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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.

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Eyes absent (eya) encodes a member of a network of nuclear transcription factors that promotes eye development in both vertebrates and invertebrates. Despite extensive studies, the molecular mechanisms whereby cell-cell signaling pathways coordinate the function of this retinal determination gene network remain unknown. Here, we report that Drosophila Eya function is positively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation and that this regulation extends to developmental contexts independent of eye determination. In vivo genetic analyses, together with in vitro kinase assay results, demonstrate that Eya is a substrate for extracellular signal-regulated kinase, the MAPK acting downstream in the receptor tyrosine kinase (RTK) signaling pathway. Thus, phosphorylation of Eya appears to provide a direct regulatory link between the RTK/Ras/MAPK signaling cascade and the retinal determination gene network.

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Signaling by DER, the Drosophila epidermal growth factor receptor tyrosine kinase (RTK), is essential for proper migration and survival of midline glial cells (MGCs) in the embryonic central nervous system (CNS) [1-4]. We recently isolated a gene called split ends (spen) in a screen designed to identify new components of the RTK/Ras pathway [5]. Drosophila Spen and its orthologs are characterized by a distinct set of RNA recognition motifs (RRMs) and a SPOC domain, a highly conserved carboxy-terminal domain of unknown function [5-7]. To investigate spen function in the context of RTK signaling, we examined the consequences of spen loss-of-function mutations on embryonic CNS development. We found that spen was required for normal migration and survival of MGCs and that embryos lacking spen had CNS defects strikingly reminiscent of those seen in mutants of several known components of the DER signaling pathway. In addition, spen interacted synergistically with the RTK effector pointed. Using MGC-targeted expression, we found that increased Ras signaling rescued the lethality associated with expression of a dominant-negative spen transgene. Therefore, spen encodes a positively acting component of the DER/Ras signaling pathway.

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The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins.

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Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein-protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration.

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Drosophila yan has been postulated to act as an antagonist of the proneural signal mediated by the sevenless/Ras1/MAPK pathway. We have mutagenized the eight MAPK phosphorylation consensus sites of yan and examined the effects of overexpressing the mutant protein in transgenic flies and transfected S2 cultured cells. Our results suggest that phosphorylation by MAPK affects the stability and subcellular localization of yan, resulting in rapid down-regulation of yan activity. Furthermore, MAPK-mediated down-regulation of yan function appears to be critical for the proper differentiation of both neuronal and nonneuronal tissues throughout development, suggesting that yan is an essential component of a general timing mechanism controlling the competence of a cell to respond to inductive signals.

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We show that the activities of two Ets-related transcription factors required for normal eye development in Drosophila, pointed and yan, are regulated by the Ras1/MAPK pathway. The pointed gene codes for two related proteins, and we show that one form is a constitutive activator of transcription, while the activity of the other form is stimulated by the Ras1/MAPK pathway. Mutation of the single consensus MAPK phosphorylation site in the second form abrogates this responsiveness. yan is a negative regulator of photoreceptor determination, and genetic data suggest that it acts as an antagonist of Ras1. We demonstrate that yan can repress transcription and that this repression activity is negatively regulated by the Ras1/MAPK signal, most likely through direct phosphorylation of yan by MAPK.

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The Notch locus of Drosophila melanogaster encodes a 2,703-amino-acid transmembrane protein required for a variety of developmental processes, including neurogenesis, oogenesis and ommatidial assembly. The Notch protein contains a large extracellular domain of 36 epidermal growth factor-like repeats as well as three Notch/Lin-12 repeats and an intracellular domain with 6 Cdc10/ankyrin repeats, motifs that are highly conserved in several vertebrate Notch homologues. Truncation of the extracellular domain of the Drosophila Notch protein produces an activated receptor, as judged by its ability to cause phenotypes similar to gain-of-function alleles of duplications of the Notch locus. Equivalent truncations of vertebrate Notch-related proteins have been associated with malignant neoplasma and other developmental abnormalities. We present here an analysis of activated Notch function at single-cell resolution in the Drosophila compound eye. We find that overexpression of full-length Notch in defined cell types has no apparent effects but that overexpression of activated Notch in the same cells transiently blocks their proper cell-fate commitment, causing them either to adopt incorrect cell fates or to differentiate incompletely. Moreover, an activated Notch protein lacking the transmembrane domain is translocated to the nucleus, raising the possibility that Notch may participate directly in nuclear events.

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The Notch gene of Drosophila plays an important role in cell fate specification throughout development. The Notch protein contains a large extracellular domain of 36 EGF-like repeats as well as 3 Notch/lin-12 repeats and an intracellular domain with 6 cdc10/ankyrin repeats, motifs which are highly conserved in several vertebrate Notch homologues [1-7]. In this review we summarize the results of two recent studies which attempt to establish structure-function relationships of the various domains of the Notch gene product [8, 9]. The functions of various structural domains of the Notch protein in vivo were investigated using a series of deletion mutants which have been ectopically expressed either under the hsp70 heat-shock promoter or under the sevenless eye-specific promoter. Truncation of the extracellular domain of Drosophila Notch produces an activated receptor as judged by its ability to cause phenotypes matching those of gain-of-function alleles or duplications of the Notch locus [8]. Equivalent truncations of vertebrate Notch-related proteins have been associated with malignant neoplasms and other developmental abnormalities [3, 6, 10, 11]. In contrast, dominant negative phenotypes result from overexpression of a protein lacking most intracellular sequences. These results were extended by an analysis of activated Notch function at single-cell resolution in the Drosophila compound eye [9]. It was shown that while overexpression of full-length Notch in defined cell types has no apparent effects, overexpression of activated Notch in the same cells transiently blocks their proper cell-fate commitment, causing them to either adopt incorrect cell fates or to differentiate incompletely. Moreover, an activated Notch protein lacking the transmembrane domain is translocated to the nucleus, raising the possibility that Notch may participate directly in nuclear events.

