Fly Embryos Research Articles

Counting Motors by Force

Properties of the microtubule motor protein kinesin-1 have been well characterized in vitro but not in the viscous environment of a cell. By measuring the force that kinesin-1 exerts on lipid droplets in fly embryos, Shubeita et al. (2008) determine the number of active motors per droplet and find unexpected differences between motor regulation in vivo and in vitro.

Activation of Wingless Targets Requires Bipartite Recognition of DNA by TCF

Specific recognition of DNA by transcription factors is essential for precise gene regulation. In Wingless (Wg) signaling in Drosophila, target gene regulation is controlled by T cell factor (TCF), which binds to specific DNA sequences through a high mobility group (HMG) domain. However, there is considerable variability in TCF binding sites, raising the possibility that they are not sufficient for target location. Some isoforms of human TCF contain a domain, termed the C-clamp, that mediates binding to an extended sequence in vitro. However, the significance of this extended sequence for the function of Wnt response elements (WREs) is unclear. In this report, we identify a cis-regulatory element that, to our knowledge, was previously unpublished. The element, named the TCF Helper site (Helper site), is essential for the activation of several WREs. This motif greatly augments the ability of TCF binding sites to respond to Wg signaling. Drosophila TCF contains a C-clamp that enhances in vitro binding to TCF-Helper site pairs and is required for transcriptional activation of WREs containing Helper sites. A genome-wide search for clusters of TCF and Helper sites identified two new WREs. Our data suggest that DNA recognition by fly TCF occurs through a bipartite mechanism, involving both the HMG domain and the C-clamp, which enables TCF to locate and activate WREs in the nucleus.

Computational prediction of RNA structural motifs involved in posttranscriptional regulatory processes

Messenger RNA molecules are tightly regulated, mostly through interactions with proteins and other RNAs, but the mechanisms that confer the specificity of such interactions are poorly understood. It is clear, however, that this specificity is determined by both the nucleotide sequence and secondary structure of the mRNA. Here, we develop RNApromo, an efficient computational tool for identifying structural elements within mRNAs that are involved in specifying posttranscriptional regulations. By analyzing experimental data on mRNA decay rates, we identify common structural elements in fast-decaying and slow-decaying mRNAs and link them with binding preferences of several RNA binding proteins. We also predict structural elements in sets of mRNAs with common subcellular localization in mouse neurons and fly embryos. Finally, by analyzing pre-microRNA stem-loops, we identify structural differences between pre-microRNAs of animals and plants, which provide insights into the mechanism of microRNA biogenesis. Together, our results reveal unexplored layers of posttranscriptional regulations in groups of RNAs and are therefore an important step toward a better understanding of the regulatory information conveyed within RNA molecules. Our new RNA motif discovery tool is available online.

The Drosophila homologue of Arf-GAP GIT1, dGIT, is required for proper muscle morphogenesis and guidance during embryogenesis

GIT1-like proteins are GTPase-activating proteins (GAPs) for Arfs and interact with a variety of signaling molecules to function as integrators of pathways controlling cytoskeletal organization and cell motility. In this report, we describe the characterization of a Drosophila homologue of GIT1, dGIT, and show that it is required for proper muscle morphogenesis and myotube guidance in the fly embryo. The dGIT protein is concentrated at the termini of growing myotubes and localizes to muscle attachment sites in late stage embryos. dgit mutant embryos show muscle patterning defects and aberrant targeting in subsets of their muscles. dgit mutant muscles fail to localize the p21-activated kinase, dPak, to their termini. dPak and dGIT form a complex in the presence of dPIX and dpak mutant embryos show similar muscle morphogenesis and targeting phenotypes to that of dgit. We propose that dGIT and dPak are part of a complex that promotes proper muscle morphogenesis and myotube targeting during embryogenesis.

