Nanoscale organization of hedgehog is essential for long-range signaling.

Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.

A crazy ants' crazy form of reproduction: Causes and consequences.

The yellow crazy ant, or the long-legged ant, Anoplolepis gracilipes (formerly Anoplolepis longipes) - named so for its meandering movements when disturbed, possibly owing to its long legs and antennae - is globallywidespread and currently classified as one of '100 of the world's worst invasive species' (Lowe et al. 2000). This status is assigned to species that are non-native in a region and cause significant negative ecological and/or socioeconomic impacts, including declines in native biodiversity, changes in native ecosystem structure and function, and the breakdown of native biogeographic realms. Possibly, themost devastating and multipronged impacts of A. gracilipes have been observed on island ecosystems, such as on Christmas Island in the Indian Ocean, where it impacted the entire island ecosystem by reducing arthropod, reptile, bird, and mammalian diversity on the forest floor and canopy, causing an 'invasional meltdown' (O'Dowd et al. 2003).

Introgressive hybridization and evolution of a novel protein phenotype: glue protein profiles in the nasuta-albomicans complex of Drosophila.

Glue proteins are tissue-specific proteins synthesized by larval salivary gland cells of Drosophila. In Drosophila nasuta nasuta and D. n. albomicans of the nasuta subgroup, the genes that encode the major glue protein fractions are X-linked. In the present study, these X-linked markers have been employed to trace the pattern of introgression of D. n. nasuta and D. n. albomicans genomes with respect to the major glue protein fractions in their interracial hybrids, called cytoraces. These cytoraces have inherited the chromosomes of both parents and have been maintained in the laboratory for over 400-550 generations. The analysis has revealed that cytoraces with D. n. albomicans X chromosome show either D. n. nasuta pattern or a completely novel pattern of glue protein fractions. Further, quantitative analysis also shows lack of correlation between the chromosomal pattern of inheritance and overall quantity of the major glue protein fractions in the cytoraces. Thus, in cytoraces the parental chromosomes are not just differentially represented but there is evidence for introgression even at the gene level.

Population Genetics of DROSOPHILA NASUTA NASUTA, DROSOPHILA NASUTA ALBOMICANA and Their Hybrids. I. Karyotypic Mosaicism in the Hybrid Populations.

D. n. nasuta and D. n. albomicana constitute a pair of chromosomal races with 2n=8 and 2n=6, respectively. The F(1) of these has 2n=7 and it is fertile. There exists a state of karyotypic mosaicism as evidenced by the presence of 26 types of chromosome combinations in F(2), F(3) and F(10) populations. In the midst of this karyotypic noise, the karyotype similar to that of F(1) reached 51% of the population. Implications of these findings are discussed.

Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms.

The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred approximately 2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.

Patterns of replication in the neo-sex chromosomes of Drosophila nasuta albomicans.

Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species of Drosophila.

Incipient sexual isolation in the nasuta-albomicans complex of Drosophila: no-choice experiments.

Drosophila nasuta nasuta and Drosophila nasuta albomicans are cross-fertile races of Drosophila. Hybridization between these races in the laboratory has given rise to new races (Cytoraces), among which karyotypic composition differs from one another and also from those of the parental races. In this study, we search for the evidence of incipient reproductive isolation among the parental races and four Cytoraces by assessing the fraction of no-matings, mating latency and copulation duration in all possible types of homo- and heterogamic crosses (N = 4184). In no-choice conditions, the latency time (time to initiation of copulation) is lower in homogamic crosses than in heterogamic crosses for both parental races and Cytoraces. Latency time and copulation duration are negatively correlated, whereas fraction of no matings is positively correlated with latency time. Thus these six closely related races of the nasuta-albomicans complex show the initiation of the earliest stages of pre-zygotic isolation, manifested as a tendency for matings to be initiated earlier and more often, and for a longer duration, among homogamic rather than heterogamic individuals

Incipient sexual isolation in the nasuta-albomicans complex of Drosophila: mating preference in male-, female- and multiple-choice mating experiments.

Interracial divergence is an important facet of speciation. The nasuta-albomicans complex of Drosophila with sixteen morphologically identical, karyotypically different but cross-fertile races is an excellent system to study a few dimensions of raciation. Drosophila nasuta nasuta, Drosophila nasuta albomicans, Cytorace 1, Cytorace 2, Cytorace 3 and Cytorace 4 of this subgroup have been subjected to male-, female- and multiple-choice mating experiments. Out of 8456 crosses conducted, 7185 had successful matings. The overall impression is that mating is far from random amongst these six closely related races of the nasuta-albomicans complex. The males of D. n. albomicans, Cytorace 1 and Cytorace 4 in male-choice, the females of Cytorace 1 and Cytorace 2 in female-choice, and the males and females of D. n. nasuta, D. n. albomicans, Cytorace 1 and Cytorace 4 against the males and females of Cytorace 2 in multiple-choice experiments, had significantly more homogamic matings than expected. Thus in this study of evolutionary experimentation on raciation under laboratory conditions, we have documented the initiation of preference for conspecific matings among closely related and independently evolving members of the nasuta-albomicans complex of Drosophila.

