Role for Wnt Signaling in Retinal Neuropil Development: Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis.

Screens for genes that orchestrate neural circuit formation in mammals have been hindered by practical constraints of germline mutagenesis. To overcome these limitations, we combined RNA-seq with somatic CRISPR mutagenesis to study synapse development in the mouse retina. Here synapses occur between cellular layers, forming two multilayered neuropils. The outer neuropil, the outer plexiform layer (OPL), contains synapses made by rod and cone photoreceptor axons on rod and cone bipolar dendrites, respectively. We used RNA-seq to identify selectively expressed genes encoding cell surface and secreted proteins and CRISPR-Cas9 electroporation with cell-specific promoters to assess their roles in OPL development. Among the genes identified in this way are Wnt5a and Wnt5b. They are produced by rod bipolars and activate a non-canonical signaling pathway in rods to regulate early OPL patterning. The approach we use here can be applied to other parts of the brain.

Developmental control of lateralized neuron size in the nematode Caenorhabditis elegans.

BACKGROUND: Nervous systems are generally bilaterally symmetric on a gross structural and organizational level but are strongly lateralized (left/right asymmetric) on a functional level. It has been previously noted that in vertebrate nervous systems, symmetrically positioned, bilateral groups of neurons in functionally lateralized brain regions differ in the size of their soma. The genetic mechanisms that control these left/right asymmetric soma size differences are unknown. The nematode Caenorhabditis elegans offers the opportunity to study this question with single neuron resolution. A pair of chemosensory neurons (ASEL and ASER), which are bilaterally symmetric on several levels (projections, synaptic connectivity, gene expression patterns), are functionally lateralized in that they express distinct chemoreceptors and sense distinct chemosensory cues. RESULTS: We describe here that ASEL and ASER also differ substantially in size (soma volume, axonal and dendritic diameter), a feature that is predicted to change the voltage conduction properties of the two sensory neurons. This difference in size is not dependent on sensory input or neuronal activity but developmentally programmed by a pathway of gene regulatory factors that also control left/right asymmetric chemoreceptor expression of the two ASE neurons. This regulatory pathway funnels via the DIE-1 Zn finger transcription factor into the left/right asymmetric distribution of nucleoli that contain the rRNA regulator Fibrillarin/FIB-1, a RNA methyltransferase implicated in the non-hereditary immune disease scleroderma, which we find to be essential to establish the size differences between ASEL and ASER. CONCLUSIONS: Taken together, our findings reveal a remarkable conservation of the linkage of functional lateralization with size differences across phylogeny and provide the first insights into the developmentally programmed regulatory mechanisms that control neuron size lateralities.

C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy.

Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion.

Analysis of multiple ethyl methanesulfonate-mutagenized Caenorhabditis elegans strains by whole-genome sequencing.

Whole-genome sequencing (WGS) of organisms displaying a specific mutant phenotype is a powerful approach to identify the genetic determinants of a plethora of biological processes. We have previously validated the feasibility of this approach by identifying a point-mutated locus responsible for a specific phenotype, observed in an ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans strain. Here we describe the genome-wide mutational profile of 17 EMS-mutagenized genomes as assessed with a bioinformatic pipeline, called MAQGene. Surprisingly, we find that while outcrossing mutagenized strains does reduce the total number of mutations, a striking mutational load is still observed even in outcrossed strains. Such genetic complexity has to be taken into account when establishing a causative relationship between genotype and phenotype. Even though unintentional, the 17 sequenced strains described here provide a resource of allelic variants in almost 1000 genes, including 62 premature stop codons, which represent candidate knockout alleles that will be of further use for the C. elegans community to study gene function.

The C. elegans Tailless/TLX transcription factor nhr-67 controls neuronal identity and left/right asymmetric fate diversification.

