Drosophila Eye Nuclei Segmentation Based on Graph Cut and Convex Shape Prior.

The rapid advance in three-dimensional (3D) confocal imaging technologies is rapidly increasing the availability of 3D cellular images. However, the lack of robust automated methods for the extraction of cell or organelle shapes from the images is hindering researchers ability to take full advantage of the increase in experimental output. The lack of appropriate methods is particularly significant when the density of the features of interest in high, such as in the developing eye of the fruit fly. Here, we present a novel and efficient nuclei segmentation algorithm based on the combination of graph cut and convex shape prior. The main characteristic of the algorithm is that it segments nuclei foreground using a graph cut algorithm and splits overlapping or touching cell nuclei by simple convex and concavity analysis, using a convex shape assumption for nuclei contour. We evaluate the performance of our method by applying it to a library of publicly-available two-dimensional (2D) images that were hand-labeled by experts. Our algorithm yields a substantial quantitative improvement over other methods for this benchmark. For example, our method achieves a decrease of 3.2 in the Hausdorff distance and an decrease of 1.8 per slice in the merged nuclei error.

The RNAi pathway initiated by Dicer-2 in Drosophila.

Injection or expression of double-stranded RNA (dsRNA) in Drosophila serves as a trigger that causes cells to specifically cleave homologous mRNA transcripts. Our approach is to identify essential components of the RNA interference (RNAi) mechanism by isolating and characterizing mutations that cause the RNAi response to be abnormal. These studies have thus far led to the identification of seven genetic loci that encode proteins acting at various steps in the RNAi process. We have molecularly identified several of these proteins. Two are members of the Dicer family. Dicer-1 and Dicer-2 are required for short interfering RNA (siRNA)-directed mRNA cleavage by facilitating distinct steps in the assembly of the RNA-induced silencing complex (RISC). AGO2 is a RISC component that both carries out transcript cleavage and facilitates RISC maturation. Other factors appear to function as regulators of RISC assembly rather than as core factors for RNAi.

Stem cell division is regulated by the microRNA pathway.

One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

Genome-wide survey of protein kinases required for cell cycle progression.

Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery.

Gene silencing by double-stranded RNA.

Eukaryotes silence gene expression in the presence of double-stranded RNA homologous to the silenced gene. Silencing occurs by the targeted degradation of mRNA. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21--23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21--23 nucleotides apart. This mechanism, termed RNAi, has functional links to viral defense and silencing phenomena, such as cosuppression. It also functions to repress the hopping of transposable elements.

Overlapping activators and repressors delimit transcriptional response to receptor tyrosine kinase signals in the Drosophila eye.

Regulated transcription of the prospero gene in the Drosophila eye provides a model for how gene expression is specifically controlled by signals from receptor tyrosine kinases. We show that prospero is controlled by signals from the EGF receptor DER and the Sevenless receptor. A direct link is established between DER activation of a transcription enhancer in prospero and binding of two transcription factors that are targets of DER signaling. Binding of the cell-specific Lozenge protein is also required for activation, and overlapping Lozenge protein distribution and DER signaling establishes expression in a subset of equivalent cells competent to respond to Sevenless. We show that Sevenless activates prospero independent of the enhancer and involves targeted degradation of Tramtrack, a transcription repressor.

Heritable gene silencing in Drosophila using double-stranded RNA.

RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants, nematodes, Drosophila, zebrafish, and mice. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene. Rapid degradation of mRNA in affected cells blocks gene expression. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.

Endocytosis: why not wait to deubiquitinate?

Deubiquitination by the Fat facets protein - a regulator of photoreceptor differentiation during Drosophila eye development - has been found to activate endocytosis, while ubiquitination inhibits endocytosis. Surprisingly, this is the opposite effect that ubiquitination has on endocytosis of many plasma membrane proteins.

ebi regulates epidermal growth factor receptor signaling pathways in Drosophila.

ebi regulates the epidermal growth factor receptor (EGFR) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes EGFR-dependent down-regulation of Tramtrack88, an antagonist of neuronal development.

Interactions between Wingless and DFz2 during Drosophila wing development.

