Bhagwati P. Gupta

Caenorhabditis briggsae Recombinant Inbred Line Genotypes Reveal Inter-Strain Incompatibility and the Evolution of Recombination

The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosomes physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae

BackgroundThe nematode C. briggsae serves as a useful model organism for comparative analysis of developmental and behavioral processes. The amenability of C. briggsae to genetic manipulations and the availability of its genome sequence have prompted researchers to study evolutionary changes in gene function and signaling pathways. These studies rely on the availability of forward genetic tools such as mutants and mapping markers.ResultsWe have computationally identified more than 30,000 polymorphisms (SNPs and indels) in C. briggsae strains AF16 and HK104. These include 1,363 SNPs that change restriction enzyme recognition sites (snip-SNPs) and 638 indels that range between 7 bp and 2 kb. We established bulk segregant and single animal-based PCR assay conditions and used these to test 107 polymorphisms. A total of 75 polymorphisms, consisting of 14 snip-SNPs and 61 indels, were experimentally confirmed with an overall success rate of 83%. The utility of polymorphisms in genetic studies was demonstrated by successful mapping of 12 mutations, including 5 that were localized to sub-chromosomal regions. Our mapping experiments have also revealed one case of a misassembled contig on chromosome 3.ConclusionsWe report a comprehensive set of polymorphisms in C. briggsae wild-type strains and demonstrate their use in mapping mutations. We also show that molecular markers can be useful tools to improve the C. briggsae genome sequence assembly. Our polymorphism resource promises to accelerate genetic and functional studies of C. briggsae genes.

New tools for investigating the comparative biology of Caenorhabditis briggsae and C. elegans.

Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.

Haloperidol-loaded intranasally administered lectin functionalized poly(ethylene glycol)-block-poly(D,L)-lactic-co-glycolic acid (PEG-PLGA) nanoparticles for the treatment of schizophrenia.

Lectin-functionalized, polyethylene glycol-block-poly-(D,L)-lactic-co-glycolic acid nanoparticles loaded with haloperidol were prepared with narrow size distributions and sizes <135nm. The nanoparticles exhibited high Solanum tuberosum lectin (STL) conjugation efficiencies, encapsulation efficiencies, and drug loading capacities. The in vitro release of haloperidol was 6-8% of the loaded amount in endo-lysosomal conditions over 96h, demonstrating minimal drug leakage and the potential for the efficient drug transport to the targeted brain tissue. The haloperidol released upon erosion was successful in displacing [(3)H] N-propylnorapomorphine and binding to bovine striatal dopamine D2 receptors. Both haloperidol-loaded nanoparticle formulations were found to be highly effective at inducing catalepsy. Intranasal administration of STL-functionalized nanoparticles increased the brain tissue haloperidol concentrations by 1.5-3-fold compared to non-STL-functionalized particles and other routes of administration. This formulation demonstrates promise in the reduction of the drug dose necessary to produce a therapeutic effect with antipsychotic drugs for the treatment of schizophrenia.

Behavior of Caenorhabditis elegans in alternating electric field and its application to their localization and control

Caenorhabditis elegans is an attractive model organism because of its genetic similarity to humans and the ease of its manipulation in the laboratory. Recently, it was shown that a direct current electric field inside microfluidic channel induces directed movement that is highly sensitive, reliable, and benign. In this letter, we describe the worm’s movement response to alternating electric fields in a similar channel setup. We demonstrate that the 1 Hz and higher frequency of alternating current field can effectively localize worms in the channel. This discovery could potentially help design microfluidic devices for high throughput automated analysis of worms.

Sexual Selection and Maintenance of Sex: Evidence from Comparisons of Rates of Genomic Accumulation of Mutations and Divergence of Sex-Related Genes in Sexual and Hermaphroditic Species of Caenorhabditis

Several hypotheses have been proposed to explain the persistence of dioecy despite the reproductive advantages conferred to hermaphrodites, including greater efficiency at purging deleterious mutations in the former. Dioecy can benefit from both mutation purging and accelerated evolution by bringing together beneficial mutations in the same individual via recombination and shuffling of genotypes. In addition, mathematical treatment has shown that sexual selection is also capable of mitigating the cost of maintaining separate sexes by increasing the overall fitness of sexual populations, and genomic comparisons have shown that sexual selection can lead to accelerated evolution. Here, we examine the advantages of dioecy versus hermaphroditism by comparing the rate of evolution in sex-related genes and the rate of accumulation of deleterious mutations using a large number of orthologs (11,493) in the dioecious Caenorhabditis remanei and the hermaphroditic Caenorhabditis briggsae. We have used this data set to estimate the deleterious mutation rate per generation, U, in both species and find that although it is significantly higher in hermaphrodites, both species are at least 2 orders of magnitude lower than the value required to explain the persistence of sex by efficiency at purging deleterious mutations alone. We also find that genes expressed in sperm are evolving rapidly in both species; however, they show a greater increase in their rate of evolution relative to genes expressed in other tissues in C. remanei, suggesting stronger sexual selection pressure acting on these genes in dioecious species. Interestingly, the persistence of a signal of rapid evolution of sperm genes in C. briggsae suggests a recent evolutionary origin of hermaphrodism in this lineage. Our results provide empirical evidence of increased sexual selection pressure in dioecious animals, supporting the possibility that sexual selection may play an important role in the maintenance of sexual reproduction.

