John F. Etchberger

Searching for neuronal left/right asymmetry: genomewide analysis of nematode receptor-type guanylyl cyclases.

Functional left/right asymmetry (“laterality”) is a fundamental feature of many nervous systems, but only very few molecular correlates to functional laterality are known. At least two classes of chemosensory neurons in the nematode Caenorhabditis elegans are functionally lateralized. The gustatory neurons ASE left (ASEL) and ASE right (ASER) are two bilaterally symmetric neurons that sense distinct chemosensory cues and express a distinct set of four known chemoreceptors of the guanylyl cyclase (gcy) gene family. To examine the extent of lateralization of gcy gene expression patterns in the ASE neurons, we have undertaken a genomewide analysis of all gcy genes. We report the existence of a total of 27 gcy genes encoding receptor-type guanylyl cyclases and of 7 gcy genes encoding soluble guanylyl cyclases in the complete genome sequence of C. elegans. We describe the expression pattern of all previously uncharacterized receptor-type guanylyl cyclases and find them to be highly biased but not exclusively restricted to the nervous system. We find that >41% (11/27) of all receptor-type guanylyl cyclases are expressed in the ASE gustatory neurons and that one-third of all gcy genes (9/27) are expressed in a lateral, left/right asymmetric manner in the ASE neurons. The expression of all laterally expressed gcy genes is under the control of a gene regulatory network composed of several transcription factors and miRNAs. The complement of gcy genes in the related nematode C. briggsae differs from C. elegans as evidenced by differences in chromosomal localization, number of gcy genes, and expression patterns. Differences in gcy expression patterns in the ASE neurons of C. briggsae arise from a difference in cis-regulatory elements and trans-acting factors that control ASE laterality. In sum, our results indicate the existence of a surprising multitude of putative chemoreceptors in the gustatory ASE neurons and suggest the existence of a substantial degree of laterality in gustatory signaling mechanisms in nematodes.

An unusual Zn-finger/FH2 domain protein controls a left/right asymmetric neuronal fate decision in C. elegans.

Gene regulatory networks that control the terminally differentiated state of a cell are, by and large, only superficially understood. In a mutant screen aimed at identifying regulators of gene batteries that define the differentiated state of two left/right asymmetric C. elegans gustatory neurons, ASEL and ASER, we have isolated a mutant, fozi-1, with a novel mixed-fate phenotype, characterized by de-repression of ASEL fate in ASER. fozi-1 codes for a protein that functions in the nucleus of ASER to inhibit the expression of the LIM homeobox gene lim-6, neuropeptide-encoding genes and putative chemoreceptors of the GCY gene family. The FOZI-1 protein displays a highly unusual domain architecture, that combines two functionally essential C2H2 zinc-finger domains, which are probably involved in transcriptional regulation, with a formin homology 2 (FH2) domain, normally found only in cytosolic regulators of the actin cytoskeleton. We demonstrate that the FH2 domain of FOZI-1 has lost its actin polymerization function but maintains its phylogenetically ancient ability to homodimerize. fozi-1 genetically interacts with several transcription factors and micro RNAs in the context of specific regulatory network motifs. These network motifs endow the system with properties that provide insights into how cells adopt their stable terminally differentiated states.

Vector-free DNA constructs improve transgene expression in C. elegans.

To the editor: One advantage of the Caenorhabditis elegans model system is the ease and speed with which transgenic worms can be generated1. This feature has been used to rescue mutant phenotypes, to express genes heterologously and to analyze gene expression patterns and cis-regulatory regions with reporter genes2. With transgenic approaches, one often relies in part on the interpretation of negative results, such as the failure to observe reporter expression after deletion of putative regulatory elements or the failure to observe a biological effect by expressing a gene in one but not in another cell type. The conventional strategy to generate expression constructs in C. elegans relies on subcloning the piece of DNA under investigation, usually into a set of standard vectors that contain a common backbone2,3. While dissecting regulatory regions of several C. elegans promoters, we found that subcloned promoter fragments consistently failed to yield detectable expression in expected cell types (Fig. 1) whereas the same reporter constructs, when injected in linear form without adjacent vector sequences (generated either by PCR or by restriction digest), yielded consistent, highly penetrant expression in expected cell types (6 different reporter constructs from the promoters of 5 different genes; Fig. 1, Supplementary Table 1 and Supplementary Methods online). We consider the observed expression of the linear, non-vector-containing constructs to be more authentic than the absence of expression of the subcloned constructs because the observed expression (i) matches the presumed site of function of the gene (for example, lim-6 acts in the ASEL neuron to repress ASER neuron fate4), (ii) matches the expression of larger, subcloned reporter gene constructs derived from the same promoter (Fig. 1) and (iii) occurs in the expected cell type based on the presence of well-characterized cis-regulatory motifs in the promoter5. The lack of expression in the vector-containing construct is unlikely to be due to known transgene silencing effects because we did not observe gfp expression in mutant backgrounds in which RNA interference–mediated transgene silencing effects are disrupted (Supplementary Table 2 online). To test whether expression of the linear reporters is due to the linear structure or the absence of the vector backbone, we linearized one of the subcloned constructs (gcy-5del1:: gfp) by single restriction digest. None of the resulting transgenic lines (6 in total) showed the reproducible gfp expression observed with the linear, vector-free construct, indicating that the absence of the vector is critical for observing reporter gene expression. Supplementary Table 2 summarizes all experimental conditions tested. One potential explanation for our observations is that the vector backbone may dictate the packaging of the DNA into higher-order chromatin structure, making regulatory elements in the promoter less accessible. Our findings indicate that negative results obtained with subcloned DNA must be interpreted with caution and should motivate, if feasible, the use of linear, vector-free expression constructs in C. elegans that are generated, for example, by PCR fusion6. If expression constructs were generated by subcloning, constructs may be PCRamplified without the vector sequence and injected directly into the gonad.

Cis-regulatory Mutations in the Caenorhabditis elegans Homeobox Gene Locus cog-1 Affect Neuronal Development

We apply here comparative genome hybridization as a novel tool to identify the molecular lesion in two Caenorhabditis elegans mutant strains that affect a neuronal cell fate decision. The phenotype of the mutant strains resembles those of the loss-of-function alleles of the cog-1 homeobox gene, an inducer of the fate of the gustatory neuron ASER. We find that both lesions map to the cis-regulatory control region of cog-1 and affect a phylogenetically conserved binding site for the C2H2 zinc-finger transcription factor CHE-1, a previously known regulator of cog-1 expression in ASER. Identification of this CHE-1-binding site as a critical regulator of cog-1 expression in the ASER in vivo represents one of the rare demonstrations of the in vivo relevance of an experimentally determined or predicted transcription-factor-binding site. Aside from the mutationally defined CHE-1-binding site, cog-1 contains a second, functional CHE-1-binding site, which in isolation is sufficient to drive reporter gene expression in the ASER but in an in vivo context is apparently insufficient for promoting appropriate ASER expression. The cis-regulatory control regions of other ASE-expressed genes also contain ASE motifs that can promote ASE neuron expression when isolated from their genomic context, but appear to depend on multiple ASE motifs in their normal genomic context. The multiplicity of cis-regulatory elements may ensure the robustness of gene expression.