Squid hnRNP protein promotes apical cytoplasmic transport and localization of Drosophila pair-rule transcripts.
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Drosophila melanogaster pair-rule segmentation gene transcripts localize apically of nuclei in blastoderm embryos. This might occur by asymmetric (vectorial) export from one side of the nucleus or by transport within the cytoplasm. We have followed fluorescently labeled pair-rule transcripts postinjection into Drosophila embryos. Naked, microinjected fushi tarazu (ftz) transcripts do not localize in blastoderm embryos, indicating that cytoplasmic mechanisms alone are insufficient for apical targeting. However, prior exposure of ftz to Drosophila or human embryonic nuclear extract leads to rapid, specific, microtubule-dependent transport, arguing against vectorial export. We present evidence that ftz transcript localization involves the Squid (Hrp40) hnRNP protein and that the activity of hnRNP proteins in promoting transcript localization is evolutionarily conserved. We propose that cytoplasmic localization machineries recognize transcripts in the context of nuclear partner proteins. (+info)
bloated tubules (blot) encodes a Drosophila member of the neurotransmitter transporter family required for organisation of the apical cytocortex.
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We have identified a novel member of the vertebrate sodium- and chloride-dependent neurotransmitter symporter family from Drosophila melanogaster. This gene, named bloated tubules (blot), shows significant sequence similarity to a subgroup of vertebrate orphan transporters. blot transcripts are maternally supplied and during embryogenesis exhibit a complex and dynamic pattern in a subset of ectodermally derived epithelia, notably in the Malpighian tubules, and in the nervous system. Animals mutant for this gene are larval lethals, in which the Malpighian tubule cells are distended with an enlarged and disorganised apical surface. Embryos lacking the maternal component of blot expression die during early stages of development. They show an inability to form actin filaments in the apical cortex, resulting in impaired syncytial nuclear divisions, severe defects in the organisation of the cortical cytoskeleton, and a failure to cellularise. For the first time, a neurotransmitter transporter-like protein has been implicated in a function outside the nervous system. The isolation of blot thus provides the basis for an analysis of the relationship between the function of this putative transporter and epithelial morphogenesis. (+info)
Dynamic rearrangement of the spectrin membrane skeleton during the generation of epithelial polarity in Drosophila.
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The origin of epithelial cell polarity during development is a fundamental problem in cell biology. Central to this process is the establishment of asymmetric membrane domains that will ultimately form the apical and basolateral surfaces. The spectrin-based membrane skeleton has long been thought to participate in the generation of this asymmetry. Drosophila melanogaster contains two known (beta)-spectrin isoforms: a conventional (beta)-spectrin chain, and the novel isoform (beta)(Heavy)-spectrin. These two proteins are restricted to the basolateral and apical membrane domains, respectively. To assay for the emergence of membrane asymmetry, we have characterized the distribution of these two (beta)-spectrins during the formation of the primary epithelium in the fly embryo. Our results show that the syncytial embryo contains a maternally established apical membrane skeleton containing (beta)(Heavy)-spectrin into which the basolateral (beta)-spectrin membrane skeleton is added. We have called this process basolateral interpolation. Although basolateral membrane skeleton addition begins during cellularization, it does not become fully established until the formation of a mature zonula adherens at mid to late gastrulation. The behavior of (beta)-spectrin is consistent with a primary role in establishing and/or maintaining the basolateral domain while the behavior of (beta)(Heavy)-spectrin suggests that its primary role is associated with a specialized DE-cadherin complex associated with the furrow canals and with the maturation of the zonula adherens. Thus, the apical spectrin membrane skeleton appears to play a distinct rather than analogous role to the basolateral spectrin membrane skeleton, during the emergence of cell polarity. We find that there are several parallels between our observations and previous studies on the establishment of primary epithelial polarity in vertebrates, suggesting that basolateral interpolation of the membrane skeleton may be a common mechanism in many organisms. (+info)
Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.
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Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin. (+info)
Mutations in centrosomin reveal requirements for centrosomal function during early Drosophila embryogenesis.
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BACKGROUND: Although centrosomes serve as the primary organizing centers for the microtubule-based cytoskeleton in animal cells, various studies question the requirements for these organelles during the formation of microtubule arrays and execution of microtubule-dependent processes. Using a genetic approach to interfere with centrosomal function, we present an assessment of this issue, in the context of early embryogenesis of the fruit fly Drosophila melanogaster. RESULTS: We identified mutant alleles of the centrosomin (cnn) locus, which encodes a core component of centrosomes in Drosophila. The cnn mutant flies were viable but sterile. The normal course of early embryonic development was arrested in all progeny of cnn mutant females. Our analysis identified a failure to form functional centrosomes and spindle poles as the primary mutant phenotype of cnn embryos. Various aspects of early development that are dependent on cytoskeletal control were disrupted in cnn mutant embryos. In particular, structural rearrangements of cortical microfilaments were strongly dependent on proper centrosomal function. CONCLUSIONS: Centrosomin is an essential core component of early embryonic centrosomes in Drosophila. Microtubule-dependent events of early embryogenesis display differential requirements for centrosomal function. (+info)
Ubiquitous expression of a Drosophila adenomatous polyposis coli homolog and its localization in cortical actin caps.
