Cbl-Associated Protein CAP contributes to correct formation and robust function of the Drosophila heart tube

The formation of a tube-like structure is a basic step in the making of functional hearts in vertebrates and invertebrates and therefore, its understanding provides important information on heart development and function. In Drosophila, the cardiac tube originates from two bilateral rows of dorsally migrating cells. On meeting at the dorsal midline, coordinated changes in cell shape and adhesive properties transform the two sheets of cells into a linear tube. ECM and transmembrane proteins linked to the cytoskeleton play an important role during these dynamic processes. Here we characterize the requirement of Cbl-Associated Protein (CAP) in Drosophila heart formation. In embryos, CAP is expressed in late migrating cardioblasts and is located preferentially at their luminal and abluminal periphery. CAP mutations result in irregular cardioblast alignment and imprecisely controlled cardioblast numbers. Furthermore, CAP mutant embryos show a strongly reduced heart lumen and an aberrant shape of lumen forming cardioblasts. Analysis of double heterozygous animals reveals a genetic interaction of CAP with Integrin- and Talin-encoding genes. In post-embryonic stages, CAP closely colocalizes with Integrin near Z-bands and at cell-cell contact sites. CAP mutants exhibit a reduced contractility in larval hearts and show a locally disrupted morphology, which correlates with a reduced pumping efficiency. Our observations imply a function of CAP in linking Integrin signaling with the actin cytoskeleton. As a modulator of the cytoskeleton, CAP is involved in the establishment of proper cell shapes during cardioblast alignment and cardiac lumen formation in the Drosophila embryo. Furthermore, CAP is required for correct heart function throughout development

High-resolution in situ hybridization analysis on the chromosomal interval 61C7-61C8 of Drosophila melanogaster reveals interbands as open chromatin domains

Eukaryotic chromatin is organized in contiguous domains that differ in protein binding, histone modifications, transcriptional activity, and in their degree of compaction. Genome-wide comparisons suggest that, overall, the chromatin organization is similar in different cells within an organism. Here, we compare the structure and activity of the 61C7-61C8 interval in polytene and diploid cells of Drosophila. By in situ hybridization on polytene chromosomes combined with high-resolution microscopy, we mapped the boundaries of the 61C7-8 interband and of the 61C7 and C8 band regions, respectively. Our results demonstrate that the 61C7-8 interband is significantly larger than estimated previously. This interband extends over 20 kbp and is in the range of the flanking band domains. It contains several active genes and therefore can be considered as an open chromatin domain. Comparing the 61C7-8 structure of Drosophila S2 cells and polytene salivary gland cells by ChIP for chromatin protein binding and histone modifications, we observe a highly consistent domain structure for the proximal 13 kbp of the domain in both cell types. However, the distal 7 kbp of the open domain differs in protein binding and histone modification between both tissues. The domain contains four protein-coding genes in the proximal part and two noncoding transcripts in the distal part. The differential transcriptional activity of one of the noncoding transcripts correlates with the observed differences in the chromatin structure between both tissues. The significance of our findings for the organization and structure of open chromatin domains will be discussed

Drosophila nuclear lamin precursor Dm0 is translated from either of two developmentally regulated mRNA species apparently encoded by a single gene.

A cDNA clone encoding a portion of Drosophila nuclear lamins Dm1 and Dm2 has been identified by screening a lambda-gt11 cDNA expression library using Drosophila lamin-specific monoclonal antibodies. Two different developmentally regulated mRNA species were identified by Northern blot analysis using the initial cDNA as a probe, and full-length cDNA clones, apparently corresponding to each message, have been isolated. In vitro transcription of both full-length cDNA clones in a pT7 transcription vector followed by in vitro translation in wheat germ lysate suggests that both clones encode lamin Dm0, the polypeptide precursor of lamins Dm1 and Dm2. Nucleotide sequence analyses confirm the impression that both cDNA clones code for the identical polypeptide, which is highly homologous with human lamins A and C as well as with mammalian intermediate filament proteins. The two clones differ in their 3'-untranslated regions. In situ hybridization of lamin cDNA clones to Drosophila polytene chromosomes shows only a single locus of hybridization at or near position 25F on the left arm of chromosome 2. Southern blot analyses of genomic DNA are consistent with the notion that a single or only a few highly similar genes encoding Drosophila nuclear lamin Dm0 exist in the genome

Observations on flower and fruit anatomy in dioecious species of Cordia (Cordiaceae, Boraginales) with evolutionary interpretations

