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Biomedical subjects

M B O'Connor

Publications and source records attributed to M B O'Connor.

At least 19 recordsLinked to original sources
PubMed2005

A role for betaFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster.

Variations in ecdysteroid titers play crucial roles in arthropods by initiating and regulating molting and metamorphosis. The recent identification of genes coding for cytochrome P450 enzymes involved in Drosophila ecdysteroidogenesis provides new molecular tools to investigate the regulation of insect hormone production. In the present study, we used an enzyme immunoassay to show that the molting hormone titer is strictly correlated with the steroidogenic capacity of the ring gland. A temporal correlation between dynamics of ecdysone production and expression of genes encoding steroidogenic enzymes was observed during the third instar, suggesting that the timing of hormone production depends on transcriptional regulation of the biosynthetic enzymes. Using clonal analysis, levels of two steroidogenic enzymes, Phantom (PHM) and Disembodied (DIB), were shown to be very reduced in ftz transcription factor 1 (ftz-f1) mutant ring gland cells whereas there was no effect of the without children (woc) mutation, suggesting that FTZ-F1 regulates phm and dib expression. Since betaFTZ-F1 is the homolog of the vertebrate steroidogenic factor 1 (SF1), which plays a key role in the differentiation of vertebrate steroidogenic organs through transcriptional regulation of steroidogenic enzymes, this study emphasizes the strong parallels between insects and vertebrates with respect to the regulatory mechanisms of steroidogenesis.

Animals↗
PubMed2002

Reticular groove contraction in dairy cattle following drenching with an anti-bloat solution or a combination of anti-bloat solution and NaCl.

AIM: To determine whether the inclusion of NaCl in an antibloat drench increased the incidence of contraction of the reticular (oesophageal) groove in cattle. METHODS: Non-lactating Friesian dairy cows aged 3-10 years (n=30) were subjected to a 13C-octanoic-acid breath test after being drenched with either an anti-bloat solution alone or a mixture of anti-bloat solution and NaCl, to determine the incidence of reticular groove contraction. RESULTS: Drenching with an anti-bloat solution alone did not result in detectable by-pass of the reticulorumen in 27/29 cows; minor by-pass occurred in 2/29 cows. The inclusion of NaCl in the anti-bloat solution increased the incidence of reticulorumen by-pass; minor by-pass occurred in 12/30 cows and substantial by-pass was detected in 5/30 cows. The incidence of by-pass did not vary significantly with cow age. CONCLUSIONS: Drenching with an anti-bloat solution alone did not result in significant by-pass of the reticulorumen. The inclusion of NaCl in the anti-bloat drench increased the incidence of reticulorumen by-pass. The proportion of anti-bloat/ NaCl fluid by-passed was considered to be of no practical significance to the protection from bloat afforded in the majority of animals, but may significantly decrease protection from bloat afforded by drenching in 10-15% of cows. The proportion of animals at risk within a herd may vary with their physiological state and the method and frequency (number of doses per drench) of drenching.

Journal Article↗
PubMed2001

Twisted gastrulation is a conserved extracellular BMP antagonist.

Bone morphogenetic protein (BMP) signalling regulates embryonic dorsal-ventral cell fate decisions in flies, frogs and fish. BMP activity is controlled by several secreted factors including the antagonists chordin and short gastrulation (SOG). Here we show that a second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signalling using anti-phospho-Smad staining shows that the tsg and sog loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, TSG and SOG together make a more effective inhibitor of BMP signalling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. We show that Tsgs from different species are functionally equivalent, and conclude that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signalling.

Animals↗
PubMed2000

Is chordin a long-range- or short-range-acting factor? Roles for BMP1-related metalloproteases in chordin and BMP4 autofeedback loop regulation.

