Helen M. Chamberlin

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.

Evolutionary innovation of the excretory system in Caenorhabditis elegans

The evolution of complexity relies on changes that result in new gene functions. Here we show that the unique morphological and functional features of the excretory duct cell in C. elegans result from the gain of expression of a single gene. Our results show that innovation can be achieved by altered expression of a transcription factor without coevolution of all target genes.

Pax2/5/8 proteins promote cell survival in C. elegans

Programmed cell death, or apoptosis, plays an important role during normal development, and is disrupted in a range of disease states. Although the key molecular events that occur during apoptosis are well characterized, less is known about the regulatory inputs that influence whether a cell will live or die. Work in mouse and human cells has shown that Pax transcription factors can influence cell death and promote cell survival, but the mechanism for their activity is not clear. Here, we show that two Pax2/5/8-related genes (egl-38 and pax-2) influence both somatic and germline cell death in C. elegans. Using genetic and molecular experiments, we show that the Pax proteins act as transcriptional regulators of ced-9, the C. elegans bcl-2 gene. These results identify a mechanism for Pax2/5/8-mediated regulation of cell death, and underscore the importance of transcriptional regulation of core apoptotic pathway genes in influencing cell survival.

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.

Transcriptional Regulation of AQP-8, a Caenorhabditis elegans Aquaporin Exclusively Expressed in the Excretory System, by the POU Homeobox Transcription Factor CEH-6

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5′ deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer sites function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6s mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney.

Evolution of regulatory elements producing a conserved gene expression pattern in Caenorhabditis

Summary Natural selection acts at the level of function, not at the logistical level of how organisms achieve a particular function. Consequently, significant DNA sequence and regulatory differences can achieve the same function, such as a particular gene expression pattern. To investigate how regulatory features underlying a conserved function can evolve, we compared the regulation of a conserved gene expression pattern in the related species Caenorhabditis elegans and C. briggsae. We find that both C. elegans and C. briggsae express the ovo‐related zinc finger gene lin‐48 in the same pattern in hindgut cells. However, the regulation of this gene by the Pax‐2/5/8 protein EGL‐38 differs in two important ways. First, specific differences in the regulatory sequences of lin‐48 result in the presence of two redundant EGL‐38 response elements in C. elegans, whereas the redundancy is absent in C. briggsae. Second, there is a single egl‐38 gene in C. briggsae. In contrast, the gene is duplicated in C. elegans, with only one copy retaining the ability to regulate lin‐48 in vivo. These results illustrate molecular changes that can occur despite maintenance of conserved gene function in different species.

The bromodomain protein LEX-1 acts with TAM-1 to modulate gene expression in C. elegans

In many organisms, repetitive DNA serves as a trigger for gene silencing. However, some gene expression is observed from repetitive genomic regions such as heterochromatin, suggesting mechanisms exist to modulate the silencing effects. From a genetic screen in C. elegans, we have identified mutations in two genes important for expression of repetitive sequences: lex-1 and tam-1. Here we show that lex-1 encodes a protein containing an ATPase domain and a bromodomain. LEX-1 is similar to the yeast Yta7 protein, which maintains boundaries between silenced and active chromatin. tam-1 has previously been shown to encode a RING finger/B-box protein that modulates gene expression from repetitive DNA. We find that lex-1, like tam-1, acts as a class B synthetic multivulva (synMuv) gene. However, since lex-1 and tam-1 mutants have normal P granule localization, it suggests they act through a mechanism distinct from other class B synMuvs. We observe intragenic (interallelic) complementation with lex-1 and a genetic interaction between lex-1 and tam-1, data consistent with the idea that the gene products function in the same biological process, perhaps as part of a protein complex. We propose that LEX-1 and TAM-1 function together to influence chromatin structure and to promote expression from repetitive sequences.

Developmental patterning in the Caenorhabditis elegans hindgut.

Developmental pattern formation allows cells within a tissue or organ to coordinate their development and establish cell types in relationship to one another. To better characterize the developmental patterning events within one organ, the C. elegans hindgut, we have analyzed the expression pattern of several genes using green fluorescent protein-based reporter transgenes. In wild-type animals, these genes are expressed in subsets of hindgut cells rather than in individual cell types. In mutant animals, we find that some, but not all, genes expressed in cells with altered development exhibit a corresponding alteration of gene expression. The results are consistent with a model where a combination of factors contribute to each cells fate, and address how developmental information converges to specify cell types.

Somatic gonad sheath cells and Eph receptor signaling promote germ-cell death in C. elegans

Programmed cell death eliminates unwanted cells during normal development and physiological homeostasis. While cell interactions can influence apoptosis as they do other types of cell fate, outside of the adaptive immune system little is known about the intercellular cues that actively promote cell death in healthy cells. We used the Caenorhabditis elegans germline as a model to investigate the extrinsic regulators of physiological apoptosis. Using genetic and cell biological methods, we show that somatic gonad sheath cells, which also act as phagocytes of dying germ cells, promote death in the C. elegans germline through VAB-1/Eph receptor signaling. We report that the germline apoptosis function of VAB-1 impacts specific cell death pathways, and may act in parallel to extracellular signal-regulated kinase MAPK signaling. This work defines a non-autonomous, pro-apoptotic signaling for efficient physiological cell death, and highlights the dynamic nature of intercellular communication between dying cells and the phagocytes that remove them.

Visualizing Multidimensional Data with Glyph SPLOMs

Scatterplot matrices or SPLOMs provide a feasible method of visualizing and representing multi‐dimensional data especially for a small number of dimensions. For very high dimensional data, we introduce a novel technique to summarize a SPLOM, as a clustered matrix of glyphs, or a Glyph SPLOM. Each glyph visually encodes a general measure of dependency strength, distance correlation, and a logical dependency class based on the occupancy of the scatterplot quadrants. We present the Glyph SPLOM as a general alternative to the traditional correlation based heatmap and the scatterplot matrix in two examples: demography data from the World Health Organization (WHO), and gene expression data from developmental biology. By using both, dependency class and strength, the Glyph SPLOM illustrates high dimensional data in more detail than a heatmap but with more summarization than a SPLOM. More importantly, the summarization capabilities of Glyph SPLOM allow for the assertion of “necessity” causal relationships in the data and the reconstruction of interaction networks in various dynamic systems.