The Paf1 complex and P-TEFb have reciprocal and antagonist roles in maintaining multipotent neural crest progenitors.

Multipotent progenitor populations are necessary for generating diverse tissue types during embryogenesis. We show the RNA polymerase-associated factor 1 complex (Paf1C) is required to maintain multipotent progenitors of the neural crest (NC) lineage in zebrafish. Mutations affecting each Paf1C component result in near-identical NC phenotypes; alyron mutant embryos carrying a null mutation in paf1 were analyzed in detail. In the absence of zygotic paf1 function, definitive premigratory NC progenitors arise but fail to maintain expression of the sox10 specification gene. The mutant NC progenitors migrate aberrantly and fail to differentiate appropriately. Blood and germ cell progenitor development is affected similarly. Development of mutant NC could be rescued by additional loss of positive transcription elongation factor b (P-TEFb) activity, a key factor in promoting transcription elongation. Consistent with the interpretation that inhibiting/delaying expression of some genes is essential for maintaining progenitors, mutant embryos lacking the CDK9 kinase component of P-TEFb exhibit a surfeit of NC progenitors and their derivatives. We propose Paf1C and P-TEFb act antagonistically to regulate the timing of the expression of genes needed for NC development.

MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq.

Forward genetic screens in model organisms are vital for identifying novel genes essential for developmental or disease processes. One drawback of these screens is the labor-intensive and sometimes inconclusive process of mapping the causative mutation. To leverage high-throughput techniques to improve this mapping process, we have developed a Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) that works without parental strain information or requiring a preexisting SNP map of the organism, and adapts to differential recombination frequencies across the genome. MMAPPR accommodates the considerable amount of noise in RNA-seq data sets, calculates allelic frequency by Euclidean distance followed by Loess regression analysis, identifies the region where the mutation lies, and generates a list of putative coding region mutations in the linked genomic segment. MMAPPR can exploit RNA-seq data sets from isolated tissues or whole organisms that are used for gene expression and transcriptome analysis in novel mutants. We tested MMAPPR on two known mutant lines in zebrafish, nkx2.5 and tbx1, and used it to map two novel ENU-induced cardiovascular mutants, with mutations found in the ctr9 and cds2 genes. MMAPPR can be directly applied to other model organisms, such as Drosophila and Caenorhabditis elegans, that are amenable to both forward genetic screens and pooled RNA-seq experiments. Thus, MMAPPR is a rapid, cost-efficient, and highly automated pipeline, available to perform mutant mapping in any organism with a well-assembled genome.

Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.

BACKGROUND: Genome editing techniques, including ZFN, TALEN, and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a polymerase chain reaction (PCR) amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of "double peaks" due to the mismatched region. RESULTS: To facilitate indel identification, we developed an online tool called Poly Peak Parser (available at http://yost.genetics.utah.edu/software.php) that is able to separate chromatogram data containing ambiguous base calls into wild-type and mutant allele sequences. This tool allows the nature of the indel to be determined from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning. CONCLUSIONS: The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals, this tool can also be used to identify heterozygous indels in many contexts.

Maternal Gdf3 is an obligatory cofactor in Nodal signaling for embryonic axis formation in zebrafish.

Zebrafish Gdf3 (Dvr1) is a member of the TGFß superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFß ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis.