Fly Embryos Research Articles

Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.

SPEX2: automated concise extraction of spatial gene expression patterns from Fly embryo ISH images

Motivation: Microarray profiling of mRNA abundance is often ill suited for temporal–spatial analysis of gene expressions in multicellular organisms such as Drosophila. Recent progress in image-based genome-scale profiling of whole-body mRNA patterns via in situ hybridization (ISH) calls for development of accurate and automatic image analysis systems to facilitate efficient mining of complex temporal–spatial mRNA patterns, which will be essential for functional genomics and network inference in higher organisms.Results: We present SPEX2, an automatic system for embryonic ISH image processing, which can extract, transform, compare, classify and cluster spatial gene expression patterns in Drosophila embryos. Our pipeline for gene expression pattern extraction outputs the precise spatial locations and strengths of the gene expression. We performed experiments on the largest publicly available collection of Drosophila ISH images, and show that our method achieves excellent performance in automatic image annotation, and also finds clusters that are significantly enriched, both for gene ontology functional annotations, and for annotation terms from a controlled vocabulary used by human curators to describe these images.Availability: Software will be available at http://www.sailing.cs.cmu.edu/Contact: epxing@cs.cmu.eduSupplementary information: Supplementary data are avilable at Bioinformatics online.

Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/ chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum ( Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.

Protein Complex of Drosophila ATRX/XNP and HP1a Is Required for the Formation of Pericentric Beta-heterochromatin in Vivo

ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked alpha-thalassemia mental retardation (ATR-X) syndrome. Here we perform biochemical and genetic analyses of the Drosophila melanogaster ortholog of ATRX. The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multisubunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity.

CenH3/CID Incorporation Is Not Dependent on the Chromatin Assembly Factor CHD1 in Drosophila

CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3CENP-A, which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3CID in Drosophila. In contrast to the findings in fission yeast and vertebrate cells, our evidence clearly argues against such a role for CHD1 in Drosophila. CHD1 does not localize to centromeres in either S2 cells or developing fly embryos. Down-regulation of CHD1 in S2 cells by RNAi reveals unchanged levels of CenH3CID at the centromeres. Most notably, ablation of functional CHD1 in Chd1 mutant fly embryos does not interfere with centromere and kinetochore assembly, as the levels and localization of CenH3CID, CENP-C and BubR1 in the mutant embryos remain similar to those seen in wild-type embryos. These results indicate that Drosophila CHD1 has no direct function in the incorporation of the centromeric H3 variant CenH3CID into chromatin. Therefore, centromeric chromatin assembly may involve different mechanisms in different organisms.

Analysis of integrin turnover in fly myotendinous junctions

Transient (short-term) cell adhesion underlies dynamic processes such as cell migration, whereas stable (long-term) cell adhesion maintains tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is not known whether turnover is also required for maintenance of long-term adhesion. We used fluorescence recovery after photobleaching to analyze the dynamics of an integrin adhesion complex (IAC) in a model of long-term cell-ECM adhesion, myotendinous junctions (MTJs), in fly embryos and larvae. We found that the IAC undergoes turnover in MTJs and that this process is mediated by clathrin-dependent endocytosis. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover. Also, altering Rab5 activity weakened MTJs, resulting in muscle defects. In addition, growth of MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover. We propose that IAC turnover is tightly regulated in long-term cell-ECM adhesions to allow normal tissue growth and maintenance.

Neighbors limit cell renovations

![Figure][1] Reducing the number of adherens junctions causes epithelial tissue to rip (right), though the cells remain linked by strands of membrane. No cell is an island, especially when it’s embedded in an epithelial layer of a fruit fly embryo. As [Martin et al.][2] reveal, forces

Analysis of integrin turnover in fly myotendinous junctions

Transient (short-term) cell adhesion underlies dynamic processes such as cell migration, whereas stable (long-term) cell adhesion maintains tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is not known whether turnover is also required for maintenance of long-term adhesion. We used fluorescence recovery after photobleaching to analyze the dynamics of an integrin adhesion complex (IAC) in a model of long-term cell-ECM adhesion, myotendinous junctions (MTJs), in fly embryos and larvae. We found that the IAC undergoes turnover in MTJs and that this process is mediated by clathrin-dependent endocytosis. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover. Also, altering Rab5 activity weakened MTJs, resulting in muscle defects. In addition, growth of MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover. We propose that IAC turnover is tightly regulated in long-term cell-ECM adhesions to allow normal tissue growth and maintenance.

Wingless Enforces Strict Segregation

Signaling by the secreted morphogen wingless controls deposition of a myosin II–containing barrier that prevents cell mixing at segmental boundaries of the fly embryo.

Developmental Evolution: How Beetles Evolved Their Shields

Beetle forewings are modified into hardened structures called elytra. A recent study indicates that the evolution of elytra involved co-opting genes for exoskeleton formation into the wing development gene network of beetles on at least three separate occasions.