Making Animals the Right Size

Classical experiments with amphibians revealed that bisected embryos develop into properly proportioned tadpoles and that transplantation of a small group of dorsal cells called "Spemann’s organizer" into the ventral side of an early embryo leads to the formation of a properly proportioned second axis. Thus, the morphogen activation gradients that drive patterning in the early embryo appear to scale with size. Two theoretical models (an inhibition-based model and the shuttling-based model) have been proposed to explain how the bone morphogenetic protein (BMP) activity gradient that drives axis formation is established. Ben-Zvi et al . provide evidence for the shuttling-based model, which previously had only experimental support in fly embryos, in frog ( Xenopus laevis ) embryos and suggest that the additional BMP ligand antidorsalizing morphogenic protein (ADMP), which is produced in the dorsal pole and which is not present in Drosophila , allows the Xenopus embryos to scale patterning with size. First, they created a mathematical model for the core patterning network containing the BMP ligands BMP (which represented BMP2, 4, and 7) and ADMP, the BMP antagonist chordin, and the protease Xlr, which degrades chordin. In this model, chordin and ADMP were produced at the dorsal pole, and BMP signaling repressed ADMP. Systematic screening for parameters that allowed the activation gradient of BMP to be robust and to scale with embryo size resulted in the identification of 21 networks that scaled properly out of the 26,000 possible networks. In each of the networks that scaled, the ligands were concentrated at the ventral pole, suggesting that activation gradients were generated by shuttling of the ligands from their site of production to the ventral pole and not simply by localized production of an inhibitor. Further, the models were consistent with experimentally proven properties of the core patterning components: Properly scaling networks required that chordin binds BMP with higher affinity than it binds ADMP, that binding of chordin facilitates diffusion of BMP ligands, and that chordin is degraded primarily when complexed with ligand. A shuttling model that relied solely on BMP and chordin was insufficient to provide scaling; however, the addition of ADMP with its BMP-mediated repression was sufficient. Experiments in which Xenopus embryos were injected with a tagged BMP4 with or without coinjection of morpholino oligonucleotides (MOs) against chordin showed that BMP4 only concentrated at the ventral pole in the presence of chordin. Axis inversion, which was also consistent with a shuttling mechanism, occurred when chordin was depleted from the organizer at the dorsal side with MOs and a second organizer was induced on the ventral side. Induction of a second organizer on the ventral side in conjunction with MOs for ADMP resulted in the expression of a ventrally expressed gene between the original dorsal organizer and the ventral organizer, which is also consistent with shuttling of BMP ligands. Thus, as in fly embryos, vertebrates also appear to use a shuttling mechanism for robust patterning that scales with embryo size. D. Ben-Zvi, B.-Z. Shilo, A. Fainsod, N. Barkai, Scaling of the BMP activation gradient in Xenopus embryos. Nature 453 , 1205-1211 (2008). [PubMed]

RAX is required for fly neuronal development and mouse embryogenesis

RAX was originally discovered as the unique cellular activator for the dsRNA-dependent, interferon-inducible protein kinase PKR. Recent findings indicate that RAX is also a critical component of the RNA-induced silencing complex and a regulator of transcription. Here we report novel phenotypes for both fruit flies carrying a transposon insertion in the 5′ UTR of dRax (independently identified as loqs/R3D1) and mice with a deletion of the entire Rax gene. In Drosophila we observe a high level of dRax expression in the developing nerve cord. Mutant fly embryos homozygous for the insertion dRax[f00791] display highly abnormal commissural axon structure of the CNS and 70% of the flies homozygous for the mutant allele die prior to adulthood. Surviving male flies have reduced fertility and female flies are sterile. Furthermore, these flies appear to have a severe defect in nervous system coordination or neuromuscular function resulting in significantly reduced locomotion. Mice were also generated that are heterozygous for a deletion of the entire Rax gene (exons 1–8). While mice that are heterozygous for the mutant allele are viable and appear normal, we are unable to obtain mice homozygous for this mutant allele. Furthermore, we have not observed any embryo obtained by mating heterozygous mice at either E3.5, 7, or 14 that is nullizygous for the Rax gene. Since Rax is expressed in preimplantation blastocysts, these data indicate that deletion of the entire Rax gene is embryonic lethal in mice at a preimplantation stage of development. Collectively, these findings in two different species illustrate the importance of RAX for embryonic development.

Jennifer Zallen: decoding the developmental dance. Interview by Ruth Williams.

Like choreographed dancers on a stage, cells must coordinate their movements during embryogenesis to build the body shape. In her laboratory at the Sloan Kettering Institute in New York City, Jennifer Zallen is figuring out the dance steps of a particularly dramatic coordinated cell movement called convergent extension. Figure 1 Jennifer Zallen During convergent extension, the fly embryo narrows in width and more than doubles in length to create the basic layout for the body plan. Zallen has discovered that extension occurs by local cell movements. Some cells simply wedge between their neighbors, and other cells come together in groups from across the width of the embryo, forming arrangements called “rosettes.” The rosettes then disassemble as the cells arrange themselves into an approximate line, thereby elongating the embryo (1). Zallen, who set up her lab three years ago, is now investigating candidate cellular choreographers using mutant screens. She has also recently started measuring cell shapes and movements to detect patterns within populations that might give clues to the underlying order of convergent extension. Zallen moved to Sloan Kettering after completing her postdoctoral work on fly morphogenesis in the lab of Nobel prize-winning developmental biologist, Eric Wieschaus (2, 3). Before that she earned her Ph.D. with esteemed neuroscientist Cori Bargmann by working on neuronal migration during worm development (4, 5, 6). And before that she studied biology at Harvard. It could be argued that Zallen has the text-book career of a developmental biologist. Her recent foray into measuring cell shapes and movements, however, has taken her not just into a different chapter, but into an entirely different book—that of condensed matter physics (7).

PROCESS FLOW FOR CLASSIFICATION AND CLUSTERING OF FRUIT FLY GENE EXPRESSION PATTERNS.

The rapidly growing collection of fruit fly embryo images makes automated Image Segmentation and classification an indispensable requirement for a large-scale analysis of in situ hybridization (ISH) - gene expression patterns (GEP). We present here such an automated process flow for Segmenting, Classification, and Clustering large-scale sets of Drosophila melanogaster GEP that is capable of dealing with most of the complications implicated in the images.

Segmenting the fly embryo: logical analysis of the role of the Segment Polarity cross-regulatory module

Initially activated by the pair-rule genes, the expression patterns of the segment polarity genes engrailed and wingless become consolidated through inter-cellular interactions between juxtaposed cells. We delineate a logical model focusing on a dozen molecular components at the core of the regulatory network controlling this process. Our model leads to the following conclusions: (1) the pair-rule signals, which activate engrailed and wingless genes independently of each other, need to be operative until the inter-cellular circuit involving these two genes is functional. This implies that the pair-rule pattern is instrumental both in determining the activation of the genes engrailed and wingless in rows of adjacent cells, and in consolidating these expression patterns; (2) the consolidation of engrailed and wingless expression patterns requires the simultaneous activation of both autocrine and paracrine Wingless-pathways, and the Hedgehog pathway; (3) protein kinase A plays at least two roles through the phosphorylation of Cubitus interruptus, the effector molecule of the Hedgehog signalling pathway and (4) the roles of Sloppy-paired and Naked in the delineation of the engrailed and wingless expression domains are emphasized as being important for segmental boundary formation. Moreover, the application of an original computational method leads to the delineation of a subset of crucial regulatory circuits enabling the coexistence of specific expression states at the cellular level, as well as specific combination of cellular states inter-connected through Wingless and Hedgehog signalling. Finally, the simulation of altered expressions of segment polarity genes leads to results consistent with the published data.

