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At least 631 records · Page 35Linked to original sources
PubMed1993

Conservation of regulatory elements controlling hairy pair-rule stripe formation.

The hairy (h) gene is one of two pair-rule loci whose striped expression is directly regulated by combinations of gap proteins acting through discrete upstream regulatory fragments, which span several kilobases. We have undertaken a comparative study of the molecular biology of h pair-rule expression in order to identify conserved elements in this complex regulatory system, which should provide important clues concerning the mechanism of stripe formation. A molecular comparison of the h locus in Drosophila virilis and Drosophila melanogaster reveals a conserved overall arrangement of the upstream regulatory elements that control individual pair-rule stripes. We demonstrate that upstream fragments from D. virilis will direct the proper expression of stripes in D. melanogaster, indicating that these are true functional homologs of the stripe-producing D. melanogaster regulatory elements, and that the network of trans-acting proteins that act upon these regulatory elements is highly conserved. We also demonstrate that the spatial relationships between specific h stripes and selected gap proteins are highly conserved. We find several tracts of extensive nucleotide sequence conservation within homologous stripe-specific regulatory fragments, which have facilitated the identification of functional subelements within the D. melanogaster regulatory fragment for h stripe 5. Some of the conserved nucleotide tracts within this regulatory fragment contain consensus binding sites for potential trans-regulatory (gap and other) proteins, while many appear devoid of known binding sites. This comparative approach, coupled with the analysis of reporter gene expression in gap mutant embryos suggests that the Kr and gt proteins establish the anterior and posterior borders of h stripe 5, respectively, through spatial repression. Other, as yet unidentified, proteins are certain to play a role in stripe activation, presumably acting through other conserved sequence tracts.

Amino Acid Sequence↗
PubMed1993

Drosophila hairy pair-rule gene regulates embryonic patterning outside its apparent stripe domains.

The hairy (h) segmentation gene of Drosophila regulates segmental patterning of the early embryo, and is expressed in a set of anteroposterior stripes during the blastoderm stage. We have used a set of h gene deletions to study the h promoter and the developmental requirements for individual h stripes. The results confirm upstream regulation of h striping but indicate that expression in the anterodorsal head domain depends on sequences downstream of the two transcription initiation sites. Surprisingly, the two anterior-most h domains appear to be dispensable for head development and embryonic viability. One partial promoter deletion expresses ectopic h, leading to misexpression of other segmentation genes and embryonic pattern defects. We demonstrate that h affects patterning outside its apparent stripe domains, supporting a model in which primary pair-rule genes act as concentration-dependent transcriptional regulators, i.e. as local morphogens.

Animals↗
PubMed1993

Control of segmental asymmetry in Drosophila embryos.

During Drosophila development, an important aspect of body patterning is the division of the embryo into repeating morphological units referred to as parasegments. The parasegmental domains are first defined at the blastoderm stage by alternating stripes of transcripts encoded by the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) and later by stripes encoded by the segment polarity genes engrailed (en) and wingless. Here, we show that the runt gene (run) is required to generate asymmetries within these parasegmental domains. Using a heat-shock-inducible run transgene, we found that ectopic run expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Unexpectedly, we also found that ectopic run was a rapid and potent repressor of odd-numbered en stripes. Two remarkably different segmental phenotypes were generated as a consequence of these effects. In solving the mechanisms underlying these phenotypes, we discovered that the positioning of en stripes is largely determined by the actions of negative regulators. Our data indicate that run is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of run and odd by the product of the eve gene. The spatial restriction of gene expression via negative and double negative pathways such as these is likely to be a common theme during development.

Animals↗
PubMed1994

Positioning adjacent pair-rule stripes in the posterior Drosophila embryo.

We present a genetic and molecular analysis of two hairy (h) pair-rule stripes in order to determine how gradients of gap proteins position adjacent stripes of gene expression in the posterior of Drosophila embryos. We have delimited regulatory sequences critical for the expression of h stripes 5 and 6 to 302 bp and 526 bp fragments, respectively, and assayed the expression of stripe-specific reporter constructs in several gap mutant backgrounds. We demonstrate that posterior stripe boundaries are established by gap protein repressors unique to each stripe: h stripe 5 is repressed by the giant (gt) protein on its posterior border and h stripe 6 is repressed by the hunchback (hb) protein on its posterior border. Interestingly, Krüppel (Kr) limits the anterior expression limits of both stripes and is the only gap gene to do so, indicating that stripes 5 and 6 may be coordinately positioned by the Kr repressor. In contrast to these very similar cases of spatial repression, stripes 5 and 6 appear to be activated by different mechanisms. Stripe 6 is critically dependent upon knirps (kni) for activation, while stripe 5 likely requires a combination of activating proteins (gap and non-gap). To begin a mechanistic understanding of stripe formation, we locate binding sites for the Kr protein in both stripe enhancers. The stripe 6 enhancer contains higher affinity Kr-binding sites than the stripe 5 enhancer, which may allow for the two stripes to be repressed at different Kr protein concentration thresholds. We also demonstrate that the kni activator binds to the stripe 6 enhancer and present evidence for a competitive mechanism of Kr repression of stripe 6.

Animals↗
PubMed1994

A processed form of the Spätzle protein defines dorsal-ventral polarity in the Drosophila embryo.

Stein et al. (1991) identified a soluble, extracellular factor that induces ventral structures at the site where it is injected in the extracellular space of the early Drosophila embryo. This factor, called polarizing activity, has the properties predicted for a ligand for the transmembrane receptor encoded by the Toll gene. Using a bioassay to follow activity, we purified a 24 x 10(3) M(r) protein that has polarizing activity. The purified protein is recognized by antibodies to the C-terminal half of the Spätzle protein, indicating that this polarizing activity is a product of the spätzle gene. The purified protein is smaller than the primary translation product of spätzle, suggesting that proteolytic processing of Spätzle on the ventral side of the embryo is required to generate the localized, active form of the protein.

Animals↗
PubMed1995

Pair-rule expression of the Drosophila fushi tarazu gene: a nuclear receptor response element mediates the opposing regulatory effects of runt and hairy.

The segmentation genes runt and hairy are required for the proper transcriptional regulation of the pair-rule gene fushi tarazu during the blastoderm stage of Drosophila embryogenesis. The expression of different fushi tarazu reporter genes was examined in runt and hairy mutant embryos, as well as in runt over-expressing embryos in order to identify DNA elements responsible for mediating these regulatory effects. The results indicated that runt and hairy act through a common 32 base-pair element. This element, designated as fDE1, contains a binding site for a small family of orphan nuclear receptor proteins that are uniformly expressed in blastoderm embryos. The pair-rule expression of reporter gene constructs containing multimerized fDE1 elements depends on activation by runt and repression by hairy. Examination of reporter genes with mutated fDE1 elements provided further evidence that this element mediates both transcriptional activation and repression. Genetic experiments indicated that the opposing effects of runt and hairy were not due solely to cross-regulatory interactions between these two genes and that fDE1-dependent expression is regulated by factors in addition to runt and hairy.

Animals↗
PubMed1996

The role of segment polarity genes during early oogenesis in Drosophila.

In the Drosophila ovary, hedgehog (hh) signaling from cells near the apical tip of the germarium stimulates the proliferation and specification of somatic cells in region 2 of the germarium, 2-5 cells away from the hh-expressing cells (A. J. Forbes, H. Lin, P. Ingham and A. Spradling (1996) Development 122, 1125-1135). This report examines the role during early oogenesis of several genes that are known to function in hh-mediated signaling during embryonic and larval development (P. Ingham (1995) Current Opin. Genetics Dev. 5, 528-534). As in imaginal discs, engrailed (en) is co-expressed with hh in the germarium, while patched (ptc) and cubitus interruptus (ci) are expressed in somatic cells throughout the germarium and in developing egg chambers, with ptc expression being elevated within 10 cell diameters of the source of the hh signal. Moreover, the somatic cell overproliferation caused by ectopic hh expression is accompanied by elevated levels of ptc and is phenocopied in ptc- somatic clones. These analyses suggest that ptc and ci are components of the hh signaling pathway in the germarium. However, unlike embryos and imaginal discs, neither wingless (wg) nor decapentaplegic (dpp) appear to mediate the ovarian hh signal. wg is expressed in 'cap cells,' a subset of hh-expressing cells located adjacent to germ-line stem cells, but is unaffected by ectopic hh expression. Nor does the ectopic expression of wg or dpp mimic the effect of ectopic hh expression. We propose that Hh diffuses from apical cells, including cap cells, and regulates the proliferation of nearby ovarian somatic cells by antagonizing the negative effects of ptc on ci activity in these cells, thereby allowing the transcription of ci-dependent genes, including ptc itself.

Animals↗
PubMed1996

Ectopic expression of wingless in imaginal discs interferes with decapentaplegic expression and alters cell determination.

We have expressed the segment polarity gene wingless (wg) ectopically in imaginal discs to examine its regulation of both ventral patterning and transdetermination. By experimentally manipulating the amount of Wg protein, we show that different thresholds of Wg activity elicit different outcomes, which are mediated by regulation of decapentaplegic (dpp) expression and result in alterations in the expression of homeotic genes. A high level of Wg activity leads to loss of all dorsal pattern elements and the formation of a complete complement of ventral pattern elements on the dorsal side of legs, and is correlated with repression of dpp expression. wg expression in dorsal cells of each disc also leads to dose-dependent transdetermination in those cells in homologous discs such as the labial, antennal and leg, but not in cells of dorsally located discs. When dpp expression is repressed by high levels of Wg, transdetermination does not occur, confirming that dpp participates with wg to induce transdetermination. These and other experiments suggest that dorsal expression of wg alters disc patterning and disc cell determination by modulating the expression of dpp. The dose-dependent effects of wg on dpp expression, ventralization of dorsal cells and transdetermination support a model in which wg functions as a morphogen in imaginal discs.

Animals↗
PubMed1996

Compartments, wingless and engrailed: patterning the ventral epidermis of Drosophila embryos.

Recent experiments on the wing disc of Drosophila have shown that cells at the interface between the anterior and posterior compartments drive pattern formation by becoming the source of a morphogen. Here we ask whether this model applies to the ventral embryonic epidermis. First, we show that interfaces between posterior (engrailed ON) and anterior (engrailed OFF) cells are required for pattern formation. Second, we provide evidence that Wingless could play the role of the morphogen, at least within part of the segmental pattern. We looked at the cuticular structures that develop after different levels of uniform Wingless activity are added back to unsegmented embryos (wingless- engrailed-). Because it is rich in landmarks, the T1 segment is a good region to analyse. There, we find that the cuticle formed depends on the amount of added Wingless activity. For example, a high concentration of Wingless gives the cuticle elements normally found near the top of the presumed gradient. Unsegmented embryos are much shorter than wild type. If Wingless activity is added in stripes, the embryos are longer than if it is added uniformly. We suggest that the Wingless gradient landscape affects the size of the embryo, so that steep slopes would allow cells to survive and divide, while an even distribution of morphogen would promote cell death. Supporting the hypothesis that Wingless acts as a morphogen, we find that these stripes affect, at a distance, the type of cuticle formed and the planar polarity of the cells.

Animals↗
PubMed1996

Expression domains of a zebrafish homologue of the Drosophila pair-rule gene hairy correspond to primordia of alternating somites.

her1 is a zebrafish cDNA encoding a bHLH protein with all features characteristic of members of the Drosophila HAIRY-E(SPL) family. During late gastrulation stages, her1 is expressed in the epibolic margin and in two distinct transverse bands of hypoblastic cells behind the epibolic front. After completion of epiboly, this pattern persists essentially unchanged through postgastrulation stages; the marginal domain is incorporated in the tail bud and, depending on the time point, either two or three paired bands of expressing cells are present within the paraxial presomitic mesoderm separated by regions devoid of transcripts. Labelling of cells within the her1 expression domains with fluorescein-dextran shows that the cells in the epibolic margin and the tail bud are not allocated to particular somites. However, allocation of cells to somites occurs between the marginal expression domain and the first expression band, anterior to it. Moreover, the her1 bands, and the intervening non-expressing zones, each represents the primordium of a somite. This expression pattern is highly reminiscent of that of Drosophila pair-rule genes. A possible participation of her1 in functions related to somite formation is discussed.

Animals↗
PubMed1997

Ftz-F1 is a cofactor in Ftz activation of the Drosophila engrailed gene.

The fushi tarazu pair-rule gene is required for the formation of alternating parasegmental boundaries in the Drosophila embryo. fushi tarazu encodes a homeodomain protein necessary for transcription of the engrailed gene in even-numbered parasegments. Here we report that, within an engrailed enhancer, adjacent and conserved binding sites for the Fushi tarazu protein and a cofactor are each necessary, and together sufficient, for transcriptional activation. Footprinting shows that the cofactor site can be bound specifically by Ftz-F1, a member of the nuclear receptor superfamily. Ftz-F1 and the Fushi tarazu homeodomain bind the sites with 4- to 8-fold cooperativity, suggesting that direct contact between the two proteins may contribute to target recognition. Even parasegmental reporter expression is dependent on Fushi tarazu and maternal Ftz-F1, suggesting that these two proteins are indeed the factors that act upon the two sites in embryos. The two adjacent binding sites are also required for continued activity of the engrailed enhancer after Fushi tarazu protein is no longer detectable, including the period when engrailed, and the enhancer, become dependent upon wingless. We also report the existence of a separate negative regulatory element that apparently responds to odd-skipped.