Diffusible morphogen models have been used widely to explain regional specification of tissues and body axes during animal development. The three-signal model for patterning the dorsal-ventral axis of the amphibian embryo proposes, in part, that a factor(s) secreted from Spemann's organizer is responsible for converting lateral marginal zone into more dorsal cell fates. We examine the possibility that chordin, a secreted inhibitor of bone morphogenetic protein (BMP) signaling and candidate "dorsalizing signal," is a long-range-acting factor. We show that chordin can, when overexpressed, act directly over distances of at least 450 microm in the early Xenopus embryo to create a gradient of BMP signaling. However, since lower levels of chordin can still induce secondary axes and these amounts of chordin act only locally to inhibit a BMP target gene, we suggest that chordin likely acts as a short-range signal in vivo. Furthermore, BMP1, a secreted metalloprotease that cleaves chordin protein in vitro, inhibits chordin's axis-inducing effects, suggesting that BMP1 functions to negatively regulate chordin's action in vivo. A dominant-negative mutant BMP1 blocks the in vitro cleavage of chordin protein by wild-type BMP1 and induces secondary axes when injected ventrally. We argue that BMP1 and Xolloid are probably functionally redundant metalloproteases and may have two roles in the early Xenopus embryo. One role may be to inhibit the action of low-level chordin protein expressed throughout the entire embryo and a possible second role may be to inhibit activation of a juxtacrine cell relay, thereby confining chordin's action to the organizer region preventing chordin from functioning as a long-range-acting factor.

Animals↗
PubMed2000

Functional analysis of repressor binding sites in the iab-2 regulatory region of the abdominal-A homeotic gene.

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. Previously, the Hunchback (HB) protein has been implicated in setting the anterior expression limit of the UBX homeotic protein in parasegment 6. Here we investigate DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that in contrast to Ubx enhancer elements, both HB and Krüppel (KR) are required to set the ABD-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that HB and KR are direct regulators of this iab-2 enhancer. The single KR site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest that KR repression occurs through a local quenching mechanism. We also show that the gap repressor Giant (GT) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. Finally, we show that this iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (PHO). These iab-2 PHO sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the PHO sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer.

Animals↗
PubMed2000

TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development.

Transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-beta/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH(2)-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.

Amino Acid Sequence↗
PubMed2000

Processing of the Drosophila Sog protein creates a novel BMP inhibitory activity.

Structurally unrelated neural inducers in vertebrate and invertebrate embryos have been proposed to function by binding to BMP4 or Dpp, respectively, and preventing these homologous signals from activating their receptor(s). In this study, we investigate the functions of various forms of the Drosophila Sog protein using the discriminating assay of Drosophila wing development. We find that misexpression of Drosophila Sog, or its vertebrate counterpart Chordin, generates a very limited vein-loss phenotype. This sog misexpression phenotype is very similar to that of viable mutants of glass-bottom boat (gbb), which encodes a BMP family member. Consistent with Sog selectively interfering with Gbb signaling, Sog can block the effect of misexpressing Gbb, but not Dpp in the wing. In contrast to the limited BMP inhibitory activity of Sog, we have identified carboxy-truncated forms of Sog, referred to as Supersog, which when misexpressed cause a broad range of dpp(-) mutant phenotypes. In line with its phenotypic effects, Supersog can block the effects of both misexpressing Dpp and Gbb in the wing. Vertebrate Noggin, on the other hand, acts as a general inhibitor of Dpp signaling, which can interfere with the effect of overexpressing Dpp, but not Gbb. We present evidence that Sog processing occurs in vivo and is biologically relevant. Overexpression of intact Sog in embryos and adult wing primordia leads to the developmentally regulated processing of Sog. This in vivo processing of Sog can be duplicated in vitro by treating Sog with a combination of the metalloprotease Tolloid (Tld) plus Twisted Gastrulation (Tsg), another extracellular factor involved in Dpp signaling. In accord with this result, coexpression of intact Sog and Tsg in developing wings generates a phenotype very similar to that of Supersog. Finally, we provide evidence that tsg functions in the embryo to generate a Supersog-like activity, since Supersog can partially rescue tsg(-) mutants. Consistent with this finding, sog(- )and tsg(-) mutants exhibit similar dorsal patterning defects during early gastrulation. These results indicate that differential processing of Sog generates a novel BMP inhibitory activity during development and, more generally, that BMP antagonists play distinct roles in regulating the quality as well as the magnitude of BMP signaling.

