Evolution and development of male-specific leg brushes in Drosophilidae.

The origin, diversification, and secondary loss of sexually dimorphic characters are common in animal evolution. In some cases, structurally and functionally similar traits have evolved independently in multiple lineages. Prominent examples of such traits include the male-specific grasping structures that develop on the front legs of many dipteran insects. In this report, we describe the evolution and development of one of these structures, the male-specific "sex brush." The sex brush is composed of densely packed, irregularly arranged modified bristles and is found in several distantly related lineages in the family Drosophilidae. Phylogenetic analysis using 250 genes from over 200 species provides modest support for a single origin of the sex brush followed by many secondary losses; however, independent origins of the sex brush cannot be ruled out completely. We show that sex brushes develop in very similar ways in all brush-bearing lineages. The dense packing of brush hairs is explained by the specification of bristle precursor cells at a near-maximum density permitted by the lateral inhibition mechanism, as well as by the reduced size of the surrounding epithelial cells. In contrast to the female and the ancestral male condition, where bristles are arranged in stereotypical, precisely spaced rows, cell migration does not contribute appreciably to the formation of the sex brush. The complex phylogenetic history of the sex brush can make it a valuable model for investigating coevolution of sex-specific morphology and mating behavior.

Secondary reversion to sexual monomorphism associated with tissue-specific loss of doublesex expression.

Animal evolution is characterized by frequent turnover of sexually dimorphic traits-new sex-specific characters are gained, and some ancestral sex-specific characters are lost, in many lineages. In insects, sexual differentiation is predominantly cell autonomous and depends on the expression of the doublesex (dsx) transcription factor. In most cases, cells that transcribe dsx have the potential to undergo sex-specific differentiation, while those that lack dsx expression do not. Consistent with this mode of development, comparative research has shown that the origin of new sex-specific traits can be associated with the origin of new spatial domains of dsx expression. In this report, we examine the opposite situation-a secondary loss of the sex comb, a male-specific grasping structure that develops on the front legs of some drosophilid species. We show that while the origin of the sex comb is linked to an evolutionary gain of dsx expression in the leg, sex comb loss in a newly identified species of Lordiphosa (Drosophilidae) is associated with a secondary loss of dsx expression. We discuss how the developmental control of sexual dimorphism affects the mechanisms by which sex-specific traits can evolve.

The steroid-hormone ecdysone coordinates parallel pupariation neuromotor and morphogenetic subprograms via epidermis-to-neuron Dilp8-Lgr3 signal induction.

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of Drosophila larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling.

Diverse Cis-Regulatory Mechanisms Contribute to Expression Evolution of Tandem Gene Duplicates.

Pairs of duplicated genes generally display a combination of conserved expression patterns inherited from their unduplicated ancestor and newly acquired domains. However, how the cis-regulatory architecture of duplicated loci evolves to produce these expression patterns is poorly understood. We have directly examined the gene-regulatory evolution of two tandem duplicates, the Drosophila Ly6 genes CG9336 and CG9338, which arose at the base of the drosophilids between 40 and 60 Ma. Comparing the expression patterns of the two paralogs in four Drosophila species with that of the unduplicated ortholog in the tephritid Ceratitis capitata, we show that they diverged from each other as well as from the unduplicated ortholog. Moreover, the expression divergence appears to have occurred close to the duplication event and also more recently in a lineage-specific manner. The comparison of the tissue-specific cis-regulatory modules (CRMs) controlling the paralog expression in the four Drosophila species indicates that diverse cis-regulatory mechanisms, including the novel tissue-specific enhancers, differential inactivation, and enhancer sharing, contributed to the expression evolution. Our analysis also reveals a surprisingly variable cis-regulatory architecture, in which the CRMs driving conserved expression domains change in number, location, and specificity. Altogether, this study provides a detailed historical account that uncovers a highly dynamic picture of how the paralog expression patterns and their underlying cis-regulatory landscape evolve. We argue that our findings will encourage studying cis-regulatory evolution at the whole-locus level to understand how interactions between enhancers and other regulatory levels shape the evolution of gene expression.

Multispecies Analysis of Expression Pattern Diversification in the Recently Expanded Insect Ly6 Gene Family.

Gene families often consist of members with diverse expression domains reflecting their functions in a wide variety of tissues. However, how the expression of individual members, and thus their tissue-specific functions, diversified during the course of gene family expansion is not well understood. In this study, we approached this question through the analysis of the duplication history and transcriptional evolution of a rapidly expanding subfamily of insect Ly6 genes. We analyzed different insect genomes and identified seven Ly6 genes that have originated from a single ancestor through sequential duplication within the higher Diptera. We then determined how the original embryonic expression pattern of the founding gene diversified by characterizing its tissue-specific expression in the beetle Tribolium castaneum, the butterfly Bicyclus anynana, and the mosquito Anopheles stephensi and those of its duplicates in three higher dipteran species, representing various stages of the duplication history (Megaselia abdita, Ceratitis capitata, and Drosophila melanogaster). Our results revealed that frequent neofunctionalization episodes contributed to the increased expression breadth of this subfamily and that these events occurred after duplication and speciation events at comparable frequencies. In addition, at each duplication node, we consistently found asymmetric expression divergence. One paralog inherited most of the tissue-specificities of the founder gene, whereas the other paralog evolved drastically reduced expression domains. Our approach attests to the power of combining a well-established duplication history with a comprehensive coverage of representative species in acquiring unequivocal information about the dynamics of gene expression evolution in gene families.