Detailed anatomical studies of flowers and fruits may render traits of phylogenetic importance, but are still rare in the Boraginales. An Old World clade of Cordia comprises many dioecious species, but the floral anatomy is largely unexplored and, hence, the structural basis for sex separation. Fresh material of buds, functionally unisexual flowers and fruits were studied using a stack shot camera. Fixed such material of Cordia crenata, C. cf. grandicalyx and C. sinensis was sectioned using a microtome and stained with safranin red and astra blue, and the gynoecial surface was studied using SEM. The stamens of the female flowers are largely intact, but do not produce pollen, whereas the gynoecium of the male flowers is highly reduced though develops ovules lacking an embryo sac. The morphologically rather unreduced stamens of the female flower may have undergone a shift of function from producing pollen towards attracting pollinators by optical signals. The outer parenchyma of the gynoecium is strongly stained and after fertilisation, one through three ovules are maturing towards seeds. The presence of more than a single seed enclosed in the undivided endocarp is rare in Cordiaceae

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10³ and 66 x 10³, on Western blots. We have used this antibody to isolate five clones (λcs1, -2, -3, -4 and λj63) from λgt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone λcs3, we have demonstrated that the fusion proteins encoded by four of these clones (λcs1-4) share at least two epitopes with the 185 x 10³ M_r centrosomal antigen. This indicates that clones λcs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10³ bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10³ and 66 x 10³, on Western blots. We have used this antibody to isolate five clones (λcs1, -2, -3, -4 and λj63) from λgt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone λcs3, we have demonstrated that the fusion proteins encoded by four of these clones (λcs1-4) share at least two epitopes with the 185 x 10³ M_r centrosomal antigen. This indicates that clones λcs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10³ bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle

Identification of Eps15 as Antigen Recognized by the Monoclonal Antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila Brain

The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies

Evolutionary conservation of lampbrush-like loops in drosophilids

<p>Abstract</p> <p>Background</p> <p>Loopin-1 is an abundant, male germ line specific protein of <it>Drosophila melanogaster</it>. The polyclonal antibody T53-F1 specifically recognizes Loopin-1 and enables its visualization on the Y-chromosome lampbrush-like loop named kl-3 during primary spermatocyte development, as well as on sperm tails. In order to test lampbrush-like loop evolutionary conservation, extensive phase-contrast microscopy and immunostaining with T53-F1 antibody was performed in other drosophilids scattered along their genealogical tree.</p> <p>Results</p> <p>In the male germ line of all species tested there are cells showing giant nuclei and intranuclear structures similar to those of <it>Drosophila melanogaster </it>primary spermatocytes. Moreover, the antibody T53-F1 recognizes intranuclear structures in primary spermatocytes of all drosophilids analyzed. Interestingly, the extent and conformation of the staining pattern is species-specific. In addition, the intense staining of sperm tails in all species suggests that the terminal localization of Loopin-1 and its orthologues is conserved. A comparison of these cytological data and the data coming from the literature about sperm length, amount of sperm tail entering the egg during fertilization, shape and extent of both loops and primary spermatocyte nuclei, seems to exclude direct relationships among these parameters.</p> <p>Conclusion</p> <p>Taken together, the data reported strongly suggest that lampbrush-like loops are a conserved feature of primary spermatocyte nuclei in many, if not all, drosophilids. Moreover, the conserved pattern of the T53-F1 immunostaining indicates that a Loopin-1-like protein is present in all the species analyzed, whose localization on lampbrush-like loops and sperm tails during spermatogenesis is evolutionary conserved.</p

Multiple network properties overcome random connectivity to enable stereotypic sensory responses

Connections between neuronal populations may be genetically hardwired or random. In the insect olfactory system, projection neurons of the antennal lobe connect randomly to Kenyon cells of the mushroom body. Consequently, while the odor responses of the projection neurons are stereotyped across individuals, the responses of the Kenyon cells are variable. Surprisingly, downstream of Kenyon cells, mushroom body output neurons show stereotypy in their responses. We found that the stereotypy is enabled by the convergence of inputs from many Kenyon cells onto an output neuron, and does not require learning. The stereotypy emerges in the total response of the Kenyon cell population using multiple odor-specific features of the projection neuron responses, benefits from the nonlinearity in the transfer function, depends on the convergence:randomness ratio, and is constrained by sparseness. Together, our results reveal the fundamental mechanisms and constraints with which convergence enables stereotypy in sensory responses despite random connectivity