Animals↗
PubMed2000

The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels.

Ecdysteroids regulate a wide variety of cellular processes during arthropod development, yet little is known about the genes involved in the biosynthesis of these hormones. Previous studies have suggested that production of 20-hydroxyecdysone in Drosophila and other arthropods involves a series of cytochrome P450 catalyzed hydroxylations of cholesterol. In this report, we show that the disembodied (dib) locus of Drosophila codes for a P450-like sequence. In addition, we find that dib mutant embryos have very low titers of ecdysone and 20-hydroxyecdysone (20E) and fail to express IMP-E1 and L1, two 20E-inducible genes, in certain tissues of the embryo. In situ hybridization studies reveal that dib is expressed in a complex pattern in the early embryo, which eventually gives way to restricted expression in the prothoracic portion of the ring gland. In larval and adult tissues, dib expression is observed in the prothoracic gland and follicle cells of the ovaries respectively, two tissues known to synthesize ecdysteroids. Phenotypic analysis reveals that dib mutant embryos produce little or no cuticle and exhibit severe defects in many late morphogenetic processes such as head involution, dorsal closure and gut development. In addition, we examined the phenotypes of several other mutants that produce defective embryonic cuticles. Like dib, mutations in the spook (spo) locus result in low embryonic ecdysteroid titers, severe late embryonic morphological defects, and a failure to induce IMP-E1. From these data, we conclude that dib and spo likely code for essential components in the ecdysone biosynthetic pathway and that ecdysteroids regulate many late embryonic morphogenetic processes such as cell movement and cuticle deposition.

Amino Acid Sequence↗
PubMed1999

The Drosophila activin receptor baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development.

The TGF-beta superfamily of growth and differentiation factors, including TGF-beta, Activins and bone morphogenetic proteins (BMPs) play critical roles in regulating the development of many organisms. These factors signal through a heteromeric complex of type I and II serine/threonine kinase receptors that phosphorylate members of the Smad family of transcription factors, thereby promoting their nuclear localization. Although components of TGF-beta/Activin signaling pathways are well defined in vertebrates, no such pathway has been clearly defined in invertebrates. In this study we describe the role of Baboon (Babo), a type I Activin receptor previously called Atr-I, in Drosophila development and characterize aspects of the Babo intracellular signal-transduction pathway. Genetic analysis of babo loss-of-function mutants and ectopic activation studies indicate that Babo signaling plays a role in regulating cell proliferation. In mammalian cells, activated Babo specifically stimulates Smad2-dependent pathways to induce TGF-beta/Activin-responsive promoters but not BMP-responsive elements. Furthermore, we identify a new Drosophila Smad, termed dSmad2, that is most closely related to vertebrate Smads 2 and 3. Activated Babo associates with dSmad2 but not Mad, phosphorylates the carboxy-terminal SSXS motif and induces heteromeric complex formation with Medea, the Drosophila Smad4 homolog. Our results define a novel Drosophila Activin/TGF-beta pathway that is analogous to its vertebrate counterpart and show that this pathway functions to promote cellular growth with minimal effects on patterning.

Activin Receptors↗
PubMed1998

Complex interactions of the protein L-isoaspartyl methyltransferase and calmodulin revealed with the yeast two-hybrid system.