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The Notch gene of Drosophila plays an important role in cell fate specification throughout development. To investigate the functions of specific structural domains of the Notch protein in vivo, a series of deletion mutants have been ectopically expressed under the hsp70 heat shock promoter. Two classes of dominant phenotypes are observed, one suggestive of Notch loss-of-function mutations and the other of Notch gain-of-function mutations. Dominant activated phenotypes result from overexpression of a protein lacking most extracellular sequences, while dominant negative phenotypes result from overexpression of a protein lacking most intracellular sequences. These results support the notion that Notch functions as a receptor whose extracellular domain mediates ligand binding, resulting in the transmission of developmental signals by the cytoplasmic domain. Finally, the phenotypes observed suggest that the cdc 10/ankyrin repeat region within the intracellular domain plays an essential role in the postulated signal transduction events.

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The neurogenic loci Notch and Delta, which both encode EGF-homologous transmembrane proteins, appear to function together in mediating cell-cell communication and have been shown to interact at the cell surface in vitro. To examine the role of the EGF repeats in this interaction, we performed an extensive deletion mutagenesis of the extracellular domain of Notch. We find that of the 36 EGF repeats of Notch, only two, 11 and 12, are both necessary and sufficient to mediate interactions with Delta. Furthermore, this Delta binding ability is conserved in the corresponding two repeats from the Xenopus Notch homolog. We report a novel molecular interaction between Notch and Serrate, another EGF-homologous transmembrane protein containing a region of striking similarity to Delta, and show that the same two EGF repeats of Notch also constitute a Serrate binding domain. These results suggest that Notch may act as a multifunctional receptor whose 36 EGF repeats form a tandem array of discrete ligand-binding units, each of which may potentially interact with several different proteins during development.

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The Notch gene in Drosophila encodes a transmembrane protein with homology to EGF that appears to mediate cell-cell interactions necessary for proper epidermal vs. neural fate decisions. In this study, we examine Notch expression in detail throughout embryonic and imaginal development using confocal laser-scanning microscopy and specific mAb probes. We find that Notch is expressed in a tissue-specific manner as early as the cellular blastoderm stage, when cells of the presumptive mesoderm clearly express less Notch than adjacent ectodermal precursors. Notch is abundantly expressed during the initial determination of neuronal lineages, such as the embryonic neuroblasts and the precursors of sensory neurons in the imaginal disc epithelia, but expression quickly decreases during subsequent differentiation. These changing patterns of Notch expression do not correlate well with cell movements, and thus do not appear to support the notion that the major function of Notch is to maintain epithelial integrity via adhesive mechanisms. Our data suggest instead that Notch may act as a cell-surface receptor, perhaps functioning in the lateral inhibition mechanism that is necessary for proper spacing of neuronal precursors.

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Genetic analyses have raised the possibility of interactions between the gene products of the neurogenic loci Notch and Delta, each of which encodes a transmembrane protein with EGF homology. To examine the possibility of intermolecular association between the products of these two genes, we studied the effects of their expression on aggregation in Drosophila S2 cells. We find that Notch-expressing cells form mixed aggregates specifically with cells that express Delta and that this process is calcium dependent. In addition, we show that Notch and Delta can associate within the membrane of a single cell, and further, that they form detergent-soluble intermolecular complexes. Our analyses suggest that Notch and Delta proteins interact at the cell surface via their extracellular domains.

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The genetic and molecular analysis of the Notch locus, which codes for a transmembrane protein sharing homology with the mammalian epidermal growth factor, suggests that the Notch protein is involved in a cell interaction mechanism essential for the differentiation of the embryonic nervous system of Drosophila. Taking advantage of the negative complementation between two Notch mutations that affect the extracellular domain of the protein, we have tried to dissect the genetic circuitry in which Notch is integrated by searching for genes whose products may interact with the Notch protein. This genetic screen has led to the identification of a surprisingly restricted set of interacting loci, including Delta and mastermind. Like Notch, both of these genes belong to a group of loci, the neurogenic loci, which have been previously identified by virtue of their similar mutant phenotype affecting early neurogenesis. We extend these studies by systematically exploring interactions between specific mutations in the Notch molecule and the other neurogenic genes. Furthermore, we show that the molecular lesions of two Notch alleles (nd and nd2), which interact dramatically with mastermind mutations, as well as with a mutation affecting the transducin homologous product of the neurogenic locus Enhancer of split, involve changes in the intracellular domain of the protein.

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