Animals↗
PubMed1997

Establishing neuroblast-specific gene expression in the Drosophila CNS: huckebein is activated by Wingless and Hedgehog and repressed by Engrailed and Gooseberry.

The Drosophila ventral neuroectoderm produces a stereotyped array of central nervous system precursors, called neuroblasts. Each neuroblast has a unique identity based on its position, pattern of gene expression and cell lineage. To understand how neuronal diversity is generated, we need to learn how neuroblast-specific gene expression is established, and how these genes control cell fate within neuroblast lineages. Here we address the first question: how is neuroblast-specific gene expression established? We focus on the huckebein gene, because it is expressed in a subset of neuroblasts and is required for aspects of neuronal and glial determination. We show that Huckebein is a nuclear protein first detected in small clusters of neuroectodermal cells and then in a subset of neuroblasts. The secreted Wingless and Hedgehog proteins activate huckebein expression in distinct but overlapping clusters of neuroectodermal cells and neuroblasts, whereas the nuclear Engrailed and Gooseberry proteins repress huckebein expression in specific regions of neuroectoderm or neuroblasts. Integration of these activation and repression inputs is required to establish the precise neuroectodermal pattern of huckebein, which is subsequently required for the development of specific neuroblast cell lineages.

Animals↗
PubMed1999

Repression of the wing vein development in Drosophila by the nuclear matrix protein plexus.

The wing of Drosophila is separated into several sectors by the wing veins. Vein primordia are specified by the positional information provided by hedgehog and decapentaplegic in the wing imaginal disc and express the key regulatory gene rhomboid. One model of this process is that boundaries of gene expression regulated by hedgehog or decapentaplegic provide reference points where rhomboid transcription is activated. We present an analysis of the gene plexus, whose loss of function causes an excess vein phenotype. Molecular cloning revealed that plexus encodes a novel 1990-amino acid protein with cysteine-rich motifs. Plexus protein was ubiquitously expressed and was tightly associated with the nuclear matrix. In plexus mutant wing imaginal discs, an anteroposterior positional coordinate was established normally as revealed by the wild-type pattern of spalt major and knirps expression. However, the expression of several vein-specific and intervein-specific genes was misregulated, as if they had neglected the positional coordinate. These results suggest that Plexus is an essential component of a global repressor of vein differentiation. Although Plexus protein was expressed in vein primordia of the wing disc, it does not appear to interfere with vein differentiation in the normal position. A genetic epistasis test between px and knirps suggests that plexus acts downstream of knirps. We propose that the vein differentiation takes place by inactivation of the plexus-mediated repression by prepattern genes such as knirps. Plexus may regulate transcription of vein-and intervein-specific genes by tethering transcriptional regulators to specific locations in the nucleus.

Amino Acid Sequence↗
PubMed1999

Hox genes differentially regulate Serrate to generate segment-specific structures.

Diversification of Drosophila segmental morphologies requires the functions of Hox transcription factors. However, there is little information describing pathways through which Hox activities effect the discrete cellular changes that diversify segmental architecture. We have identified the Drosophila signaling protein Serrate as the product of a Hox downstream gene that acts in many segments as a component of such pathways. In the embryonic epidermis, Serrate is required for morphogenesis of normal abdominal denticle belts and maxillary mouth hooks, both Hox-dependent structures. The Hox genes Ultrabithorax and abdominal-A are required to activate an early stripe of Serrate transcription in abdominal segments. In the abdominal epidermis, Serrate promotes denticle diversity by precisely localizing a single cell stripe of rhomboid expression, which generates a source of EGF signal that is not produced in thoracic epidermis. In the head, Deformed is required to activate Serrate transcription in the maxillary segment, where Serrate is required for normal mouth hook morphogenesis. However, Serrate does not require rhomboid function in the maxillary segment, suggesting that the Hox-Serrate pathway to segment-specific morphogenesis can be linked to more than one downstream function.

Animals↗
PubMed2001

Recognition and long-range interactions of a minimal nanos RNA localization signal element.

