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M Affolter

Publications and source records attributed to M Affolter.

At least 19 recordsLinked to original sources
PubMed2001

Nuclear interpretation of Dpp signaling in Drosophila.

Signaling by Decapentaplegic (Dpp), a member of the TGFbeta superfamily of signaling molecules similar to vertebrate BMP2 and BMP4, has been implicated in many developmental processes in Drosophila melanogaster. Notably, Dpp acts as a long-range morphogen during imaginal disc growth and patterning. Genetic approaches led to the identification of a number of gene products that constitute the core signaling pathway. In addition to the ligand-activated heteromeric receptor complex and the signal-transducing intracellular Smad proteins, Dpp signaling requires two nuclear proteins, Schnurri (Shn) and Brinker (Brk), to prime cells for Dpp responsiveness. A complex interplay between the nuclear factors involved in Dpp signaling appears to control the transcriptional readout of the Dpp morphogen gradient. It remains to be seen whether similar molecular mechanisms operate in the nucleus in vertebrate systems.

Animals↗
PubMed2001

A HOX complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer.

A central theme during development and homeostasis is the generation of cell type-specific responses to the action of a limited number of extant signaling cascades triggered by extracellular ligands. The molecular mechanisms by which information from such signals are integrated in responding cells in a cell-type specific manner remain poorly understood. We have undertaken a detailed characterization of an enhancer that is regulated by DPP signaling and by the homeotic protein Labial and its partners, Extradenticle and Homothorax. The expression driven by this enhancer (lab550) and numerous deletions and point mutants thereof was studied in wild-type and mutant Drosophila embryos as well as in cultured cells. We find that the lab550 enhancer is composed of two elements, a Homeotic Response Element (HOMRE) and a DPP Response Element (DPPRE) that synergize. None of these two elements can reproduce the expression of lab550, either with regard to expression level or with regard to spatial restriction. The isolated DPPRE of lab550 responds extremely weakly to DPP. Interestingly, we found that the inducibility of this DPPRE is weak because it is tuned down by the action of a repressor element. This repressor element and an additional 50 bp element appear to be crucial for the cooperation of the HOMRE and the DPPRE, and might tightly link the DPP response to the homeotic input. The cooperation between the different elements of the enhancer leads to the segmentally restricted activity of lab550 in the endoderm and provides a mechanism to create specific responses to DPP signaling with the help of a HOX protein complex.

Animals↗
PubMed2001

Specificity of FGF signaling in cell migration in Drosophila.

We wanted to investigate the relationship between receptor tyrosine kinase (RTK) activated signaling pathways and the induction of cell migration. Using Drosophila tracheal and mesodermal cell migration as model systems, we find that the intracellular domain of the fibroblast growth factor receptors (FGFRs) Breathless (Btl) and Heartless (Htl) can be functionally replaced by the intracellular domains of Torso (Tor) and epidermal growth factor receptor (EGFR). These hybrid receptors can also rescue cell migration in the absence of Downstream of FGFR (Dof), a cytoplasmic protein essential for FGF signaling. These results demonstrate that tracheal and mesodermal cells respond during a specific time window to a receptor tyrosine kinase (RTK) signal with directed migration, independent of the presence or absence of Dof. We discuss our findings in the light of the recent findings that RTKs generate a generic signal that is interpreted in responding cells according to their developmental history.

Animals↗
PubMed2000

Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker.

The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence-specific DNA binding protein. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.

Animals↗
PubMed2000

Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14.

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

Amino Acid Sequence↗
PubMed2000

Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development.

The Drosophila serum response factor (DSRF) is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the DSRF regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signalling pathway, is sufficient to induce DSRF expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signalling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory.

Animals↗
PubMed2000

Genetic control of branching morphogenesis during Drosophila tracheal development.

