Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. Here, we sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. We compared the activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster and found strong functional conservation among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, we found three novel wg enhancer activities in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. Our results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. We suggest that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers.enhancers | novelty | gene regulation | development | pigmentation

Vulval differentiation in Caenorhabditis elegans is controlled by a conserved signal transduction pathway mediated by Ras and a kinase cascade that includes Raf, Mek and MAPK. Activation of this cascade is positively regulated by a number of proteins such as KSR (kinase suppressor of Ras), SUR‐8/SOC‐2, SUR‐6/PP2A‐B and CDF‐1. We describe the functional characterization of sur‐7 and several genes that regulate signaling downstream of ras. We identified sur‐7 by isolating a mutation that suppresses an activated ras allele, and showed that SUR‐7 is a divergent member of the cation diffusion facilitator family of heavy metal ion transporters that is probably localized to the endoplosmic recticulum membrane and regulates cellular Zn2+ concentrations. Genetic double mutant analyses suggest that the SUR‐7‐mediated effect is not a general toxic response. Instead, Zn2+ ions target a specific step of the pathway, probably regulation of the scaffolding protein KSR. Biochemical analysis in mammalian cells indicates that high Zn2+ concentration causes a dramatic increase of KSR phosphorylation. Genetic analysis also indicates that PP2A phosphatase and PAR‐1 kinase act downstream of Raf to positively and negatively regulate KSR activity, respectively.

Sexual dimorphism is widespread throughout the metazoa and plays important roles in mate recognition and preference, sexbased niche partitioning, and sex-specific coadaptation. One notable example of sex-specific differences in insect body morphology is presented by the higher diptera, such as Drosophila, in which males develop fewer abdominal segments than females. Because diversity in segment number is a distinguishing feature of major arthropod clades, it is of fundamental interest to understand how different numbers of segments can be generated within the same species. Here we show that sex-specific and segment-specific regulation of the Wingless (Wg) morphogen underlies the development of sexually dimorphic adult segment number in Drosophila. Wg expression is repressed in the developing terminal male abdominal segment by the combination of the Hox protein Abdominal-B (Abd-B) and the sex-determination regulator Doublesex (Dsx). The subsequent loss of the terminal male abdominal segment during pupation occurs through a combination of developmental processes including segment compartmental transformation, apoptosis, and suppression of cell proliferation. Furthermore, we show that ectopic expression of Wg is sufficient to rescue this loss. We propose that dimorphic Wg regulation, in concert with monomorphic segment-specific programmed cell death, are the principal mechanisms of sculpting the sexually dimorphic abdomen of Drosophila.morphogenesis | segmentation | homeotic | epithelia B rachycera, higher diptera that include drosophilidae, exhibit an evolutionary trend toward reduced abdominal size that contributes to swift, maneuverable flight (1). Such reduction is especially pronounced within the infraorder Muscomorpha. Within this group of flies, abdominal reduction is sexually dimorphic such that adult males have fewer segments than females. Lower diptera, which includes mosquitoes and midges, retain ancestral morphology with respect to segment number; both adult males and females generate eight abdominal segments. In Muscomorpha the most posterior adult abdominal segments (all or a subset of segments A5-A8) are modified in females, usually as a telescoping ovipositor, whereas corresponding segments are absent in males (2). In all diptera, segment number is monomorphic during embryogenesis and larval development, reflecting the basal insect body plan of three head, three thoracic, and 11 abdominal segments. For most diptera, only embryonic abdominal segments 1-8 generate adult abdominal tissue (only segments 1-7 in the drosophilidae). The more posterior embryonic segments contribute to the adult genitalia. During pupation, sex-specific developmental programs are deployed that sculpt sexually dimorphic segment morphology and number.The posterior abdomen of Drosophila melanogaster serves as an excellent model to study the development of these sex-specific morphologies. Posterior abdominal segment identity, morphology, and number in both sexes is regulated by the Hox protein Abdominal-B (Abd-B) (3, 4). Abd-B ex...