Localization of nanos (nos) mRNA to the germ plasm at the posterior pole of the Drosophila embryo is essential to activate nos translation and thereby generate abdominal segments. nos RNA localization is mediated by a large cis-acting localization signal composed of multiple, partially redundant elements within the nos 3' untranslated region. We identify a protein of approximately 75 kDa (p75) that interacts specifically with the nos +2' localization signal element. We show that the function of this element can be delimited to a 41 nucleotide domain that is conserved between D. melanogaster and D. virilis, and confers near wild-type localization when present in three copies. Two small mutations within this domain eliminate both +2' element localization function and p75 binding, consistent with a role for p75 in nos RNA localization. In the intact localization signal, the +2' element collaborates with adjacent localization elements. We show that different +2' element mutations not only abolish collaboration between the +2' and adjacent +1 element but also produce long-range deleterious effects on localization signal function. Our results suggest that higher order structural interactions within the localization signal, which requires factors such as p75, are necessary for association of nos mRNA with the germ plasm.

3' Untranslated Regions↗
PubMed1992

Cloning and expression of Drosophila HP1 homologs from a mealybug, Planococcus citri.

The mealybug chromosome cycle is one of the most dramatic examples of genomic imprinting known. In embryos that are to become male the entire paternal chromosome set becomes heterochromatic and inactive at the blastoderm stage, while the maternal set remains active and euchromatic. HP1 is a protein from Drosophila melanogaster, which binds preferentially to heterochromatin on polytene chromosomes and is likely to be a modifier of position effect variegation. This paper describes the isolation and sequencing of two cDNA clones encoding HP1 homologs from the mealybug, Planococcus citri. The protein product of the cDNA clone that was closer to HP1 in sequence was expressed as a fusion protein in Escherichia coli, and polyclonal rat antibodies were raised against it. Immunohistochemistry to mealybug squash preparations showed that this protein was a male-specific nuclear protein, but that it was not specifically associated with the heterochromatic set of chromosomes.

Amino Acid Sequence↗
PubMed1998

Species-specific action and distribution of tachykinin-related peptides in the foregut of the cockroaches Leucophaea maderae and Periplaneta americana.

Nine tachykinin-related peptides (TRPs) have been isolated from the brain and intestine of the cockroach Leucophaea maderae. In the present investigation, two of the nine TRPs, LemTRP 1 and 5, were tested for their ability to stimulate contractions in the foregut of the cockroaches L. maderae and Periplaneta americana in vitro. The two LemTRPs and the related locust peptide locustatachykinin I (LomTK I) induced contractions in the foregut of P. americana in a dose-dependent manner, but had no myostimulatory action in L. maderae. A half-maximal response for the LemTRPs and LomTK I was obtained at 5x10(-9)mol l-1. In both species, the neuropeptide proctolin stimulated foregut contractions. Using an antiserum to LomTK I, we demonstrated that in both species there are LomTK-like-immunoreactive (LomTK-LI) cell bodies and fibers within the ganglia and nerves of the stomatogastric nervous system. However, correlated with the species-specific action of the TRPs, we found efferent LomTK-LI nerve fibers supplying muscle fibers in the foregut of P. americana, but not in L. maderae. In both cockroach species, there is a rich supply of proctolin-immunoreactive fibers to the foregut muscle. Some of the LomTK-LI fibers supplying the P. americana foregut muscle contain co-localized proctolin immunoreactivity. These fibers appear to be derived from a large cell body in the frontal ganglion which also displayed co-localized immunoreactivities. Since TRP-containing neurons are restricted to the nerves and ganglia of the stomatogastric nervous system both in P. americana and L. maderae, TRPs may be involved in the control of foregut movements in both species, but in P. americana the control may be more complex with the additional peripherally projecting LomTK-LI neurons.

Animals↗
PubMed1995

Purification and cDNA cloning of an antifungal protein from the hemolymph of Holotrichia diomphalia larvae.

An antifungal protein (AFP), holotricin 3, was purified from the hemolymph of the coleopteran insect Holotrichia diomphalia larvae. Analysis of its cDNA showed that holotricin 3 is a novel Gly- and His-rich protein consisting of 84 amino acid residues. This protein was similar to AFP, an antifungal protein of Sarcophaga peregrina that was reported previously, in terms of molecular size and high content of Gly and His residues. However, no appreciable sequence similarity was detected between the two proteins.

Amino Acid Sequence↗