RNA polymerase caught loitering

Instead of transcribing an entire gene, RNA polymerase sometimes starts to work and then pauses, finishing the job at a later time. According to a pair of papers, this herky-jerky transcription pattern affects more than 10% of fly genes, including some developmental movers and shapers. That polymerase stalling occurs on a few genes is old news. However, no one had surveyed the entire genome to determine its prevalence. The two groups used chromatin immunoprecipitation microarrays to detect genes with polymerase stuck near the promoter. When Ginger Muse, Karen Adelman (National Institute of Environmental Health Sciences, Research Triangle Park, NC), and colleagues applied the technique to a line of embryonic Drosophila cells, they pinpointed stalling on more than 1,000 genes. Many were involved in development or responses to environmental stimuli. Julia Zeitlinger, Michael Levine (University of California, Berkeley), and colleagues also identified lots of genes with idling polymerase in fly embryos. The list of 1,600 genes, which overlapped with that of the other group, included some architects of development, such as Hox, Wnt, and Notch. Having RNA polymerase poised for action might speed synthesis of needed proteins, help coordinate gene expression, or prepare genes for later activation. And the commonness of this effect “opens the door to a whole new cast of characters in regulating developmental genes,” says Levine.

Localized mRNA is the norm

![Graphic][1] Many different mRNAs (green) stake out specific locations in fly embryo cells. KRAUSE/ELSEVIER Location, location, location. It's critical for real estate, proteins, and—according to work by Eric Lecuyer, Henry Krause, and colleagues (University of Toronto, Canada

Plasma and Cavitation Dynamics during Pulsed Laser Microsurgeryin vivo

We compare the plasma and cavitation dynamics underlying pulsed laser microsurgery in water and in fruit fly embryos (in vivo)--specifically for nanosecond pulses at 355 and 532 nm. We find two key differences. First, the plasma-formation thresholds are lower in vivo--especially at 355 nm--due to the presence of endogenous chromophores that serve as additional sources for plasma seed electrons. Second, the biological matrix constrains the growth of laser-induced cavitation bubbles. Both effects reduce the disrupted region in vivo when compared to extrapolations from measurements in water.

TheDrosophilahomolog ofMCPH1,a human microcephaly gene, is required for genomic stability in the early embryo

Mutation of human microcephalin (MCPH1) causes autosomal recessive primary microcephaly, a developmental disorder characterized by reduced brain size. We identified mcph1, the Drosophila homolog of MCPH1, in a genetic screen for regulators of S-M cycles in the early embryo. Embryos of null mcph1 female flies undergo mitotic arrest with barrel-shaped spindles lacking centrosomes. Mutation of Chk2 suppresses these defects, indicating that they occur secondary to a previously described Chk2-mediated response to mitotic entry with unreplicated or damaged DNA. mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. In contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. Components of this checkpoint, however, appear to cooperate with MCPH1 to regulate embryonic cell cycles in a manner independent of Cdk1 phosphorylation. We propose a model in which MCPH1 coordinates the S-M transition in fly embryos: in the absence of mcph1, premature chromosome condensation results in mitotic entry with unreplicated DNA, genomic instability, and Chk2-mediated mitotic arrest. Finally, brains of mcph1 adult male flies have defects in mushroom body structure, suggesting an evolutionarily conserved role for MCPH1 in brain development.

Transgenesis upgrades forDrosophila melanogaster

Drosophila melanogaster is a highly attractive model system for the study of numerous biological questions pertaining to development, genetics, cell biology, neuroscience and disease. Until recently, our ability to manipulate flies genetically relied heavily on the transposon-mediated integration of DNA into fly embryos. However, in recent years significant improvements have been made to the transgenic techniques available in this organism, particularly with respect to integrating DNA at specific sites in the genome. These new approaches will greatly facilitate the structure-function analyses of Drosophila genes, will enhance the ease and speed with which flies can be manipulated, and should advance our understanding of biological processes during normal development and disease.