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We report the expression pattern of a new adenomatous polyposis coli (APC) homolog called E-APC during Drosophila development. E-APC protein is expressed in all embryonic and larval cells we have examined. In the early blastoderm embryo, we see a striking concentration of E-APC in the cortical actin caps. Microtubules are closely associated with these caps. Since human APC has been reported to bind to microtubules, we investigated whether the cortical E-APC co-localizes with tubulin. However, this was not the case, implying that the putative tubulin-binding property of human APC is not well conserved. (+info)
Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node.
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Invariant patterning of left-right asymmetry during embryogenesis depends upon a cascade of inductive and repressive interactions between asymmetrically expressed genes. Different cascades of asymmetric genes distinguish the left and right sides of the embryo and are maintained by a midline barrier. As such, the left and right sides of an embryo can be viewed as distinct and autonomous fields. Here we describe a series of experiments that indicate that the initiation of these programs requires communication between the two sides of the blastoderm. When deprived of either the left or the right lateral halves of the blastoderm, embryos are incapable of patterning normal left-right gene expression at Hensen's node. Not only are both flanks required, suggesting that there is no single signaling source for LR pattern, but the blastoderm must be intact. These results are consistent with our previously proposed model in which the orientation of LR asymmetry in the frog, Xenopus laevis, depends on large-scale partitioning of LR determinants through intercellular gap junction channels (M. Levin and M. Mercola (1998) Developmental Biology 203, 90-105). Here we evaluate whether gap junctional communication is required for the LR asymmetry in the chick, where it is possible to order early events relative to the well-characterized left and right hierarchies of gene expression. Treatment of cultured chick embryos with lindane, which diminishes gap junctional communication, frequently unbiased normal LR asymmetry of Shh and Nodal gene expression, causing the normally left-sided program to be recapitulated symmetrically on the right side of the embryo. A survey of early expression of connexin mRNAs revealed that Cx43 is present throughout the blastoderm at Hamburger-Hamilton stage 2-3, prior to known asymmetric gene expression. Application of antisense oligodeoxynucleotides or blocking antibody to cultured embryos also resulted in bilateral expression of Shh and Nodal transcripts. Importantly, the node and primitive streak at these stages lack Cx43 mRNA. This result, together with the requirement for an intact blastoderm, suggests that the path of communication through gap junction channels circumvents the node and streak. We propose that left-right information is transferred unidirectionally throughout the epiblast by gap junction channels in order to pattern left-sided Shh expression at Hensen's node. (+info)
Flik, a chick follistatin-related gene, functions in gastrular dorsalisation/neural induction and in subsequent maintenance of midline Sonic hedgehog signalling.
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We have targetted the chick gene Flik with antisense oligodeoxynucleotide treatment at gastrular stages, when it is expressed in organiser-derived structures of the midline (K. Patel et al., 1996, Dev. Biol. 178, 327-342). A specific syndrome of deficient axial patterning and holoprosencephaly is produced. Most aspects of this syndrome can be understood as due to attenuation of dorsalising and neural-inducing signals during gastrulation, followed by failure to maintain the later signals from chordamesoderm/neural midline that pattern the mesodermal and neural cross sections during subsequent stages. Anatomical effects are first apparent at early neurula stages and correspond with what might be expected from a reduced counteraction of the ventralising Bone morphogenetic protein (BMP) pathway at the earlier stages, coupled with inadequate Sonic hedgehog (Shh) signalling subsequently. Delay in the clearing of BMP-4 RNA expression from the presumptive neural region at gastrulation is indeed seen, though chordin RNA expression within organiser derivatives remains normal. Subsequently, specific attenuation of chordamesoderm and neural midline Shh expression is observed. Brief preincubation of stage 4 chick blastoderms in supernatant from Xenopus oocytes that have been injected with Flik RNA prolongs and enhances the competence of their peripheral epiblast to respond to neural inductive signals from grafted Hensen's nodes. This effect specifically mimics that recently observed using microg/ml solutions of recombinant Follistatin (D. J. Connolly et al., 1999, Int. J. Dev. Biol., in press), further suggesting that Flik protein might act in vivo by somehow modulating activity of signalling pathways through BMP or other TGFbeta-related ligands. We discuss the significance of the observations in relation to recent ideas about neural induction, about possible redundancy in gene action, and about subsequent patterning of the axial cross section, suggesting that a Flik function in autocrine/paracrine maintenance of later midline Shh signalling represents a role of the gene separate from that in primary dorsalisation/neural induction. (+info)