New Tools for Investigating the Comparative Biology of Caenorhabditis  briggsae and C. elegans

Abstract

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. C OMPARATIVE analysis of genes between related species is a powerful experimental approach, allowing one to exploit nature's experiments to illuminate the sequence basis of function. Commonly, conservation of sequence is used to delineate functional elements in a genome and is particularly effective with protein-coding sequence. However, in many instances differences in sequence may also be informative. For example, the small size of transcription factor binding sites allows for generation of new sites and loss of old sites, masking the conservation of functions (Andolfatto 2005). In other cases, a change in sequence is associated with a change in function that sheds light on the underlying mechanisms of gene action. In these instances, simple sequence comparisons are inadequate; they need to be supplemented with functional analysis.For comparative functional analysis of Caenorhabditis elegans, C. briggsae remains the preferred species. Although it shows an estimated 1.6 substitutions per neutral site with C. elegans (Stein et al. 2003), it remains the closest identified living relative. Both are selffertilizing hermaphrodites and have very similar morphology. Recent evidence suggests that the developmental cell lineage is remarkably constant between the two species (Zhao et al. 2008). However, changes in cellular signaling underlying these patterns have been revealed by quantitative study of development after cell ablation or manipulation of the signaling molecules (Felix 2007)...

A Method for Rapid and Simultaneous Mapping of Genetic Loci and Introgression Sizes in Nematode Species

“…We then tested the mapping using the SNP-based oligonucleotide microarray we had developed previously [19]. The mapping results agreed well with those using the current method (data not shown).…”

cel-mir-237 and its homologue, hsa-miR-125b, modulate the cellular response to ionizing radiation

“…Mutants were backcrossed to N2 6 times before being analyzed. The unc-119(ed3);[Cbr unc-119::mir-237]#7 and unc-119(ed3);[Cbr unc-119::mir-237]#44 strains were generated via micro-particle bombardment 73,74 of Cbr unc-119::mir-237 constructs into the C. elegans unc-119(ed3) starvation-induced lethal reporter system 75 . Primers for cloning and characterization are shown in Table S4.…”

Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis

“…Images were consecutively collected for both GFP and mCherry channels every 90 s from 41 focal planes for a total of 6 h. For each gene perturbation, images were collected for at least three replicate embryos. Automated lineage and gene expression profiling were performed as described (22). Wild-type embryos were curated up to ϳ350-cell stage for all wild-type embryos and to the equivalent stage or to the last editable stage for all the perturbed embryos by RNAi.…”