Drosophila Wingless (Wg) is a secreted signaling protein of the Wnt family. Mutations in the wg gene disrupt the patterning of embryonic segments and their adult derivatives. Wg protein has been shown in cell culture to functionally interact with DFz2, a receptor that is structurally related to the tissue polarity protein Frizzled (Fz). However, it has not been determined if DFz2 functions in the Wg signaling pathway during fly development. Here we demonstrate that overexpression of DFz2 increases Wg-dependent signaling to induce ectopic margin bristle formation in developing Drosophila wings. Overexpression of a truncated form of DFz2 acts in a dominant-negative manner to block Wg signaling at the wing margin, and this block is rescued by co-expression of full-length DFz2 but not full-length Fz. Our results suggest that DFz2 and not Fz acts in the Wg signaling pathway for wing margin development. However, a truncated form of Fz also blocks Wg signaling in embryo and wing margin development, and the truncated form of DFz2 affects ommatidial polarity during eye development. These observations suggest that a single dominant-negative form of Fz or DFz2 can block more than one type of Wnt signaling pathway and imply that truncated proteins of the Fz family lose some aspect of signaling specificity.

Proteasomal regulation of nuclear receptor corepressor-mediated repression.

Repression of gene transcription is a fundamental property of nuclear hormone receptors. We report here that cell-specific repression by nuclear receptors correlates with levels of nuclear receptor corepressor (N-CoR) protein. N-CoR protein levels are regulated by mSiah2, a mammalian homolog of Drosophila Seven in absentia that targets N-CoR for proteasomal degradation. mSiah2 expression is cell-type specific and differentially regulates the repressive activities of nuclear receptors. These findings establish targeted proteolysis of transcriptional coregulators as a mechanism for cell-specific regulation of gene transcription.

Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.

We investigated the potential of double-stranded RNA to interfere with the function of genes in Drosophila. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. In contrast, single-stranded RNA weakly interfered with gene activity. The method was used to determine the reception mechanism of the morphogen Wingless. Interference of the frizzled and Drosophila frizzled 2 genes together produced defects in embryonic patterning that mimic loss of wingless function. Interference of either gene alone had no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. Our results demonstrate that dsRNA interference can be used to analyze many aspects of gene function.

Photoreceptor cell differentiation requires regulated proteolysis of the transcriptional repressor Tramtrack.

The transcription repressor Tramtrack (TTK) is found in cone cells but not photoreceptor cells of the Drosophila eye. We show that down-regulation of TTK expression occurs in photoreceptor cells and is required for their fate determination. Down-regulation requires the presence of Phyllopod (PHYL), which is induced by the RAS pathway, and Seven In Absentia (SINA). Loss of either gene causes accumulation of TTK in photoreceptor cells, and TTK does not accumulate in cone cells if both PHYL and SINA are present. We report that SINA and PHYL promote ubiquitination and rapid degradation of TTK by the proteasome pathway in cell culture, and both SINA and PHYL bind to the N-terminal domain of TTK. These results argue that photoreceptor differentiation is regulated by the RAS pathway through targeted proteolysis of the TTK repressor.

Ras1 signaling and transcriptional competence in the R7 cell of Drosophila.

The receptor tyrosine kinase Sevenless determines R7 cell fate by activation of the Ras1 pathway in a subset of equivalent cells competent to respond in the Drosophila eye. We show that the prospero gene becomes transcriptionally activated at a low level in all Sevenless-competent cells prior to Sevenless signaling, and this requires the activities of Ras1 and two Ras1/MAP kinase-responsive ETS transcription factors. Restriction of high-level prospero expression to the R7 cell appears as a subsequent event, which requires Sevenless activation of the Ras1/MAP kinase pathway. We show that Phyllopod, a nuclear factor whose expression is induced by Sevenless, interacts with another nuclear factor, Sina, to form a complex, and that both factors are involved in upregulating transcription of the prospero gene in the eye. Ultimately, prospero expression is required for proper connectivity of R7 photoreceptor axons to their synaptic targets. Our results suggest that specific transcriptional responses are linked to the mode of activation of the Ras1/MAP kinase signal transduction pathway.

Activated Drosophila Ras1 is selectively suppressed by isoprenyl transferase inhibitors.