Evolution of sex-specific traits through changes in HOX-dependent doublesex expression.

Almost every animal lineage is characterized by unique sex-specific traits, implying that such traits are gained and lost frequently in evolution. However, the genetic mechanisms responsible for these changes are not understood. In Drosophila, the activity of the sex determination pathway is restricted to sexually dimorphic tissues, suggesting that spatial regulation of this pathway may contribute to the evolution of sex-specific traits. We examine the regulation and function of doublesex (dsx), the main transcriptional effector of the sex determination pathway, in the development and evolution of Drosophila sex combs. Sex combs are a recent evolutionary innovation and show dramatic diversity in the relatively few Drosophila species that have them. We show that dsx expression in the presumptive sex comb region is activated by the HOX gene Sex combs reduced (Scr), and that the male isoform of dsx up-regulates Scr so that both genes become expressed at high levels in this region in males but not in females. Precise spatial regulation of dsx is essential for defining sex comb position and morphology. Comparative analysis of Scr and dsx expression reveals a tight correlation between sex comb morphology and the expression patterns of both genes. In species that primitively lack sex combs, no dsx expression is observed in the homologous region, suggesting that the origin and diversification of this structure were linked to the gain of a new dsx expression domain. Two other, distantly related fly lineages that independently evolved novel male-specific structures show evolutionary gains of dsx expression in the corresponding tissues, where dsx may also be controlled by Scr. These findings suggest that changes in the spatial regulation of sex-determining genes are a key mechanism that enables the evolution of new sex-specific traits, contributing to some of the most dramatic examples of phenotypic diversification in nature.

Distinct developmental mechanisms underlie the evolutionary diversification of Drosophila sex combs.

Similar selective pressures can lead to independent origin of similar morphological structures in multiple evolutionary lineages. Developmental mechanisms underlying convergent evolution remain poorly understood. In this report, we show that similar sex comb morphology in closely related Drosophila species is produced by different cellular mechanisms. The sex comb is a recently evolved, male-specific array of modified bristles derived from transverse bristle rows found on the first thoracic legs in both sexes. "Longitudinal" sex combs oriented along the proximo-distal leg axis evolved independently in several Drosophila lineages. We show that in some of these lineages, sex combs originate as one or several transverse bristle rows that subsequently rotate 90 degrees and align to form a single longitudinal row. In other species, bristle cells that make up the sex combs arise in their final longitudinal orientation. Thus, sex combs can develop through either sex-specific patterning of bristle precursor cells or male-specific morphogenesis of sexually monomorphic precursors. Surprisingly, the two mechanisms produce nearly identical morphology in some species. Phylogenetic analysis shows that each of these mechanisms has probably evolved repeatedly in different Drosophila lineages, suggesting that selection can recruit different cellular processes to produce similar functional solutions.

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta.

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.

Development of the adult leg epidermis in Manduca sexta: contribution of different larval cell populations.

During metamorphosis of the tobacco hornworm Manduca sexta, the simple thoracic legs of the larva are remodeled into the more complex adult legs. Most of the adult leg epidermis derives from the adult primordia, small sets of epidermal cells located in specific regions of the larval leg, which proliferate rapidly in the final larval instar. In contrast, the contribution of the epidermal cells outside the primordia is unknown. In this study we have determined their contribution to the adult leg by labeling them with 5-bromodeoxyuridine (BUdR) and following their fate. Although the labeled cells diminished drastically in number, small groups of these cells persisted into the midpupal stage suggesting that they do contribute to the adult leg epidermis. We also found that during the wandering stage the adult primordia went through active proliferation and very little cell death, while the cells outside the primordia went through extensive cell death accounting for the decrease in their number. Our results indicate that two distinct cell populations exist outside the adult primordia. Most cells belong to the first population, which is larval-specific and disappears through apoptosis early in metamorphosis. The second population consists of polymorphic cells that contribute to the larval, pupal and adult leg epidermis.

Synthesis and characterization of dibenzodioxadiselenafulvalene.

Dibenzodioxadiselenafulvalene (DBOSF) was prepared and the cis and trans isomers were separated. X-ray crystallographic analysis revealed the presence of Se- - -H and O- - -H hydrogen bonds in both crystals. The electrochemical properties of cis- and trans-DBOSFs together with dibenzotetraoxafulvalene (DBTOF) and cis- and trans-dibenzodioxadithiafulvalenes were investigated by means of cyclic voltammetry and DFT calculation. The electron-donating ability and the stability of oxidized forms of DBOSFs are improved compared with those of DBTOF.