The widely distributed protein-L-isoaspartyl, D-aspartyl carboxylmethyltransferase (EC 2.1.1.77) is hypothesized to play a role in the repair or metabolism of deamidated and isomerized proteins that are spontaneously generated during the aging of proteins in cells. The yeast two-hybrid system was used to identify proteins that potentially interact with the methyltransferase in a cellular processing pathway. Two cDNAs, both encoding calmodulin, were isolated from a human fetal brain cDNA library using the human methyltransferase as the bait. Enzymatic assays with purified components revealed a complex set of interactions between the methyltransferase and calmodulin. Calmodulin weakly stimulated protein carboxylmethyltransferase activity in vitro at concentrations of the two proteins reflecting their representation in mammalian brain. Calmodulin stimulation of methyltransferase was observed in both the presence and absence of calcium, although the effect was greater in the presence of calcium. Native calmodulin was not a substrate for the carboxylmethyltransferase, but deamidated variants of calmodulin act as substrates for the methyltransferase, with calculated Km values of 3.6 and 8.6 microM for calcium-liganded and unliganded calmodulin, respectively. Both the effector and substrate interactions of calmodulin with the protein isoaspartyl methyltransferase likely contributed to the positive results obtained with the two-hybrid system.

Calmodulin↗
PubMed1998

Synergistic signaling by two BMP ligands through the SAX and TKV receptors controls wing growth and patterning in Drosophila.

In Drosophila wing discs, a morphogen gradient of DPP has been proposed to determine the transcriptional response thresholds of the downstream genes sal and omb. We present evidence that the concentration of the type I receptor TKV must be low to allow long-range DPP diffusion. Low TKV receptor concentrations result, however, in low signaling activity. To enhance signaling at low DPP concentrations, we find that a second ligand, GBB, augments DPP/TKV activity. GBB signals primarily through the type I receptor SAX, which synergistically enhances TKV signaling and is required for proper OMB expression. We show that OMB expression in wing discs requires synergistic signaling by multiple ligands and receptors to overcome the limitations imposed on DPP morphogen function by receptor concentration levels.

Animals↗
PubMed1997

Production of a DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins.

During early Drosophila embryogenesis, several zygotic gene products act to establish a posttranscriptional activity gradient of the morphogen DPP. Among these molecules, Tolloid, a putative metalloprotease related to BMP-1, enhances DPP function, while SOG, an ortholog of the Xenopus organizer Chordin, inhibits DPP function. Using epistasis tests and a Xenopus secondary axis induction assay, we show that TLD negates the inhibitory effects of SOG/CHD on DPP/BMP-type ligands. In transient transfection assays, we demonstrate that TLD cleaves SOG and that cleavage is stimulated by DPP. We propose that formation of the embryonic DPP activity gradient involves the opposing effects of SOG inhibiting DPP and TLD processing SOG to release DPP from the inhibitory complex.

Animals↗
PubMed1997

Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase.

An epitope-tagged calmodulin (CaM), capable of interacting with CaM-binding proteins in cellular extracts, would be a valuable tool for identifying proteins in signal transduction pathways involving calcium. A bacterial overexpression vector for epitope-tagged CaM has been constructed by inserting the coding sequence for a nine amino acid portion of the influenza virus hemagglutinin (HA) protein into the initiation site of an overexpression vector for chicken CaM. The HA-CaM fusion produced in bacteria was compared to native CaM for its ability to activate smooth muscle myosin light chain kinase (MLCK), one of the best understood CaM-dependent enzymes. MLCK activity was tested in both a purified system and a CaM-depleted "native actomyosin" preparation maintaining many of the regulatory properties of the intact smooth muscle. HA-CaM behaves identically to unmodified CaM in both systems, indicating that the HA epitope does not adversely affect CaM function. The recombinant HA-CaM was used to sensitively detect CaM interactions with smooth muscle proteins in a modified gel overlay assay, using a monoclonal antibody against the HA epitope as the secondary reagent. Enzymatically active complexes of HA-CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and protein G-Sepharose beads.

Actomyosin↗
PubMed1997

Structural organization and developmental expression of the protein isoaspartyl methyltransferase gene from Drosophila melanogaster.