Ras CAAX (C = cysteine, A = aliphatic amino acid, and X = any amino acid) peptidomimetic inhibitors of farnesyl protein transferase suppress Ras-dependent cell transformation by preventing farnesylation of the Ras oncoprotein. These compounds are potential anticancer agents for tumors associated with Ras mutations. The peptidomimetic FTI-254 was tested for Ras1-inhibiting activity in whole animals by injection of activated Ras1val12 Drosophila larvae. FTI-254 decreased the ability of Ras1val12 to form supernumerary R7 photoreceptor cells in the compound eye of transformed flies. In contrast, it had no effect on the related supernumerary R7 phenotypes of flies transformed with either the activated sevenless receptor tyrosine kinase, Raf kinase, or a chimeric Ras1val12 protein that is membrane associated through myristylation instead of isoprenylation. Therefore, FTI-254 acts as an isoprenylation inhibitor to selectively inhibit Ras1val12 signaling activity in a whole-animal model system.

frizzled regulates mirror-symmetric pattern formation in the Drosophila eye.

Coordinated morphogenesis of ommatidia during Drosophila eye development establishes a mirror-image symmetric pattern across the entire eye bisected by an anteroposterior equator. We have investigated the mechanisms by which this pattern formation occurs and our results suggest that morphogenesis is coordinated by a graded signal transmitted bidirectionally from the presumptive equator to the dorsal and ventral poles. This signal is mediated by frizzled, which encodes a cell surface transmembrane protein. Mosaic analysis indicates that frizzled acts non-autonomously in an equatorial to polar direction. It also indicates that relative levels of frizzled in photoreceptor cells R3 and R4 of each ommatidium affect their positional fate choices such that the cell with greater frizzled activity becomes an R3 cell and the cell with less frizzled activity becomes an R4 cell. Moreover, this bias affects the choice an ommatidium makes as to which direction to rotate. Equator-outwards progression of elav expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. We propose that frizzled mediates a bidirectional signal emanating from the equator.

Identification of genes that interact with the sina gene in Drosophila eye development.

The sina gene encodes a nuclear protein that is required for the correct development of R7 photoreceptor cells in the Drosophila eye. We conducted a genetic screen for mutations that reduce the activity of sina and found mutations that define nine genes whose products may be required for normal sina activity. Three of these genes also appear to be essential for signaling by the Sevenless-Ras pathway in R7 cells, of which one gene corresponds to the rolled locus (rl). The rl gene is known to encode a mitogen-activated protein kinase necessary for signaling by Ras. These results suggest that the products of these three genes may participate in a signaling pathway involving both Ras and Sina, possibly by functionally linking these two proteins.

Mutations on the second chromosome affecting the Drosophila eye.

In the developing eye of Drosophila, cell interactions appear to be responsible for organising undifferentiated cells into unit eyes, or ommatidia. Extensive mutagenesis has been used to search for mutations affecting the development and differentiation of ommatidia. These mutations have been characterized using sections of adults and immunocytochemistry of imaginal discs. Fourteen loci on the second chromosome are described that affect the spacing of the preclusters, the differentiation of ommatidial cells, orientation of the ommatidia, or architecture of the adult retina, that cause retinal degeneration in larval or pupal eye discs, or that cause homeotic transformation of part of the head.

Evolution of gene position: chromosomal arrangement and sequence comparison of the Drosophila melanogaster and Drosophila virilis sina and Rh4 genes.

The seven in absentia (sina) gene of Drosophila encodes a nuclear protein required for normal eye development. In Drosophila melanogaster, the sina gene is located within an intron of the Rh4 opsin gene. We examine here the nucleotide sequences and chromosomal arrangements of these genes in Drosophila virilis. An interspecies comparison between D. melanogaster and D. virilis reveals that the protein-coding sequences of the sina and Rh4 genes are highly conserved, but the relative chromosomal position and structural arrangement of these genes differ between the two species. In particular, the sina and Rh4 genes are widely separated in D. virilis, and there is no intron in the Rh4 gene. Our results suggest that the Rh4 gene was translocated to another chromosomal location by a retrotransposition event.