Morphogenesis of the vulva and the vulval-uterine connection

The C. elegans hermaphrodite vulva is an established model system to study mechanisms of cell fate specification and tissue morphogenesis. The adult vulva is a tubular shaped organ composed of seven concentric toroids that arise from selective fusion between differentiated vulval progeny. The dorsal end of the vulval tubule is connected to the uterus via a multinucleate syncytium utse (uterine-seam) cell. The vulval tubule and utse are formed as a result of changes in morphogenetic processes such as cell polarity, adhesion, and invagination. A number of genes controlling these processes are conserved all the way up to human and function in similar developmental contexts. This makes it possible to extend the findings to other metazoan systems. Gene expression studies in the vulval and uterine cells have revealed the presence of regulatory networks specifying distinct cell fates. Thus, these two cell types serve as a good system to understand how gene networks confer unique cell identities both experimentally and computationally. This chapter focuses on morphogenetic processes during the formation of the vulva and its connection to uterus.

Microfluidic Approaches for Manipulating, Imaging, and Screening C. elegans

The nematode C. elegans (worm) is a small invertebrate animal widely used in studies related to fundamental biological processes, disease modelling, and drug discovery. Due to their small size and transparent body, these worms are highly suitable for experimental manipulations. In recent years several microfluidic devices and platforms have been developed to accelerate worm handling, phenotypic studies and screens. Here we review major tools and briefly discuss their usage in C. elegans research.

Conserved mechanism of Wnt signaling function in the specification of vulval precursor fates in C. elegans and C. briggsae.

The C. elegans hermaphrodite vulva serves as a paradigm for understanding how signaling pathways control organ formation. Previous studies have shown that Wnt signaling plays important roles in vulval development. To understand the function and evolution of Wnt signaling in Caenorhabditis nematodes we focused on C. briggsae, a species that is substantially divergent from C. elegans in terms of the evolutionary time scale yet shares almost identical morphology. We isolated mutants in C. briggsae that display multiple pseudo-vulvae resulting from ectopic VPC induction. We cloned one of these loci and found that it encodes an Axin homolog, Cbr-PRY-1. Our genetic studies revealed that Cbr-pry-1 functions upstream of the canonical Wnt pathway components Cbr-bar-1 (beta-catenin) and Cbr-pop-1(tcf/lef) as well as the Hox target Cbr-lin-39 (Dfd/Scr). We further characterized the pry-1 vulval phenotype in C. briggsae and C. elegans using 8 cell fate markers, cell ablation, and genetic interaction approaches. Our results show that ectopically induced VPCs in pry-1 mutants adopt 2° fates independently of the gonad-derived inductive and LIN-12/Notch-mediated lateral signaling pathways. We also found that Cbr-pry-1 mutants frequently show a failure of P7.p induction. A similar, albeit low penetrant, defect is also observed in C. elegans pry-1 mutants. The genetic analysis of the P7.p induction defect revealed that it was caused by altered regulation of lin-12 and its transcriptional target lip-1 (MAP kinase phosphatase). Thus, our results provide evidence for LIN-12/Notch-dependent and independent roles of Wnt signaling in promoting 2 degrees VPC fates in both nematode species.

Effect of pulse direct current signals on electrotactic movement of nematodes Caenorhabditis elegans and Caenorhabditis briggsae

The nematodes (worms) Caenorhabditiselegans and Caenorhabditisbriggsae are well-known model organisms to study the basis of animal development and behaviour. Their sinusoidal pattern of movement is highly stereotypic and serves as a tool to monitor defects in neurons and muscles that control movement. Until recently, a simple yet robust method to initiate movement response on-demand did not exist. We have found that the electrical stimulation in a microfluidic channel, using constant DC electric field, induces movement (termed electrotaxis) that is instantaneous, precise, sensitive, and fully penetrant. We have further characterized this behaviour and, in this paper, demonstrate that electrotaxis can also be induced using a pulse DC electric signal. Worms responded to pulse DC signals with as low as 30% duty cycle by moving towards the negative electrode at the same speed as constant DC fields (average speed of C. elegans = 296 ± 43 μm/s and C. briggsae = 356 ± 20 μm/s, for both constant and pulse DC electric fields with various frequencies). C. briggsae was found to be more sensitive to electric signals compared to C. elegans. We also investigated the turning response of worms to a change in the direction of constant and pulse DC signals. The response for constant DC signal was found to be instantaneous and similar for most worms. However, in the case of pulse DC signal, alterations in duty cycle affected the turning response time as well as the number of responding worms. Our findings show that pulse DC method allows quantitative measurement of response behaviour of worms and suggest that it could be used as a tool to study the neuronal basis of such a behaviour that is not observed under constant DC conditions.