An understanding of the molecular mechanisms of cell fate determination in the nervous system requires the elucidation of transcriptional regulatory programs that ultimately control neuron-type-specific gene expression profiles. We show here that the C. elegans Tailless/TLX-type, orphan nuclear receptor NHR-67 acts at several distinct steps to determine the identity and subsequent left/right (L/R) asymmetric subtype diversification of a class of gustatory neurons, the ASE neurons. nhr-67 controls several broad aspects of sensory neuron development and, in addition, triggers the expression of a sensory neuron-type-specific selector gene, che-1, which encodes a zinc-finger transcription factor. Subsequent to its induction of overall ASE fate, nhr-67 diversifies the fate of the two ASE neurons ASEL and ASER across the L/R axis by promoting ASER and inhibiting ASEL fate. This function is achieved through direct expression activation by nhr-67 of the Nkx6-type homeobox gene cog-1, an inducer of ASER fate, that is inhibited in ASEL through the miRNA lsy-6. Besides controlling bilateral and asymmetric aspects of ASE development, nhr-67 is also required for many other neurons of diverse lineage history and function to appropriately differentiate, illustrating the broad and diverse use of this type of transcription factor in neuronal development.

Comparing platforms for C. elegans mutant identification using high-throughput whole-genome sequencing.

BACKGROUND: Whole-genome sequencing represents a promising approach to pinpoint chemically induced mutations in genetic model organisms, thereby short-cutting time-consuming genetic mapping efforts. PRINCIPAL FINDINGS: We compare here the ability of two leading high-throughput platforms for paired-end deep sequencing, SOLiD (ABI) and Genome Analyzer (Illumina; "Solexa"), to achieve the goal of mutant detection. As a test case we used a mutant C. elegans strain that harbors a mutation in the lsy-12 locus which we compare to the reference wild-type genome sequence. We analyzed the accuracy, sensitivity, and depth-coverage characteristics of the two platforms. Both platforms were able to identify the mutation that causes the phenotype of the mutant C. elegans strain, lsy-12. Based on a 4 MB genomic region in which individual variants were validated by Sanger sequencing, we observe tradeoffs between rates of false positives and false negatives when using both platforms under similar coverage and mapping criteria. SIGNIFICANCE: In conclusion, whole-genome sequencing conducted by either platform is a viable approach for the identification of single-nucleotide variations in the C. elegans genome.

Caenorhabditis elegans mutant allele identification by whole-genome sequencing.

Identification of the molecular lesion in Caenorhabditis elegans mutants isolated through forward genetic screens usually involves time-consuming genetic mapping. We used Illumina deep sequencing technology to sequence a complete, mutant C. elegans genome and thus pinpointed a single-nucleotide mutation in the genome that affects a neuronal cell fate decision. This constitutes a proof-of-principle for using whole-genome sequencing to analyze C. elegans mutants.

Genetic screens for Caenorhabditis elegans mutants defective in left/right asymmetric neuronal fate specification.

We describe here the results of genetic screens for Caenorhabditis elegans mutants in which a single neuronal fate decision is inappropriately executed. In wild-type animals, the two morphologically bilaterally symmetric gustatory neurons ASE left (ASEL) and ASE right (ASER) undergo a left/right asymmetric diversification in cell fate, manifested by the differential expression of a class of putative chemoreceptors and neuropeptides. Using single cell-specific gfp reporters and screening through a total of almost 120,000 haploid genomes, we isolated 161 mutants that define at least six different classes of mutant phenotypes in which ASEL/R fate is disrupted. Each mutant phenotypic class encompasses one to nine different complementation groups. Besides many alleles of 10 previously described genes, we have identified at least 16 novel "lsy" genes ("laterally symmetric"). Among mutations in known genes, we retrieved four alleles of the miRNA lsy-6 and a gain-of-function mutation in the 3'-UTR of a target of lsy-6, the cog-1 homeobox gene. Using newly found temperature-sensitive alleles of cog-1, we determined that a bistable feedback loop controlling ASEL vs. ASER fate, of which cog-1 is a component, is only transiently required to initiate but not to maintain ASEL and ASER fate. Taken together, our mutant screens identified a broad catalog of genes whose molecular characterization is expected to provide more insight into the complex genetic architecture of a left/right asymmetric neuronal cell fate decision.

Uncovering novel cell cycle players through the inactivation of securin in budding yeast.

Budding yeast securin/Pds1p, an inhibitor of the anaphase activator separase/Esp1p, is involved in several checkpoint pathways and in promoting Esp1p's nuclear localization. Using a modified synthetic genetic array (SGA) screen for genes that become essential in the absence of Pds1p, we uncovered roles for uncharacterized genes in cell cycle processes, including Esp1p activation.

Systematic mutagenesis of the Saccharomyces cerevisiae MLH1 gene reveals distinct roles for Mlh1p in meiotic crossing over and in vegetative and meiotic mismatch repair.

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.

Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over.

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.