Modeling homeoprotein intercellular transfer unveils a parsimonious mechanism for gradient and boundary formation in early brain development

Morphogens are molecules inducing morphogenetic responses from cells and cell ensembles. The concept of morphogen is related to that of positional value, as the generation of morphological and physiological characteristics is function of position. Based on the observation that homeoproteins, a category of transcription factors with morphogenetic functions, traffic between abutting cells and, very often, regulate their own expression, we develop here a biophysical model of homeoprotein propagation and study the associated mathematical equations. This mode of cell signaling can generate domains of homeoprotein expression. We study both the transient and steady-state regimes and, in this latter regime, we obtain various morphogenetic gradients, depending on the value of some parameters, such as morphogen synthesis, degradation rates and efficiency of intercellular passage. The same equations, applied to pairs of homeoproteins with auto-activation and reciprocal inhibition properties, account for border formation. They also allow us to compute how specific perturbations can either be buffered or lead to modifications in the position of borders between adjacent areas. The model developed here, based on experimental data, and avoids theoretical obstacles associated with pluricellularity. It extends the idea that Bicoid homeoprotein is a morphogen in the fly embryo syncitium to most homeoproteins and to pluricellular systems. Because the position of borders between brain areas is of primary physiological importance, our model might lead to original views regarding epigenetic inter-individual variations and the origin of neurological and psychiatric diseases. In addition, it provides new hypotheses regarding the molecular basis of brain evolution.

Cells' precise pattern readout

![][1] Fly embryo nuclei detect a 10% difference in Bicoid (blue) concentration that either does or doesn't activate the head gene hunchback (green). GREGOR Running from head to tail in a fly embryo, the Bicoid (Bcd) gradient is the blueprint for anterior–posterior development.

Automatic image analysis for gene expression patterns of fly embryos

BackgroundStaining the mRNA of a gene via in situ hybridization (ISH) during the development of a D. melanogaster embryo delivers the detailed spatio-temporal pattern of expression of the gene. Many biological problems such as the detection of co-expressed genes, co-regulated genes, and transcription factor binding motifs rely heavily on the analyses of these image patterns. The increasing availability of ISH image data motivates the development of automated computational approaches to the analysis of gene expression patterns.ResultsWe have developed algorithms and associated software that extracts a feature representation of a gene expression pattern from an ISH image, that clusters genes sharing the same spatio-temporal pattern of expression, that suggests transcription factor binding (TFB) site motifs for genes that appear to be co-regulated (based on the clustering), and that automatically identifies the anatomical regions that express a gene given a training set of annotations. In fact, we developed three different feature representations, based on Gaussian Mixture Models (GMM), Principal Component Analysis (PCA), and wavelet functions, each having different merits with respect to the tasks above. For clustering image patterns, we developed a minimum spanning tree method (MSTCUT), and for proposing TFB sites we used standard motif finders on clustered/co-expressed genes with the added twist of requiring conservation across the genomes of 8 related fly species. Lastly, we trained a suite of binary-classifiers, one for each anatomical annotation term in a controlled vocabulary or ontology that operate on the wavelet feature representation. We report the results of applying these methods to the Berkeley Drosophila Genome Project (BDGP) gene expression database.ConclusionOur automatic image analysis methods recapitulate known co-regulated genes and give correct developmental-stage classifications with 99+% accuracy, despite variations in morphology, orientation, and focal plane suggesting that these techniques form a set of useful tools for the large-scale computational analysis of fly embryonic gene expression patterns.

Bicoid by the Numbers: Quantifying a Morphogen Gradient

Morphogen gradients are typically analyzed from static images of fixed embryonic tissues. Two papers in this issue of Cell now report live imaging of the Bicoid gradient in developing fruit fly embryos (Gregor et al., 2007a, 2007b). Their findings indicate that the gradient is highly reproducible from embryo to embryo and reveal that the nuclear dynamics of Bicoid are critical for maintaining precision within the gradient.