Branching morphogenesis is a widely used strategy to increase the surface area of a given organ. A number of tissues undergo branching morphogenesis during development, including the lung, kidney, vascular system and numerous glands. Until recently, very little has been known about the genetic principles underlying the branching process and about the molecules participating in organ specification and branch formation. The tracheal system of insects represents one of the best-characterised branched organs. The tracheal network provides air to most tissues and its development during embryogenesis has been studied intensively at the morphological and genetic level. More than 30 genes have been identified and ordered into sequential steps controlling branching morphogenesis. These studies have revealed a number of important principles that might be conserved in other systems.

Animals↗
PubMed2000

Schnurri mediates Dpp-dependent repression of brinker transcription.

Signalling by Decapentaplegic (Dpp), a member of the TGFbeta superfamily of signalling molecules, controls many aspects of Drosophila development by activating and repressing target genes. Several essential components of the Dpp signalling pathway have been identified, including the Dpp receptors Punt and Thick veins (Tkv) as well as the cytoplasmic mediators Mad and Medea. For target genes to be activated, Dpp signalling must suppress transcription of a repressor encoded by the brinker (brk) gene. Here we show that Schnurri (Shn), a large zinc-finger protein, is essential for Dpp-mediated repression of brk transcription; in contrast, Shn is not required for target-gene activation. Thus, the Dpp signalling pathway bifurcates, downstream of the signal-mediating SMAD proteins, into a Shn-dependent pathway leading to brk repression and a Shn-independent pathway leading to gene activation. The existence of several Shn-like proteins in vertebrates and the observation that Brk functions in BMP signalling in Xenopus indicates that a similar regulatory cascade may be conserved in higher organisms.

Animals↗
PubMed2000

Schnurri interacts with Mad in a Dpp-dependent manner.

BACKGROUND: Decapentaplegic (Dpp) is a member of the transforming growth factor-beta superfamily. Dpp governs various developmental processes in Drosophila through the transcriptional regulation of a variety of genes. Signals of Dpp are transmitted from the cell membrane to the nucleus by Medea and Mad, both belonging to the Smad protein family. Mad was shown to bind to the Dpp-responsive element in genes such as vestigial, labial, and Ultrabithorax. The DNA binding affinity of Smad proteins is relatively low, and requires other nuclear factor(s) to form stable DNA binding complexes. schnurri (shn) was identified as a candidate gene acting downstream of Dpp receptors, but its relevance to Mad has remained unknown. RESULTS: We characterized the biochemical functions of Shn. Shn forms homo-oligomers. Shn is localized in the nucleus, and is likely to have multiple nuclear localizing signals. Finally, we found that Shn interacts with Mad in a Dpp-dependent manner. CONCLUSIONS: The present results argue that Shn may act as a nuclear component of the Dpp signalling pathway through direct interaction with Mad.

Animals↗
PubMed2000

Purification, cloning, and characterization of a second arylalkylamine N-acetyltransferase from Drosophila melanogaster.

In insects, amine acetylation by the enzyme arylalkylamine N-acetyltransferase (AANAT) is involved in melatonin formation, sclerotization, and neurotransmitter inactivation. This wide spectrum of activities suggests that several AANAT enzymes are present. We recently purified a protein fraction with AANAT activity from Drosophila melanogaster and cloned the corresponding gene, aaNAT1. Following the same strategy, we now report the purification of an additional AANAT from D. melanogaster, AANAT2, and the cloning of the corresponding cDNA. The isolated protein differs from AANAT1a and AANAT1b in its molecular weight and isoelectric point. The AANAT2 shares about 30% identity with the products of the aaNAT1 gene. The enzyme does not follow one-site Michaelis-Menten kinetics when assayed with various concentrations of the arylalkylamine tryptamine and a constant concentration (0.5 mM) of the cofactor acetyl coenzyme A. The data can be interpreted in terms of an enzyme with two kinetic regimes (K(m1) = 7.2 microM, K(m2) = 0.6 mM, and v(max2) = 2.7 v(max1)) that are governed by binding of the substrate to a regulatory site (K(r) = 6.2 mM). These findings demonstrate the presence of a second expressed gene encoding an AANAT in D. melanogaster. Northern blot analysis revealed no diurnal variation of aaNAT2 transcription, similar to the results obtained for aaNAT1a and aaNAT1b.