Hybridization, transgressive segregation and evolution of new genetic systems in Drosophila.

Introgressive hybridization facilitates incorporation of genes from one species into the gene pool of another. Studies on long-term effects of introgressive hybridization in animal systems are sparse. Drosophila nasuta (2n = 8) and D. albomicans (2n = 6)-a pair of allopatric, morphologically almost identical, cross-fertile members of the nasuta subgroup of the immigrans species group-constitute an excellent system to analyse the impact of hybridization followed by transgressive segregation of parental characters in the hybrid progeny. Hybrid populations of D. nasuta and D. albomicans maintained for over 500 generations in the laboratory constitute new recombinant hybrid genomes, here termed cytoraces. The impact of hybridization, followed by introgression and transgressive segregation, on chromosomal constitution and karyotypes, some fitness parameters, isozymes, components of mating behaviour and mating preference reveals a complex pattern of interracial divergence among parental species and cytoraces. This assemblage of characters in different combinations in a laboratory hybrid zone allows us to study the emergence of new genetic systems. Here, we summarize results from our ongoing studies comparing these hybrid cytoraces with the parental species, and discuss the implications of these findings for our understanding of the evolution of new genetic systems.

Pattern of sexual isolation between parental races (Drosophila nasuta nasuta and D.n. albomicans) and the newly evolved races (Cytorace I and II).

D.n. nasuta, D.n. albomicans, Cytorace I and Cytorace II [Chromosoma, 93 (1986) 243] are closely related strains of the nasuta subgroup. Through male choice and female choice experiments, the pattern of sexual isolation if any, among them was analysed. Differential mating preference of males and females of these races suggests that they are passing through the process of anagenesis. Therefore, this, as an evolutionary experiment under laboratory conditions, offers a rare opportunity to witness different pattern and levels of divergence among four cytogentically parsimonious races of Drosophila.

Karyotype instability in the ponerine ant genus Diacamma.

The queenless ponerine ant Diacamma ceylonense and a population of Diacamma from the Nilgiri hills which we refer to as 'nilgiri', exhibit interesting similarities as well as dissimilarities. Molecular phylogenetic study of these morphologically almost similar taxa has shown that D. ceylonense is closely related to 'nilgiri' and indicates that 'nilgiri' is a recent diversion in the Diacamma phylogenetic tree. However, there is a striking behavioural difference in the way reproductive monopoly is maintained by the respective gamergates (mated egg laying workers), and there is evidence that they are genetically differentiated, suggesting a lack of gene flow. To develop a better understanding of the mechanism involved in speciation of Diacamma, we have analysed karyotypes of D. ceylonense and 'nilgiri'. In both, we found surprising inter-individual and intra-individual karyotypic mosaicism. The observed numerical variability, both at intra-individual and inter-individual levels, does not appear to have hampered the sustainability of the chromosomal diversity in each population under study. Since the related D. indicum displays no such intra-individual or inter-individual variability whatsoever under identical experimental conditions, these results are unlikely to be artifacts. Although no known mechanisms can account for the observed karyotypic variability of this nature, we believe that the present findings on the ants under study would provide opportunities for exciting new discoveries concerning the origin, maintenance and significance of intra-individual and inter-individual karyotypic mosaicism.

The complex spatio-temporal regulation of the Drosophila myoblast attractant gene duf/kirre.

A key early player in the regulation of myoblast fusion is the gene dumbfounded (duf, also known as kirre). Duf must be expressed, and function, in founder cells (FCs). A fixed number of FCs are chosen from a pool of equivalent myoblasts and serve to attract fusion-competent myoblasts (FCMs) to fuse with them to form a multinucleate muscle-fibre. The spatial and temporal regulation of duf expression and function are important and play a deciding role in choice of fibre number, location and perhaps size. We have used a combination of bioinformatics and functional enhancer deletion approaches to understand the regulation of duf. By transgenic enhancer-reporter deletion analysis of the duf regulatory region, we found that several distinct enhancer modules regulate duf expression in specific muscle founders of the embryo and the adult. In addition to existing bioinformatics tools, we used a new program for analysis of regulatory sequence, PhyloGibbs-MP, whose development was largely motivated by the requirements of this work. The results complement our deletion analysis by identifying transcription factors whose predicted binding regions match with our deletion constructs. Experimental evidence for the relevance of some of these TF binding sites comes from available ChIP-on-chip from the literature, and from our analysis of localization of myogenic transcription factors with duf enhancer reporter gene expression. Our results demonstrate the complex regulation in each founder cell of a gene that is expressed in all founder cells. They provide evidence for transcriptional control--both activation and repression--as an important player in the regulation of myoblast fusion. The set of enhancer constructs generated will be valuable in identifying novel trans-acting factor-binding sites and chromatin regulation during myoblast fusion in Drosophila. Our results and the bioinformatics tools developed provide a basis for the study of the transcriptional regulation of other complex genes.

