SILAC-based quantitative proteomic analysis of Drosophila gastrula stage embryos mutant for fibroblast growth factor signalling

Quantitative proteomic analyses in combination with genetics provide powerful tools in developmental cell signalling research. Drosophila melanogaster is one of the most widely used genetic models for studying development and disease. Here we combined quantitative proteomics with genetic selection to determine changes in the proteome upon depletion of Heartless (Htl) Fibroblast-Growth Factor (FGF) receptor signalling in Drosophila embryos at the gastrula stage. We present a robust, single generation SILAC (stable isotope labelling with amino acids in cell culture) protocol for labelling proteins in early embryos. For the selection of homozygously mutant embryos at the pre-gastrula stage, we developed an independent genetic marker. Our analyses detected quantitative changes in the global proteome of htl mutant embryos during gastrulation. We identified distinct classes of downregulated and upregulated proteins, and network analyses indicate functionally related groups of proteins in each class. In addition, we identified changes in the abundance of phosphopeptides. In summary, our quantitative proteomic analysis reveals global changes in metabolic, nucleoplasmic, cytoskeletal and transport proteins in htl mutant embryos

extradenticle determines segmental identities throughout Drosophila development

extradenticle (exd) and the homeotic selector proteins together establish segmental identities by coordinately regulating the expression of downstream target genes. The inappropriate expression of these targets in exd mutant embryos results in homeotic transformations and aberrant morphogenesis. Here we examine the role of exd in adult development by using genetic mosaics and a hypomorphic exd allele caused by a point mutation in the homeodomain. exd continues to be essential for the specification of segmental identities, consistent with a continuing requirement for exd as cofactor of the homeotic selector proteins. Loss of exd results in the homeotic transformation of abdominal segments to an A5 or A6 segmental identity, the antenna and arista to leg, and the head capsule to dorsal thorax or notum. Proximal leg structures are particularly sensitive to the loss of exd, although exd does not affect the allocation of proximal positional values of the leg imaginal disc. Using heat-shocks to induce expression of a hsp70-exd fusion gene, we show that, in contrast to the homeotic selector genes, ubiquitously high levels of exd expression do not cause pattern abnormalities or segmental transformations

A Small Genomic Region Containing Several Loci Required for Gastrulation in Drosophila

Genetic screens in Drosophila designed to search for loci involved in gastrulation have identified four regions of the genome that are required zygotically for the formation of the ventral furrow. For three of these, the genes responsible for the mutant phenotypes have been found. We now describe a genetic characterization of the fourth region, which encompasses the cytogenetic interval 24C3-25B, and the mapping of genes involved in gastrulation in this region. We have determined the precise breakpoints of several existing deficiencies and have generated new deficiencies. Our results show that the region contains at least three different loci associated with gastrulation effects. One maternal effect gene involved in ventral furrow formation maps at 24F but could not be identified. For a second maternal effect gene which is required for germ band extension, we identify a candidate gene, CG31660, which encodes a G protein coupled receptor. Finally, one gene acts zygotically in ventral furrow formation and we identify it as Traf4

Estimating the age of Calliphora vicina eggs (Diptera: Calliphoridae): determination of embryonic morphological landmarks and preservation of egg samples

ORCID No. 0000-0002-8917-9646© The Author(s) 2016. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The attached file is the published version of the article

In Vivo Monitoring of mRNA Movement in Drosophila Body Wall Muscle Cells Reveals the Presence of Myofiber Domains

Background: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. Methodology/Principal Findings: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. Conclusions/Significance: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cellbase

