Precellular Embryo Research Articles

P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

Multifunctional RNA processing protein SRm160 induces apoptosis and regulates eye and genital development in Drosophila.

SRm160 is an SR-like protein implicated in multiple steps of RNA processing and nucleocytoplasmic export. Although its biochemical functions have been extensively described, its genetic interactions and potential participation in signaling pathways remain largely unknown, despite the fact that it is highly phosphorylated in both mammalian cells and Drosophila. To begin elucidating the functions of the protein in signaling and its potential role in developmental processes, we characterized mutant and overexpression SRm160 phenotypes in Drosophila and their interactions with the locus encoding the LAMMER protein kinase, Doa. SRm160 mutations are recessive lethal, while its overexpression generates phenotypes including roughened eyes and highly disorganized internal eye structure, which are due at least in part to aberrantly high levels of apoptosis. SRm160 is required for normal somatic sex determination, since its alleles strongly enhance a subtle sex transformation phenotype induced by Doa kinase alleles. Moreover, modification of SRm160 by DOA kinase appears to be necessary for its activity, since Doa alleles suppress phenotypes induced by SRm160 overexpression in the eye and enhance those in genital discs. Modification of SRm160 may occur through direct interaction because DOA kinase phosphorylates it in vitro. Remarkably, SRm160 protein was concentrated in the nuclei of precellular embryos but was very rapidly excluded from nuclei or degraded coincident with cellularization. Also of interest, transcripts are restricted almost exclusively to the developing nervous system in mature embryos.

Precision of Hunchback Expression in the Drosophila Embryo

Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to understand this process have focused on the classical Bicoid target enhancer located immediately upstream of the P2 promoter. However, hb is also regulated by a recently identified distal shadow enhancer as well as a neglected "stripe" enhancer, which mediates expression in both central and posterior regions of cellularizing embryos. Here, we employ BAC transgenesis and quantitative imaging methods to investigate the individual contributions of these different enhancers to the dynamic hb expression pattern. These studies reveal that the stripe enhancer is crucial for establishing the definitive border of the anterior Hb expression pattern, just beyond the initial border delineated by Bicoid. Removal of this enhancer impairs dynamic expansion of hb expression and results in variable cuticular defects in the mesothorax (T2) due to abnormal patterns of segmentation gene expression. The stripe enhancer is subject to extensive regulation by gap repressors, including Kruppel, Knirps, and Hb itself. We propose that this repression helps ensure precision of the anterior Hb border in response to variations in the Bicoid gradient.

Red Flour Beetle (Tribolium) Germline Transformation and Insertional Mutagenesis

INTRODUCTIONThe procedure for introducing transgenes into the Tribolium genome is similar to that for Drosophila and is of comparable efficiency. Purified transposon vector and helper plasmids are injected through the posterior pole of precellular embryos. The injected eggs are incubated under humidified conditions until they hatch. Adults resulting from injected eggs are mated inter se, and transformants are identified among their offspring with the help of visible genetic markers. The helper plasmids express the respective transposase under control of the Drosophila hsp70 promoter, but no heat shock is required. Transgenic animals are identified by fluorescent proteins that are expressed in the eye (and several other tissues including the central nervous system [CNS]) under the control of the artificial 3xP3 promoter. Among the fluorescent proteins enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), and dsRed, it is EGFP and EYFP that provide the strongest signals. An alternative marker system based on rescue of the white-eye mutation in the eye pigmentation gene vermilion makes scoring transgenic animals even easier. Using these dominant markers, transgenic lines tagged with different markers can be crossbred, and offspring carrying both constructs can be easily identified, which is useful for remobilization experiments and misexpression experiments using the Gal4/UAS system. This protocol provides a detailed method for the generation of transgenic lines, which also can be applied toward the generation of improved mutator and helper strains.

Troponin I and Tropomyosin regulate chromosomal stability and cell polarity

The Troponin-Tropomyosin (Tn-Tm) complex regulates muscle contraction through a series of Ca(2+)-dependent conformational changes that control actin-myosin interactions. Members of this complex in Drosophila include the actin-binding protein Troponin I (TnI), and two Tropomyosins (Tm1 and Tm2), which are thought to form heterodimers. We show here that pre-cellular embryos of TnI, Tm1 and Tm2 mutants exhibit abnormal nuclear divisions with frequent loss of chromosome fragments. During cellularization, apico-basal polarity is also disrupted as revealed by the defective location of Discs large (Dlg) and its ligand Rapsynoid (Raps; also known as Partner of Inscuteable, Pins). In agreement with these phenotypes in early development, on the basis of RT-PCR assays of unfertilized eggs and germ line mosaics of TnI mutants, we also show that TnI is part of the maternal deposit during oogenesis. In cultures of the S2 cell line, native TnI is immunodetected within the nucleus and immunoprecipitated from nuclear extracts. SUMOylation at an identified site is required for the nuclear translocation. These data illustrate, for the first time, a role for TnI in the nucleus and/or the cytoskeleton of non-muscle cells. We propose that the Tn-Tm complex plays a novel function as regulator of motor systems required to maintain nuclear integrity and apico-basal polarity during early Drosophila embryogenesis.