Light Sheet Microscopy Optimized for Depth Penetration to Study Embryogenesis

Fluorescence light sheet microscopy (FLSM) has gained widespread recognition in recent years, due to its distinct advantages for the 3-dimensional (3D) imaging of living biological samples. FLSM uses a planar sheet of light to illuminate a sample, generating fluorescence over an optical section of the sample that is collected by a wide-field microscope camera oriented orthogonal to the light sheet. The orthogonal geometry between the illumination and detection pathways enables massive parallelization in both illumination and detection; futhermore, it permits optical and physical access to samples (3D cell cultures or whole embryos) in ways that are impossible in the collinear geometry of standard microscopes. Because of these features, FLSM significantly outperforms standard laser-scanning confocal microscopy in imaging speed, phototoxicity, and signal to noise in many imaging applications. An important aspect of any 3D imaging technique is its imaging depth limit (how deep into a sample useful information can be collected). In this respect, standard FLSM fares only slightly better than confocal microscopy. To overcome this hurdle, we have optimized FLSM for imaging of live thick samples by minimizing the degradation of the light sheet due to scattering, while preserving acceptable axial resolution. Using this approach we have imaged whole, live fruit fly embryos and zebrafish embryos. We achieve higher depth penetration than standard FLSM, while maintaining sub-cellular resolution, at imaging speed of about ten times faster than standard confocal microscopy.

Combining Laser Microsurgery and Finite Element Modeling to Assess Cell-Level Epithelial Mechanics

Laser microsurgery and finite element modeling are used to determine the cell-level mechanics of the amnioserosa—a morphogenetically crucial epithelium on the dorsal surface of fruit fly embryos (Drosophila melanogaster). In the experiments, a tightly focused laser ablates a subcellular hole (1 μm in diameter) that passes clean through the epithelium. The surrounding cells recoil from the wound site with a large range of initial recoil velocities. These depend on the embryo's developmental stage and the subcellular wound site. The initial recoil (up to 0.1 s) is well reproduced by a base finite element model, which assumes a uniform effective viscosity inside the cells, a constant tension along each cell-cell boundary, and a large, potentially anisotropic, far-field stress—one that far exceeds the stress equivalent of the cell-edge tensions. After 0.1 s, the experimental recoils slow dramatically. This observation can be reproduced by adding viscoelastic rods along cell edges or as a fine prestressed mesh parallel to the apical and basal membranes of the cell. The mesh also reproduces a number of double-wounding experiments in which successive holes are drilled in a single cell.

Development and fertility studies on post-bio-electrosprayed Drosophila melanogaster embryos

Bio-electrosprays (BESs) provide a means of precisely manipulating cells and thus have the potential for many clinical uses such as the generation of artificial tissuesorgans. Previously we tested the biological safety of this technology with a variety of living cells and also embryos from the vertebrate model organisms Danio rerio (zebrafish) and Xenopus tropicalis (frog). However, the viability and fertility of the treated embryos could not be fully assessed due to animal licensing laws. Here we assay the viability and fertility of Drosophila melanogaster (fruit fly) embryos in conjunction with the bio-electrospray procedure. Bio-electrosprayed Drosophila embryos developed into fully fertile adult flies that were indistinguishable from wild-type. Thus, we demonstrate that the bio-electrospray procedure does not induce genetic or physical damage that significantly affects the development or fertility of a multicellular organism. This study along with our previous investigations demonstrates the potential of this approach to be developed for the precise manipulation of sensitive biological materials.

Stripe formation in the early fly embryo: principles, models, and networks

Early development of animal embryos begins from spatially distributed products of gene expression, i.e., gradients. While maternal and early zygotic genes form broad and/or terminal gradients, their direct targets appear later on as relatively narrow stripes, which foreshadow presumptive germ layers or future segments. Evidently, stripe expression of the zygotic genes is among the key mechanisms of embryo patterning. In this paper, known qualitative and quantitative models for the stripe formation are considered on the example of early embryogenesis of Drosophila. The current model analysis emphasizes the role of spatial information flow in development. Discussion is given on frequent network motifs, pointing to spatial stripe formation solutions.

Spatial and temporal expression of dADAR mRNA and protein isoforms during embryogenesis in Drosophila melanogaster

