Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

AbstractDuring Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction

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

A Short Receptor Downregulates JAK/STAT Signalling to Control the Drosophila Cellular Immune Response

Regulation of JAK/STAT signalling by a short, nonsignalling receptor in Drosophila modulates response to specific immune challenges such as parasitoid infestations

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

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

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

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

Stability and Nuclear Dynamics of the Bicoid Morphogen Gradient

SummaryPatterning in multicellular organisms results from spatial gradients in morphogen concentration, but the dynamics of these gradients remain largely unexplored. We characterize, through in vivo optical imaging, the development and stability of the Bicoid morphogen gradient in Drosophila embryos that express a Bicoid-eGFP fusion protein. The gradient is established rapidly (∼1 hr after fertilization), with nuclear Bicoid concentration rising and falling during mitosis. Interphase levels result from a rapid equilibrium between Bicoid uptake and removal. Initial interphase concentration in nuclei in successive cycles is constant (±10%), demonstrating a form of gradient stability, but it subsequently decays by approximately 30%. Both direct photobleaching measurements and indirect estimates of Bicoid-eGFP diffusion constants (D ≤ 1 μm2/s) provide a consistent picture of Bicoid transport on short (∼min) time scales but challenge traditional models of long-range gradient formation. A new model is presented emphasizing the possible role of nuclear dynamics in shaping and scaling the gradient