Dual regulation by the Hunchback gradient in the Drosophila embryo

The regulation of segmentation gene expression is investigated by computational modeling using quantitative expression data. Previous tissue culture assays and transgene analyses raised the possibility that Hunchback (Hb) might function as both an activator and repressor of transcription. At low concentrations, Hb activates gene expression, whereas at high concentrations it mediates repression. Under the same experimental conditions, transcription factors encoded by other gap genes appear to function as dedicated repressors. Models based on dual regulation suggest that the Hb gradient can be sufficient for establishing the initial Kruppel (Kr) expression pattern in central regions of the precellular embryo. The subsequent refinement of the Kr pattern depends on the combination of Hb and the Giant (Gt) repressor. The dual-regulation models developed for Kr also explain some of the properties of the even-skipped (eve) stripe 3+7 enhancer. Computational simulations suggest that repression results from the dimerization of Hb monomers on the DNA template.

Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array.

Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. Here, we attempt to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the approximately 100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units were identified, including two that contain known microRNAs, miR-1 and -9a. We discuss the potential functions of these recently identified genes and suggest that intronic enhancers are a common feature of the DV gene network.

Linear signaling in the Toll-Dorsal pathway of Drosophila: activated Pelle kinase specifies all threshold outputs of gene expression while the bHLH protein Twist specifies a subset.

Differential activation of the Toll receptor leads to the formation of a broad Dorsal nuclear gradient that specifies at least three patterning thresholds of gene activity along the dorsoventral axis of precellular embryos. We investigate the activities of the Pelle kinase and Twist basic helix-loop-helix (bHLH) transcription factor in transducing Toll signaling. Pelle functions downstream of Toll to release Dorsal from the Cactus inhibitor. Twist is an immediate-early gene that is activated upon entry of Dorsal into nuclei. Transgenes misexpressing Pelle and Twist were introduced into different mutant backgrounds and the patterning activities were visualized using various target genes that respond to different thresholds of Toll-Dorsal signaling. These studies suggest that an anteroposterior gradient of Pelle kinase activity is sufficient to generate all known Toll-Dorsal patterning thresholds and that Twist can function as a gradient morphogen to establish at least two distinct dorsoventral patterning thresholds. We discuss how the Dorsal gradient system can be modified during metazoan evolution and conclude that Dorsal-Twist interactions are distinct from the interplay between Bicoid and Hunchback, which pattern the anteroposterior axis.

DCtBP mediates transcriptional repression by Knirps, Krüppel and Snail in the Drosophila embryo.

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Previous studies have shown that two of the repressors, Hairy and Dorsal, recruit a common co-repressor protein, Groucho. Here we present evidence that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H. This motif is essential for the repression activity of these proteins in transgenic embryos. We propose that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression.

Maternally Expressed γTub37CD inDrosophilaIs Differentially Required for Female Meiosis and Embryonic Mitosis

We report functional analysis of γTub37CD, a maternally synthesized γ-tubulin that is highly expressed during oogenesis and utilized at centrosomes in precellular embryos. TwoγTub37CDmutants contained missense mutations that altered residues conserved in all γ-tubulins and α- and/or β-tubulins. A thirdγTub37CDmissense mutant identified a conserved motif unique to γ-tubulins. A fourthγTub37CDmutant contained a nonsense mutation and the corresponding premature stop codon generated a protein null allele. Immunofluorescence analysis of laid eggs and activated oocytes derived from the mutants revealed microtubules and meiotic spindles that were close to normal even in the absence of γTub37CD. Eggs lacking the maternal γ-tubulin were arrested in meiosis, indicative of a deficiency in activation. Analysis of meiosis within vitroactivation techniques showed that the cortical microtubule cytoskeleton of mature wild-type eggs was reorganized upon activation and expressed as transient assembly of cortical asters, and this cortical reorganization was altered inγTub37CDmutants. In precellular embryos of partial loss of function mutants, spindles were frequently abnormal and cell cycle progression was inhibited. Thus, γTub37CD functions differentially in female meiosis and in the early embryo; while involved in oocyte activation, it is apparently not required or plays a subtle role in formation of the female meiotic spindle which is acentriolar, but is essential for assembly of a discrete bipolar mitotic spindle which is directed by centrosomes organized about centrioles.

Sequence and expression of Drosophila myristoyl-CoA: protein N-myristoyl transferase: evidence for proteolytic processing and membrane localisation.

