Expression data from mir-35-41(nDf50) mutant embryos grown at 20 degrees, compared to wild type

Gene expression in early animal embryogenesis is in large part controlled post-transcriptionally. Maternally-contributed microRNAs may therefore play important roles in early development. We have elucidated a major biological role of the nematode mir-35 family of maternally-contributed, essential microRNAs. We show that this microRNA family regulates the sex determination pathway at multiple levels, acting both upstream and downstream of her-1 to prevent aberrantly activated male developmental programs in hermaphrodite embryos. The predicted target genes that act downstream of the mir-35 family in this process, sup-26 and nhl-2, both encode RNA binding proteins, thus delineating a previously unknown post-transcriptional regulatory subnetwork within the well-studied sex determination pathway of C. elegans. Repression of nhl-2 by the mir-35 family is not only required for proper sex determination but also for viability, showing that a single microRNA target site can be essential. Since sex determination in C. elegans requires zygotic gene expression to read the sex chromosome karyotype, early embryos must remain gender-nave; our findings show that the mir-35 family microRNAs act in the early embryo to function as a developmental timer that preserves navet and prevents premature deleterious developmental decisions. The mir-35 family of microRNAs is essential for development. The mir-35-41(nDf50) allele deleted 7 of 8 mir-35 family members, and presents a hypomorphic phenotype in which embryonic lethality is temperature sensitive. To characterize the molecular phenotype associated with loss of mir-35 family function, we profiled gene expression in mir-35-41(nDf50) mutant embryos at both permissive (20) and restrictive (25) temperatures. (Refer to A microRNA family exerts maternal control on sex determination in C. elegans)

Developmental transcriptome profiling of the C. elegans pocket protein ortholog, lin-35

LIN-35 is the single C. elegans ortholog of the mammalian pocket protein family members, pRb, p107, and p130. To gain insight into the roles of pocket proteins during development, a microarray analysis was performed with lin-35 mutants. Stage-specific regulation patterns were revealed, indicating that LIN-35 plays diverse roles at distinct developmental stages. LIN-35 was found to repress the expression of many genes involved in cell proliferation in larvae, an activity that is carried out in conjunction with E2F. In addition, LIN-35 was found to regulate neuronal genes during embryogenesis and targets of the intestinal-specific GATA transcription factor, ELT-2, at multiple developmental stages. Additional findings suggest that LIN-35 functions in cell cycle regulation in embryos in a manner that is independent of E2F. A comparison of LIN-35-regulated genes with known fly and mammalian pocket-protein targets revealed a high degree of overlap, indicating strong conservation of pocket protein functions in diverse phyla. Based on microarray results and our refinement of the C. elegans E2F consensus sequence, we were able to generate a comprehensive list of putative E2F-regulated genes in C. elegans. These results implicate a large number of genes previously unconnected to cell cycle control as having potential roles in this process. Keywords: time course

Deep sequencing of the Caenorhabditis elegans transcriptome using RNA isolated from various developmental stages under various experimental conditions RW0001

Illumina Genome Analyzer II paired end sequencing; Illumina sequencing of C. elegans N2 larval L1 (lin-35) L1LIN35-1 array capture 1 experiment polyA+ RNAseq random fragment library RW0001

Deep sequencing of the Caenorhabditis elegans transcriptome using RNA isolated from various developmental stages under various experimental conditions RW0001

Illumina Genome Analyzer II paired end sequencing; Illumina sequencing of C. elegans N2 larval L1 (lin-35) L1LIN35-1 array capture 1 experiment 36bp polyA+ RNAseq random fragment library RW0001

Promotion of oogenesis and embryogenesis in the C. elegans gonad by EFL-1/DPL-1(E2F)does not require LIN-35(pRB)

Microarray-based expression profiling of dissected gonads from efl-1, dpl-1 and lin-35 mutants reveals that EFL-1 and DPL-1 promote expression of an extensively overlapping set of target genes, consistent with the expectation that these two proteins function as a heterodimer. Regulatory regions upstream of many of these target genes have a canonical E2F binding site, suggesting that their regulation by EFL-1/DPL-1 is direct. Many EFL-1/DPL-1 responsive genes encode proteins required for oogenesis and early embryogenesis, rather than cell cycle components. By contrast, LIN-35 appears to function primarily as a repressor of gene expression in the germ line, and the genes that it acts on are for the most part distinct from those regulated by EFL-1 and/or DPL-1 Keywords: Mutant analysis of dpl-1, efl-1, and lin-35 in dissected C. elegans gonads

RNA interference and retinoblastoma related genes are required for repression of endogenous siRNA targets in C. elegans

Expession data from L1-L2 stage nematodes (C. elegans), wild type and four mutants (alg-1, zfp-1, rde-4, lin-35). In C. elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Based on this microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1 and alg-1 and the Retinoblastoma (Rb) mutant lin-35, we found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated previously in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) upregulated in the rde-4 and zfp-1 mutants and b) upregulated in the lin-35(Rb) mutant, but not the downregulated genes are highly represented in the set of genes with corresponding endogenous short interfering RNAs (endo-siRNAs). Keywords: wildtype-mutant comparison, RNAi, Rb, endo-siRNA

miRNA expression in C. elegans fem-2(b245ts) and fem-3(q20ts) mutants

We compared animals that produce oocytes but no sperm fem-2(b245ts) to animals that produce sperm but no oocytes fem-3(q20ts). Using miRNA microarrays and we found that the mir-35-41 cluster genes were expressed to much greater extent in oogenesis than in spermatogenesis; furthermore, these miRNAs were the only C. elegans miRNAs that showed such differential germline expression.

Transcriptome profiling of slr-2, C.elegans C2H2 Zn-finger

Our slr-2 dataset showed strong overrepresentation of genes previously identified in a serial analysis of gene expression (SAGE) intestinal library (McGhee et al., 2006) (p << 0.01); 812 genes were common to both data sets. Consistent with the deregulation of intestinal genes, we observed repression of several important metabolic pathways, including the TOR and insulin signaling networks, suggesting that slr-2(ku297) mutants experience metabolic stress. We also compared differentially regulated genes in slr-2 and lin-35 single mutants. Again, we saw a statistically significant overlap (p-value < 0.01); 261 genes were present in both data sets. Strikingly, > 75% of genes common both datasets showed expression changes in the same direction, although the common dataset contained an approximately equal mixture of up and downregulated genes. Furthermore, more than fifty genes common to the lin-35 and slr-2 datasets are known to have intestinal-associated functions. That some of these common intestinal genes were absent from the gut SAGE library could be due to differences in the developmental stage of the animals assayed (adults versus L1s) as well as experimental approaches (SAGE versus microarrays) Keywords: analysis of staged mutant worms