Sister chromatid cohesion is fundamental for the faithful transmission of chromosomes during both meiosis and mitosis. Proteins involved in this process are highly conserved from yeasts to humans. In screenings for sterile animals with abnormal vulval morphology, mutations in the Caenorhabditis elegans evl-14 and scc-3 genes were isolated. Defects in cell divisions were observed in germ line as well as in vulval and somatic gonad lineages. Through positional cloning of these genes, we have shown that EVL-14 and SCC-3 are likely the only C. elegans homologs of the yeast sister chromatid cohesion proteins Pds5 and Scc3, respectively. Both evl-14 and scc-3 mutants displayed defects in the meiotic germ line. In evl-14 mutants, synaptonemal complexes (SCs) were detectable but more than the usual six DAPI (4,6-diamidino-2-phenylindole)-positive structures were seen at diakinesis, suggesting that EVL-14/PDS-5 is important for the maintenance of sister chromatid cohesion in late prophase. In scc-3 mutant animals, normal SCs were not visible and ϳ24 DAPI-positive structures were seen at diakinesis, indicating that SCC-3 is necessary for sister chromatid cohesion. Immunostaining revealed that localization of REC-8, a homolog of the yeast meiotic cohesin subunit Rec8, to the chromosomes depends on the presence of SCC-3 but not that of EVL-14/PDS-5. scc-3 RNA interference (RNAi)-treated embryos were 100% lethal and displayed defects in cell divisions. evl-14 RNAi caused a range of phenotypes. These results indicate that EVL-14/PDS-5 and SCC-3 have functions in both mitosis and meiosis.In eukaryotes, the faithful transmission of chromosomes during both meiosis and mitosis is critical for species propagation and the survival of individual organisms. Sister chromatid cohesion is fundamental to this process. In mitosis and meiosis II, sister chromatid cohesion ensures proper binding of sister kinetochores to spindle pole microtubules. In meiosis I, it is also required to arrange kinetochores associated with homologous chromosomes to face the appropriate spindle poles. Sister chromatid cohesion is established during the S phase and persists until the onset of anaphase (for reviews, see references 27, 35, and 47).A multiprotein complex called cohesin is a major effector of sister chromatid cohesion. The complex consists of at least four conserved subunits, homologs of which have been identified in all major phyla. In Saccharomyces cerevisiae they are Scc1/ Mcd1, Scc3, Smc1, and Smc3, which colocalize to chromatin in an interdependent manner (18,31,46). Scc1 is replaced in meiosis by its paralogue Rec8 (7, 25). In Caenorhabditis elegans there are four Scc1 homologs of which one, REC-8, is the likely worm ortholog of yeast Rec8 (34). Scc3 has multiple homologs in other species as well. Fission yeast contains two homologs of Scc3: Psc3 and the predicted meiosis-specific member Rec11 (13, 26,45). Three Scc3 homologs, SA1 to SA3 (stromal antigen 1 to 3) (of which one, SA3, joins the meiotic cohesin complex) (8, 28,36,42), have been fo...

We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations in dli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of the dli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-cell dli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective in dli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1 mutant background, indicating this is not an essential domain for DLI-1 function. INTRODUCTIONCytoplasmic dynein is a large multisubunit complex composed of two motor proteins, the heavy chains, and several associated subunits that have been named light, light intermediate, and intermediate chains. These subunits have no sequence homology; instead, the names reflect their relative molecular weights. Cytoplasmic dynein is the major microtubule minus-end-directed motor protein and has been implicated in a variety of cellular processes, during both interphase and mitosis. Membranous vesicle transport, endoplasmic reticulum-to-Golgi transport, and axonal retrograde transport are all dynein-dependent processes (Lacey and Haimo, 1992;Dillman et al., 1996;Presley et al., 1997).Dynein's mitotic roles are numerous. Function blocking antibody experiments against heavy chain in vertebrate cells have shown the motor protein is required for proper spindle formation and centrosome separation (Vaisberg et al., 1993). Similar results were observed in Drosophila heavy chain mutants with additional observations suggesting dynein is required to maintain centrosome association with the nuclear envelope (Robinson et al., 1999). In both Aspergillus nidulans and Neurospora crassa, dynein heavy chain mutations reveal a role for the complex in nuclear migration (Plamann et al., 1994;Xiang et al., 1995). Other recent work suggests dynein may mediate microtubule binding at the kinetochore (Wordeman and Mitchison, 1995).The one-cell Caenorhabditis elegans embryo is also an excellent model system for investigating the roles of dynein, its subunits, and the proteins with which it interacts. When cytoplasmic dynein heavy chain (dhc-1) was eliminated through RNA-mediated gene interference (RNAi), all of the above-mentioned phenotypes were o...