Metaphase karyotype differentiation in eight species of the montium subgroup of Drosophila.

The metaphase karyotypes of eight species of the montium species subgroup of Drosophila are described, of which the information pertaining to six species is new to the literature. Each of these species studied was distinctly different from the others with respect to the metaphase chromosomes, this remarkable degree of differentiation being mainly due to the extensive variability of the 4th and of the Y chromosomes.

Population genetics of Drosophila nasuta nasuta, Drosophila nasuta albomicana and their hybrids. VI. Pattern of meiotic F1 chromosome segregation.

Interracial hybridization experiments between D. n. nasuta and D. n. albomicana were made. During gametogenesis there was selective elimination of abnormal gametes in F1 females; hence, only normal gametes were produced. F1 males yielded both normal and abnormal gametes. Fertility tests on F2 and backcross progeny showed males are more often sterile than females. The implications of these findings are discussed.

The chromosomes of two Drosophila races: D. nasuta nasuta and D. n. albomicana. I. Distribution and differentiation of heterochromatin.

Heterochromatin distribution and differentiation in metaphase chromosomes of two morphologically identical Drosophila races, D. nasuta nasuta and D. n. albomicana, have been studied by C- and N-banding methods. -- The total heterochromatin values differ only slightly between these races. However, homologous chromosomes of the two Drosophila forms show striking differences in the size of heterochromatin regions and there is an alternating pattern in D. n. nasuta and D. n. albomicana of chromosomes which contain more, or respectively less heterochromatin than their counterparts in the other race. -- Three different N-banding patterns could be obtained depending on the conditions of the method employed: One banding pattern occurs which corresponds to the C-banding pattern. Another pattern is the reverse of the C-band pattern; the euchromatic chromosome regions and the centromeres are stained whereas the pericentric heterochromatin regions remain unstained. In the Y chromosomes of both races and in chromosome 4 of D. n. albomicana, however, the heterochromatin is further differentiated. In the third N-banding pattern only the centromeres are deeply stained. Furthermore, between the races, subtle staining differences in the pericentric heterochromatin regions can be observed as verified in F1 hybrids. On the basis of C- and N-banding results specific aspects of chromosomal differences between D. n. nasuta and D. n. albomicana are discussed.

The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicana. II. Differences between their microchromosomes.

The microchromosomes of the totally cross fertile Drosophila races, D. nasuta nasuta and D. nasuta albomicana have been studied in metaphase and polytene nuclei. In metaphase the microchromosome of D. n. albomicana is nearly five times longer than the homologous chromosome in D. n. nasuta. As shown by C-banding these length differences are mainly due to a massive addition of heterochromatin to the D. n. albomicana chromosome. In polytene nuclei these striking heterochromatin differences between the microchromosomes of the two Drosophila races cannot be observed. Analysis of the polytene banding pattern shows that the microchromosomes of both races differ by an inversion and by a duplication, present only in D. n. albomicana. the location and orientation of the duplicated regions in D. n. albomicana leads to a specific loop like chromosome configuration. On the basis of these differences within the Drosophila races studied it is assumed that the karyotype of D. n. albomicana is a more recent evolutionary product.

Biochemical phylogeny of seven Indian species of the montium subgroup of Drosophila.

Seven species of the montium subgroup of Drosophila found in South India were analysed with polyacrylamide disc electrophoresis. Eleven loci coding for enzymes were surveyed. The genetic distances between species have been measured. The dendrogram revealed three clusters. The first cluster includes D. nagarholensis, D. agumbensis, D. jambulina and D. kikkawai; the second group consists of D. truncata and D. anomelani; while D. mysorensis was the only species of the third group.

The chromosomes of three species of the Montium subgroup of Drosophila.

Chromosomes of Drosophila truncata, D. nagarholensis, and D. agumbensis of the montium subgroup were analyzed to record the divergence attained at the level of metaphase complement and the extent of inversion polymorphism. Results are presented in this article.