Functional dissection of the Drosophila Kallmann's syndrome protein DmKal-1

BACKGROUND: Anosmin-1, the protein implicated in the X-linked Kallmann's syndrome, plays a role in axon outgrowth and branching but also in epithelial morphogenesis. The molecular mechanism of its action is, however, widely unknown. Anosmin-1 is an extracellular protein which contains a cysteine-rich region, a whey acidic protein (WAP) domain homologous to some serine protease inhibitors, and four fibronectin-like type III (FnIII) repeats. Drosophila melanogaster Kal-1 (DmKal-1) has the same protein structure with minor differences, the most important of which is the presence of only two FnIII repeats and a C-terminal region showing a low similarity with the third and the fourth human FnIII repeats. We present a structure-function analysis of the different DmKal-1 domains, including a predicted heparan-sulfate binding site. RESULTS: This study was performed overexpressing wild type DmKal-1 and a series of deletion and point mutation proteins in two different tissues: the cephalopharyngeal skeleton of the embryo and the wing disc. The overexpression of DmKal-1 in the cephalopharyngeal skeleton induced dosage-sensitive structural defects, and we used these phenotypes to perform a structure-function dissection of the protein domains. The reproduction of two deletions found in Kallmann's Syndrome patients determined a complete loss of function, whereas point mutations induced only minor alterations in the activity of the protein. Overexpression of the mutant proteins in the wing disc reveals that the functional relevance of the different DmKal-1 domains is dependent on the extracellular context. CONCLUSION: We suggest that the role played by the various protein domains differs in different extracellular contexts. This might explain why the same mutation analyzed in different tissues or in different cell culture lines often gives opposite phenotypes. These analyses also suggest that the FnIII repeats have a main and specific role, while the WAP domain might have only a modulator role, strictly connected to that of the fibronectins

Mutations in the Catalytic Loop HRD Motif Alter the Activity and Function of Drosophila Src64

The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele

The Formation of the Bicoid Morphogen Gradient Requires Protein Movement from Anteriorly Localized mRNA

New quantitative data show that the Bicoid morphogen gradient is generated from a dynamic localized source and that protein gradient formation requires protein movement along the anterior-posterior axis

STAT Is an Essential Activator of the Zygotic Genome in the Early Drosophila Embryo

In many organisms, transcription of the zygotic genome begins during the maternal-to-zygotic transition (MZT), which is characterized by a dramatic increase in global transcriptional activities and coincides with embryonic stem cell differentiation. In Drosophila, it has been shown that maternal morphogen gradients and ubiquitously distributed general transcription factors may cooperate to upregulate zygotic genes that are essential for pattern formation in the early embryo. Here, we show that Drosophila STAT (STAT92E) functions as a general transcription factor that, together with the transcription factor Zelda, induces transcription of a large number of early-transcribed zygotic genes during the MZT. STAT92E is present in the early embryo as a maternal product and is active around the MZT. DNA–binding motifs for STAT and Zelda are highly enriched in promoters of early zygotic genes but not in housekeeping genes. Loss of Stat92E in the early embryo, similarly to loss of zelda, preferentially down-regulates early zygotic genes important for pattern formation. We further show that STAT92E and Zelda synergistically regulate transcription. We conclude that STAT92E, in conjunction with Zelda, plays an important role in transcription of the zygotic genome at the onset of embryonic development

A Modifier Screen for Bazooka/PAR-3 Interacting Genes in the Drosophila Embryo Epithelium

The development and homeostasis of multicellular organisms depends on sheets of epithelial cells. Bazooka (Baz; PAR-3) localizes to the apical circumference of epithelial cells and is a key hub in the protein interaction network regulating epithelial structure. We sought to identify additional proteins that function with Baz to regulate epithelial structure in the Drosophila embryo.The baz zygotic mutant cuticle phenotype could be dominantly enhanced by loss of known interaction partners. To identify additional enhancers, we screened molecularly defined chromosome 2 and 3 deficiencies. 37 deficiencies acted as strong dominant enhancers. Using deficiency mapping, bioinformatics, and available single gene mutations, we identified 17 interacting genes encoding known and predicted polarity, cytoskeletal, transmembrane, trafficking and signaling proteins. For each gene, their loss of function enhanced adherens junction defects in zygotic baz mutants during early embryogenesis. To further evaluate involvement in epithelial polarity, we generated GFP fusion proteins for 15 of the genes which had not been found to localize to the apical domain previously. We found that GFP fusion proteins for Drosophila ASAP, Arf79F, CG11210, Septin 5 and Sds22 could be recruited to the apical circumference of epithelial cells. Nine of the other proteins showed various intracellular distributions, and one was not detected.Our enhancer screen identified 17 genes that function with Baz to regulate epithelial structure in the Drosophila embryo. Our secondary localization screen indicated that some of the proteins may affect epithelial cell polarity by acting at the apical cell cortex while others may act through intracellular processes. For 13 of the 17 genes, this is the first report of a link to baz or the regulation of epithelial structure