Background: Hox transcription factors are deeply conserved proteins that guide development through regulation of diverse target genes. Furthermore, alteration in Hox target cis-regulation has been proposed as a major mechanism of animal morphological evolution. Crucial to understanding how homeotic genes sculpt the developing body and contribute to the evolution of form is identification and characterization of regulatory targets. Because target specificity is achieved through physical or genetic interactions with cofactors or co-regulators, characterizing interactions between homeotic genes and regulatory partners is also critical. In Drosophila melanogaster, sexually dimorphic abdominal morphology results from sex-specific gene regulation mediated by the Hox protein Abdominal-B (Abd-B) and products of the sex-determination gene doublesex (dsx). Together these transcription factors regulate numerous sex-specific characters, including pigmentation, cuticle morphology, and abdominal segment number. Results: We show Dsx expression in the developing D. melanogaster pupal abdomen is spatiotemporally dynamic, correlating with segments that undergo sexually dimorphic morphogenesis. Furthermore, our genetic analyses show Dsx expression is Abd-B dependent. Conclusions: Doublesex and Abd-B are not only requisite co-regulators of sexually dimorphic abdominal morphology. We propose that dsx is itself a transcriptional target of Abd-B. These data present a testable hypothesis about the evolution of sexually dimorphic segment number in Diptera.

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies [1][2][3] . During pupation, which spans approximately 96 hours (at 25 °C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4 . Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells-LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy. Video LinkThe video component of this article can be found at https://www.jove.com/video/3139/ Protocol Day 1 Before you start:A healthy population of flies should be maintained using standard culturing protocols: remove adults from bottles or vials after 3-4 days of egglay and allow development to proceed at a constant temperature until wandering 3 rd instar larvae initiate pupariation. The larval/pupal transition is marked by the formation of the prepupae (considered 0 hours after puparium formation-APF). Immobile pupae are distinguished from older pupae by their white coloration and from larvae that have not yet begun pupariation by their oblong,rounded shape and protrusion of the anterior spiracles. You will need:• A paint brush for collecting pupae • A humid chamber for culturing pupae: A Petri dish lined with wetted paper towel and covered with filter paper marked for the various time points of collection, genotype or sex of the pupae • 1X Phosphate buffered saline (PBS) for washing pupae Collection, culturing and staging pupae 1. Using a wetted paintbrush gently remove 0hr APF pupae from culture bottles/vials and place them in the lid of the humid chamber. 2. Using the paintbrush and 1X PBS gently wash the pupae to remove debris from the pupa case 3. If nece...

It is hypothesized that butterfly wing scale geometry and surface patterning may function to improve aerodynamic efficiency. In order to investigate this hypothesis, a method to measure butterfly flapping kinematics optically over long uninhibited flapping sequences was developed. Statistical results for the climbing flight flapping kinematics of 11 butterflies, based on a total of 236 individual flights, both with and without their wing scales, are presented. Results show, that for each of the 11 butterflies, the mean climbing efficiency decreased after scales were removed. Data was reduced to a single set of differences of climbing efficiency using are paired t-test. Results show a mean decrease in climbing efficiency of 32.2% occurred with a 95% confidence interval of 45.6%-18.8%. Similar analysis showed that the flapping amplitude decreased by 7% while the flapping frequency did not show a significant difference. Results provide strong evidence that butterfly wing scale geometry and surface patterning improve butterfly climbing efficiency. The authors hypothesize that the wing scale's effect in measured climbing efficiency may be due to an improved aerodynamic efficiency of the butterfly and could similarly be used on flapping wing micro air vehicles to potentially achieve similar gains in efficiency.