A protein carboxyl methyltransferase activity (PCMT) with a specificity for age-damaged protein D-aspartyl and L-isoaspartyl residues (E.C. 2.1.1.77) has been identified and cloned in Drosophila. The Drosophila gene was localized by chromosome in-situ hybridization to region 83AB of the third chromosome. The methyltransferase coding sequence is distributed among four exons within a 1.4-kb segment of the genome; it predicts a polypeptide of 226 amino acids that is 55% identical to the mouse enzyme. When expressed in bacteria, the Drosophila protein exhibits PCMT activity. A single 1.4-kb Pcmt transcript is detected in RNA preparations from embryos, larvae, pupae and adults. The abundance of the transcript, which is lowest in larvae and highest in adults, parallels the specific activity of the enzyme measured in extracts from the same developmental stages. It has been proposed that the PCMT initiates the repair of structurally damaged cellular proteins. The constitutive expression of PCMT and the relatively high level of expression in postmitotic adult cells suggest that PCMT activity is required through development, but acquires additional significance in aging tissues.

Animals↗
PubMed1997

Defects in glucuronate biosynthesis disrupt Wingless signaling in Drosophila.

In vitro experiments suggest that glycosaminoglycans (GAGs) and the proteins to which they are attached (proteoglycans) are important for modulating growth factor signaling. However, in vivo evidence to support this view has been lacking, in part because mutations that disrupt the production of GAG polymers and the core proteins have not been available. Here we describe the identification and characterization of Drosophila mutants in the suppenkasper (ska) gene. The ska gene encodes UDP-glucose dehydrogenase which produces glucuronic acid, an essential component for the synthesis of heparan and chondroitin sulfate. ska mutants fail to put heparan side chains on proteoglycans such as Syndecan. Surprisingly, mutant embryos produced by germ-line clones of this general metabolic gene exhibit embryonic cuticle phenotypes strikingly similar to those that result from loss-of-function mutations in genes of the Wingless (Wg) signaling pathway. Zygotic loss of ska leads to reduced growth of imaginal discs and pattern defects similar to wg mutants. In addition, genetic interactions of ska with wg and dishevelled mutants are observed. These data demonstrate the importance of proteoglycans and GAGs in Wg signaling in vivo and suggest that Wnt-like growth factors may be particularly sensitive to perturbations of GAG biosynthesis.

Amino Acid Sequence↗
PubMed1996

The Xenopus dorsalizing factor noggin ventralizes Drosophila embryos by preventing DPP from activating its receptor.

noggin is expressed in the Spemann organizer region of the Xenopus embryo and can promote dorsal cell fates within the mesoderm and neural development within the overlying ectoderm. Here, we show that noggin promotes ventral development in Drosophila, specifying ventral ectoderm and CNS in the absence of all endogenous ventral-specific zygotic gene expression. We utilize constitutively active forms of the dpp receptors to demonstrate that noggin blocks dpp signaling upstream of dpp receptor activation. These results suggest a mechanistic basis for the action of Spemann's organizer during Xenopus development and provide further support for the conservation of dorsal-ventral patterning mechanisms between arthropods and chordates.

Activin Receptors↗
PubMed1996

MADR1, a MAD-related protein that functions in BMP2 signaling pathways.

Components of the signaling pathways that lie downstream of Ser/Thr kinase receptors and are required for signaling by the TGF beta superfamily have been poorly defined. The Drosophila gene Mothers against dpp (MAD) and the C. elegans sma genes are implicated in these signaling pathways. We show that MAD functions downstream of DPP receptors and is required for receptor signaling. Phosphorylation of MADR1, a human homolog of MAD, is tightly regulated and rapidly induced by BMP2, but not TGF beta or activin. This phosphorylation is necessary for function, since a point mutant that yields a null phenotype in Drosophila is not phosphorylated. BMP2 treatment results in accumulation of MADR1 in the nucleus. MAD proteins may thus define a novel class of signaling molecules with nuclear function in Ser/Thr kinase receptor signaling pathways.

Activins↗