The enzyme N-myristoyl transferase transfers the 14 carbon fatty acid myristate to an N-terminal glycine residue in a small subset of cytoplasmic proteins. Many myristoyl proteins are components of cellular signalling pathways, some of which play important roles during embryonic development, for example protein kinase A. Thus, the function of N-myristoyl transferase is probably essential for embryogenesis and it is of some interest to study the enzyme in an organism with well understood developmental biology. In this paper we report the purification of a processed form of the Drosophila enzyme from peripheral membrane fractions of embryos by affinity chromatography to a protein containing leucine rich repeats. We have also isolated the Drosophila N-myristoyl transferase gene and determined its nucleotide sequence. The predicted amino acid sequence of the Drosophila enzyme is closely related to that of mammalian and fungal N-myristoyl transferases and residues essential for enzyme function are conserved. Our findings indicate that a fraction of Drosophila NMT is bound to the membrane and they are consistent with recent results for the human enzyme. We suggest that N-myristoyl transferase may be recruited to the membrane as part of a translational complex, perhaps by binding to p34 ribosome binding protein, a leucine rich repeat receptor of the microsomal membranes. We have also studied the expression pattern of the gene in the embryo by northern blot analysis and in situ hybridization. The transcripts appear to be uniformly distributed in the pre-cellular embryo but at later stages the RNA is barely detectable with these methods. However, from about stage 14, high levels of transcript are detected in a small number of randomly distributed cells of the central and peripheral nervous system.

Threshold responses to the dorsal regulatory gradient and the subdivision of primary tissue territories in the Drosophila embryo

Dorsoventral patterning in Drosophila is initiated by the maternal regulatory factor dorsal (dl), which is a member of the Rel family of transcription factors. dl functions as a transcriptional activator and repressor to establish different territories of gene expression in the precellular embryo. Differential regulation of dl target genes may be essential for subdividing each tissue territory (the presumptive mesoderm, neuroectoderm, and dorsal ectoderm) into multiple cell types in older embryos. Different paterns of snail (sna) and decapentaplegic (dpp) expression help define the limits of inductive interactions between the mesoderm and dorsal ectoderm after gastrulation. Similarly, the differential regulation of short gastrulation (sog) and dpp may be decisive in the initial subdivision of the dorsal ectoderm, whereas different limits of gene expression within the neuroectoderm might provide the basis for the subsequent subdivision of this tissue into ventral and lateral regions.

Spatial regulation of the gap gene giant during Drosophila development

We describe the regulated expression of the segmentation gene giant (gt) during early embryogenesis. The gt protein is expressed in two broad gradients in precellular embryos, one in anterior regions and the other in posterior regions. Double immunolocalization studies show that the gt patterns overlap with protein gradients specified by the gap genes hunchback (hb) and knirps (kni). Analysis of all known gap mutants, as well as mutations that disrupt each of the maternal organizing centers, indicate that maternal factors are responsible for initiating gt expression, while gap genes participate in the subsequent refinement of the pattern. The maternal morphogen bicoid (bcd) initiates the anterior gt pattern, while nanos (nos) plays a role in the posterior pattern. Gene dosage studies indicate that different thresholds of the bcd gradient might trigger hb and gt expression, resulting in overlapping but noncoincident patterns of expression. We also present evidence that different concentrations of hb protein are instructive in defining the limits of kni and gt expression within the presumptive abdomen. These results suggest that gt is a bona fide gap gene, which acts with hb, Krüppel and kni to initiate striped patterns of gene expression in the early embryo.

Spatial regulation of zerknüllt: a dorsal-ventral patterning gene in Drosophila.

zerknüllt (zen) is one of approximately 10 zygotically active genes that control the differentiation of the dorsal-ventral (D/V) pattern during early embryogenesis in Drosophila. Past genetic analyses suggest that maternal factors repress the expression of zen in ventral regions, thereby restricting zen products to dorsal and dorsal-lateral regions of precellular embryos. Subsequent interactions with other zygotic D/V regulatory genes refine the zen pattern, restricting expression to the dorsal-most ectoderm. Here we describe the use of zen promoter fusions and P-element transformation to identify cis elements that are responsible for the complex spatial pattern of zen expression. The zen promoter shows a two-tier organization: Distal sequences mediate its initial response to maternal factors, whereas proximal sequences are responsible for the refinement of the pattern in older embryos. The distal regulatory element has the property of a silencer (or anti-enhancer) element and can act over a distance to repress ventral expression of a heterologous promoter. Also, we discuss evidence that proximal promoter sequences interact with factors that may be modulated by a cell-cell communication pathway.

The engrailed locus of D. melanogaster provides an essential zygotic function in precellular embryos.

Early embryonic development in Drosophila depends on genes expressed during oogenesis or after zygote formation. We show that the engrailed gene is needed for the processes that organize the embryo during the nuclear divisions that precede cellularization. During the precellular blastoderm stages engrailed mutant embryos show several notable anomalies: the pole cells form at a position slightly displaced from the posterior pole; yolk nuclei continue to divide after the tenth nuclear division cycle, when wild-type yolk nuclei have stopped dividing mitotically; and somatic nuclei are not positioned uniformly along the embryo periphery and do not undergo mitotic divisions in regular waves. This early requirement for engrailed does not appear to be a maternal function, and only genetically engrailed embryos displayed these precellular phenotypes. Synthesis of a 2.7 kb poly(A)+ transcript of the engrailed region was found in precellular embryos.