UNC-6/netrin, UNC-40/DCC guidance systems for ventral guidance of axons in the amphid commissure, a major route of axon entry into the nerve ring. In addition, SAX-3/Robo and the VAB-1/Eph receptor both function to prevent aberrant axon crossing at the ventral midline. Together, these genes define pathways required for axon growth, guidance and maintenance during nervous system development. (+info)
Increased bradykinin and "normal" angiotensin peptide levels in diabetic Sprague-Dawley and transgenic (mRen-2)27 rats.
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BACKGROUND: The transgenic (mRen-2)27 rat (TGR) is a high tissue renin, high angiotensin (Ang) II model of hypertension. When administered streptozotocin (STZ), TGRs develop a rapidly progressive diabetic nephropathy with renal failure over 12 weeks. Bradykinin (BK) and Ang II are potent vasoactive peptides that may participate in the vascular and metabolic abnormalities of diabetes. METHODS: TGR and Sprague-Dawley (SD) rats were administered STZ (diabetic) or citrate buffer (nondiabetic) at six weeks of age. Diabetic rats received daily ultralente insulin to maintain moderate hyperglycemia ( approximately 18 mM). Rats were sacrificed four- and eight-weeks post-STZ or vehicle. RESULTS: Diabetes did not modify the blood pressure of either SD rats or TGRs. Diabetes increased levels of BK-(1-9) and its metabolite BK-(1-7) in kidney, aorta, and heart of both SD rats and TGRs. Diabetes did not influence Ang II levels in plasma, kidney, aorta, heart, or adrenal gland of SD rats, but reduced to normal the elevated Ang II levels in plasma, kidney, aorta, and adrenal gland of TGRs. CONCLUSIONS: STZ-induced diabetes was associated with elevated tissue levels of BK-(1-9) and "normal" circulating and tissue levels of Ang II. The increased BK-(1-9) levels were consistent with the participation of this peptide in the vascular and metabolic abnormalities of diabetes. However, the rapidly progressive nephropathy of diabetic TGRs was not associated with BK-(1-9) and Ang II levels in target organs that differed from those of diabetic SD rats. (+info)
MIG-13 positions migrating cells along the anteroposterior body axis of C. elegans.
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The C. elegans Q neuroblasts and their descendants migrate along the anteroposterior (A/P) body axis to positions that are not associated with any obvious landmarks. We find that a novel protein, MIG-13, is required to position these cells correctly. MIG-13 is a transmembrane protein whose expression is restricted to the anterior and central body regions by Hox gene activity. MIG-13 functions non-cell autonomously within these regions to promote migration toward the anterior: loss of mig-13 activity shifts the Q descendants toward the posterior, whereas increasing the level of MIG-13 shifts them anteriorly in a dose-dependent manner. Our findings suggest that MIG-13 is a component of a global A/P migration system, and that the level of MIG-13 determines where along the body axis these migrating cells stop. (+info)
Stabilization of chromatin structure by PRC1, a Polycomb complex.
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The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones. (+info)
Cytotoxic T-cell-mediated response against Yersinia pseudotuberculosis in HLA-B27 transgenic rat.
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Yersinia-induced reactive arthritis is highly associated with HLA-B27, the role of which in defense against the triggering bacteria remains unclear. The aim of this study was to examine the capacity of rats transgenic for HLA-B27 to mount a cytotoxic T-lymphocyte (CTL) response against Y. pseudotuberculosis and to determine the influence of the HLA-B27 transgene on this response. Rats transgenic for HLA-B*2705 and human beta(2)-microglobulin of the 21-4L line, which do not spontaneously develop disease, and nontransgenic syngeneic Lewis (LEW) rats were infected with Y. pseudotuberculosis. Lymph node cells were restimulated in vitro, and the presence of for Y. pseudotuberculosis-specific CTLs against infected targets was determined. Infection of 21-4L rats triggered a CD8(+) T cell-mediated cytotoxic response specific for Y. pseudotuberculosis. Analysis of this response demonstrated restriction by an endogenous major histocompatibility complex molecule. However, no restriction by HLA-B27 was detected. In addition, kinetics studies revealed a weaker anti-Yersinia CTL response in 21-4L rats than in nontransgenic LEW rats, and the level of cytotoxicity against 21-4L lymphoblast targets sensitized with Y. pseudotuberculosis was lower than that against nontransgenic LEW targets. We conclude that HLA-B27 transgenic rats mount a CTL response against Y. pseudotuberculosis that is not restricted by HLA-B27. Yet, HLA-B27 exerts a negative effect on the level of this response, which could contribute to impaired defense against Yersinia. (+info)
Competence and commitment of Caenorhabditis elegans vulval precursor cells.
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Multipotent Caenorhabditis elegans vulval precursor cells (VPCs) choose among three fates (1 degrees, 2 degrees, and 3 degrees ) in response to two intercellular signals: the EGF family growth factor LIN-3 induces 1 degrees fates at high levels and 2 degrees fates at low levels; and a signal via the receptor LIN-12 induces 2 degrees fates. If the level of LIN-3 signal is reduced by a lin-3 hypomorphic mutation, the daughters of the VPC closest to the anchor cell (AC), P6.p, are induced by the AC. By expressing LIN-3 as a function of time in LIN-3-deficient animals, we find that both VPCs and the daughters of VPCs are competent to respond to LIN-3, and VPC daughters lose competence after fusing with the hypodermis. We also demonstrate that the daughters of VPCs specified to be 2 degrees can respond to LIN-3, indicating that 2 degrees VPCs are not irreversibly committed. We propose that maintenance of VPC competence after the first cell cycle and the prioritization of the 1 degrees fate help ensure that P6.p will become 1 degrees. This mechanism of competence regulation might have been maintained from ancestral nematode species that used induction both before and after VPC division and serves to maximize the probability that a functional vulva is formed. (+info)