Acetylation↗
PubMed1999

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex.

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

Amino Acid Sequence↗
PubMed1998

Biochemical and biophysical characterization of refolded Drosophila DPP, a homolog of bone morphogenetic proteins 2 and 4.

The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperatures (5-15 degreesC). The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affinity tags to obtain a highly enriched preparation in one chromatographic step. A subsequent C4 reverse phase high pressure liquid chromatography step provides high purity, salt-free protein that is amenable to biophysical and structural studies at a yield of approximately 3 mg/liter of bacterial culture. The dimeric protein is correctly folded as determined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell culture, the refolded Escherichia coli-expressed protein is not glycosylated at a conserved N-linked site and is therefore homogeneous. The C-terminal domain dimer is more hydrophobic and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250-400 amino acids) N-terminal prodomains of transforming growth factor-beta superfamily members, shown to act as intramolecular chaperones in vivo.

Amino Acid Sequence↗
PubMed1998

Hox proteins meet more partners.

The Hox genes are clustered sets of homeobox-containing genes that play a central role in animal development. Recent genetic and molecular data suggest that Hox proteins interact with pre-existing homeodomain protein complexes. These complexes may help to regulate Hox activity and Hox specificity, and help cells to interpret signaling cascades during development.

Animals↗
PubMed1998

The Drosophila protein Dof is specifically required for FGF signaling.

Receptor tyrosine kinases (RTKs) transmit signals to the cell nucleus via the MAP kinase (MAPK) cascade, using specific molecules to link the activated receptors to the MAPK cascade activator, Ras. We have identified a component of the FGF receptor (FGFR) signal transduction pathway, Downstream of FGFR (Dof). Dof is an intracellular protein that is essential for signal transmission by the FGFR and acts downstream of the receptor and upstream of Ras. Unlike other signaling molecules that act downstream of RTKs, Dof is not expressed ubiquitously but is present exclusively in cells that express FGFRs. Dof is needed in these cells for activation of the MAPK cascade via FGF signaling, but not for activation via other RTK ligands. Dof therefore appears to be committed exclusively to FGFR-mediated signal transduction.

Amino Acid Sequence↗
PubMed1998

Molecular and biochemical characterization of the aaNAT1 (Dat) locus in Drosophila melanogaster: differential expression of two gene products.

In insects, arylalkylamine N-acetyltransferases (AANATs) have been implicated in several physiological processes, including sclerotization, inactivation of certain neurotransmitters, and, similar to the function in vertebrates, catalysis of the rate-limiting step in melatonin biosynthesis. Here, we report an extensive biochemical and functional analysis of the products of the aaNAT1 gene of Drosophila melanogaster. The aaNAT1 gene generates two transcripts through alternative first-exon usage. These transcripts are under tissue-specific and developmental control and encode proteins which differ in their N-terminus with respect to their starting methionine. The more abundant isoform, AANATlb, is first expressed during late embryogenesis in the brain, the ventral nerve cord, and the midgut; in adults, AANATlb is still detectable in the brain and midgut. The less abundant isoform, AANATla, appears only during late pupal stages and in adults is found predominantly in the brain. We demonstrate that the mutation Dat(lo) represents a hypomorphic allele of aaNAT1b, in which an insertion of two transposable elements, MDG412 and blastopia, has occurred within the first intron of the gene. Using a deficiency which removes the aaNAT1 gene, we provide evidence that aaNAT1 is not essential for the process of sclerotization. Furthermore, neither of the two enzyme isoforms shows circadian regulation of RNA or protein levels. The differing levels of abundance and distinct developmental control of AANAT1a and AANAT1b suggest different in vivo functions for these two enzymes.

Alternative Splicing↗