The Nuclear Option

The redistribution of the phospholipid phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is one of the mechanisms that targets an apoptotic cell for engulfment by a phagocyte. As well as presenting bridging molecules, such as annexin V, that bind to PS on the apoptotic cell, phagocytes are endowed with a PS receptor (PSR). Whereas antibody binding to PSR blocks engulfment of apoptotic cells in vitro, conflicting phenotypes have been observed in a number of species in which the genes for a PSR and its homologs have been disrupted. Controversy also remains over the localization of PSR, with some studies demonstrating expression of PSRs at the cell surface and others showing a nuclear localization of PSRs. Krieser et al . investigated a role for the Drosophila homolog of PSR (dPSR) in development and in mediating the engulfment of apoptotic cells by macrophages. The authors used acridine orange (AO) to stain the embryos of flies in which the dPSR gene was disrupted. AO stains apoptotic cells, and the authors found no difference in the number of AO-stained clusters between wild-type and dPSR-deficient embryos. Microscopic analysis using TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) staining revealed that there was also no difference in the number of apoptotic cells per macrophage in wild-type and dPSR-deficient flies, leading the authors to conclude that dPSR is not required for engulfment of apoptotic cells. Confocal microscopy showed that a fusion protein of cyan fluorescent protein and dPSR was localized to the nucleus of transfected S2 cells, a Drosophila cell line. In transgenic flies, overexpression of dPSR resulted in developmental defects similar to those seen in flies with mutations in the hid gene. Hid promotes apoptosis necessary for the correct development of various tissues in the fly. Using different fly strains in which the relative abundance of dPSR and Hid were altered, the authors determined that dPSR inhibited Hid-induced apoptosis and promoted cell survival in lattice cells of the developing eye. Puckered (Puc) inhibits c-Jun N-terminal kinase (JNK) activity, and a Drosophila strain in which one copy of puc is lost exhibits increased JNK activity. JNK activity is needed to mediate the pro-apoptotic effects of Hid. The authors found that the loss of one copy of puc almost completely prevented the phenotype caused by the overexpression of dPSR . Together, these data suggest that dPSR is localized to the nucleus and plays no direct role in mediating the engulfment of apoptotic cells but instead protects against apoptosis during development, possibly by inhibiting JNK activity. The authors speculate that because of its role in cell survival, the decrease in apoptotic cell engulfment observed when PSR is deleted in some animal models may be due to increased apoptosis of phagocytic cells. R. J. Krieser, F. E. Moore, D. Dresnek, B. J. Pellock, R. Patel, A. Huang, C. Brachmann, K. White, The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis. Development 134 , 2407-2414 (2007). [Abstract] [Full Text]

Automatic recognition and annotation of gene expression patterns of fly embryos

Gene expression patterns obtained by in situ mRNA hybridization provide important information about different genes during Drosophila embryogenesis. So far, annotations of these images are done by manually assigning a subset of anatomy ontology terms to an image. This time-consuming process depends heavily on the consistency of experts.We develop a system to automatically annotate a fruitfly's embryonic tissue in which a gene has expression. We formulate the task as an image pattern recognition problem. For a new fly embryo image, our system answers two questions: (1) Which stage range does an image belong to? (2) Which annotations should be assigned to an image? We propose to identify the wavelet embryo features by multi-resolution 2D wavelet discrete transform, followed by min-redundancy max-relevance feature selection, which yields optimal distinguishing features for an annotation. We then construct a series of parallel bi-class predictors to solve the multi-objective annotation problem since each image may correspond to multiple annotations.The complete annotation prediction results are available at: http://www.cs.niu.edu/~jzhou/papers/fruitfly and http://research.janelia.org/peng/proj/fly_embryo_annotation/. The datasets used in experiments will be available upon request to the correspondence author.