Adenosine Deaminases Acting on RNA (ADARs) function to co-transcriptionally deaminate specific (or non-specific) adenosines to inosines within pre-mRNAs, using double-stranded RNAs as substrate. In both Drosophila and mammals, the best-studied ADAR functions are to catalyze specific nucleotide conversions within mRNAs encoding various ligand- or voltage-gated ion channel proteins within the adult brain. In contrast, ADARs within developing fly embryos have scarcely been studied, in part because they contain little or no editase activity, raising interesting questions as to their functional significance. Quantitative RT-PCR shows that two major developmentally regulated mRNA isoform classes are produced (full-length and truncated), which arise by alternative splicing and also alternative 3′-end formation. In situ localization of specific dADAR mRNA isoforms during embryogenesis reveals that the full-length class is found primarily within the developing germ band and central nervous system, whereas the truncated isoform is mostly located in gut endothelium. Developmental Western immunoblots show that both isoform classes are expressed into protein during embryogenesis. Both the rnp-4 f 5′-UTR unspliced isoform and the full-length dADAR mRNA primarily localize in the embryonic germ band and subsequently throughout the developing central nervous system. Previous studies have shown that some rnp-4f pre-mRNAs are extensively edited by dADAR in the adult brain. Computer predictions suggest that intron–exon pairing promotes formation of an evolutionarily conserved secondary structure in the rnp-4 f 5′-UTR, forming a 177-nt RNA duplex resembling an editing site complementary sequence, which is shown to be associated with splicing failure and to generate a long isoform. Taken together, these observations led us to explore the possibility that interaction between rnp-4 f pre-mRNA and nuclear full-length dADAR protein may occur during embryogenesis. In dADAR null mutants, rnp-4 f 5′-UTR alternative splicing is significantly diminished, suggesting a non-catalytic role for dADAR in splicing regulation. A working model is proposed which provides a possible molecular mechanism.

02-P015 Dcamsap (ssp4) is a member of a family of membrane-skeleton proteins essential for the life of animals

The CAMSAP1 gene in vertebrates is a spectrin associated protein that is regulated by Ca2+/calmodulin. It is a member of a family of protein in animals, of unknown function, that contain a calponin homology (CH) domain and a conserved C-terminal DUF1781 domain. Invertebrates have single CAMSAP genes and vertebrates generally have three. The Drosophila homologue of CAMSAP1 is Dcamsap (ssp4). Transposon insertion mutations in Dcamsap are lethal. Fly embryos die before hatching with defects in head involution. We have generated an antibody to the C-terminus of Dcamsap and investigated the expression of the gene using western blotting and by immunocytochemistry to whole-mount embryos. Expression is elevated in discrete cells that may be undergoing apoptosis. Analysis of both the insertion mutations and also dsRNAi knockdown of Dcamsap in the eye indicate that Dcamsap is required for regulation of cell number during development.

Signaling Activity in Vivo

FRET analysis reveals temporal and spatial patterns of Cdc42 activation in individual cells and the whole fly embryo.

Boudinis required for septate junction organisation inDrosophilaand codes for a diffusible protein of the Ly6 superfamily

The Ly6 superfamily, present in most metazoan genomes, codes for different cell-surface proteins and secreted ligands containing an extracellular motif called a Ly6 domain or three-finger domain. We report the identification of 36 novel genes coding for proteins of this family in Drosophila. One of these fly Ly6 proteins, coded by the gene boudin (bou), is essential for tracheal morphogenesis in the fly embryo and contributes to the maintenance of the paracellular barrier and the organisation of the septate junctions in this tissue. Bou, a glycosylphosphatidylinositol anchored membrane protein, is also required for septate junction organisation in epithelial tissues and in the chordotonal organ glial cells, but not in the central nervous system. Our study reveals interesting parallelisms between the Ly6 proteins of flies and vertebrates, such as the CD59 antigen. Similarly to this human protein, Bou travels from cell to cell associated with extracellular particles and, consistently, we show that it is required in a non-cell-autonomous fashion. Our work opens the way for future studies addressing the function of Ly6 proteins using Drosophila as a model system.

The Bcd Morphogenetic Concentration Gradient is Formed by Diffusion

The morphogenic protein Bicoid is an essential activator of cellular differentiation and pattern formation in the fruit fly Drosophila melanogaster. It forms an exponential concentration gradient along the anterior-posterior axis of fly embryo and acts as a transcription factor that activates a cascade of target genes. The currently accepted model, known as the Synthesis, Diffusion & Degradation (SDD) model, assumes that the protein spreads across the embryo by simple diffusion, as was initially proposed by Francis Crick in 1970. This Model, however, has been called into question by several recent studies. To test the validity of the SDD model, we studied the localization and dynamics of a Bcd-EGFP fusion protein in live embryos using complementary fluorescence techniques: Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Correlation spectroscopy (FCS). We observed that Bcd-EGFP concentration decayed exponentially along the anterior-posterior axis of the embryo with a characteristic length of ∼100 μm, as previously reported by other groups, and we estimated the absolute nuclear and cytoplasmic Bcd-EGFP concentrations at the anterior pole to be 120 nM and 15 nM, respectively. In the cytoplasm, we found that the overwhelming majority of Bcd molecules were undergoing diffusive motion, with an average diffusion coefficient D∼5 μm2/s. This is an important result, because it provides the first experimental evidence that the mobility of cytoplasmic Bcd is high enough to support the establishment of a concentration gradient across the embryo before the beginning of cellularization, as envisioned in the SDD model. We also observed that 35% of the nuclear Bcd population was engaged in transient binding to immobile structures, with an average binding time τB=1/koff=120 ms, a result consistent with the fact that Bcd functions as a transcription factor.

Plasma Membrane Polarity and Compartmentalization Are Established before Cellularization in the Fly Embryo

Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). We used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. We propose that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo.