C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times during oocyte meiotic spindle assembly and promote both spindle pole coalescence and stability.

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.

A genetic screen for temperature-sensitive morphogenesis-defective Caenorhabditis elegans mutants.

Morphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs, and organize the body plan. Cell adhesion and the cytoskeleton are important for executing morphogenesis, but their regulation remains poorly understood. As genes required for embryonic morphogenesis may have earlier roles in development, temperature-sensitive embryonic-lethal mutations are useful tools for investigating this process. From a collection of ∼200 such Caenorhabditis elegans mutants, we have identified 17 that have highly penetrant embryonic morphogenesis defects after upshifts from the permissive to the restrictive temperature, just prior to the cell shape changes that mediate elongation of the ovoid embryo into a vermiform larva. Using whole genome sequencing, we identified the causal mutations in seven affected genes. These include three genes that have roles in producing the extracellular matrix, which is known to affect the morphogenesis of epithelial tissues in multicellular organisms: the rib-1 and rib-2 genes encode glycosyltransferases, and the emb-9 gene encodes a collagen subunit. We also used live imaging to characterize epidermal cell shape dynamics in one mutant, or1219ts, and observed cell elongation defects during dorsal intercalation and ventral enclosure that may be responsible for the body elongation defects. These results indicate that our screen has identified factors that influence morphogenesis and provides a platform for advancing our understanding of this fundamental biological process.

Identification of rfk-1, a Meiotic Driver Undergoing RNA Editing in Neurospora.

Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1's MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.

A critical component of meiotic drive in Neurospora is located near a chromosome rearrangement.

Neurospora fungi harbor a group of meiotic drive elements known as Spore killers (Sk). Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3) are two Sk elements that map to a region of suppressed recombination. Although this recombination block is limited to crosses between Sk and Sk-sensitive (Sk(S)) strains, its existence has hindered Sk characterization. Here we report the circumvention of this obstacle by combining a classical genetic screen with next-generation sequencing technology and three-point crossing assays. This approach has allowed us to identify a novel locus called rfk-1, mutation of which disrupts spore killing by Sk-2. We have mapped rfk-1 to a 45-kb region near the right border of the Sk-2 element, a location that also harbors an 11-kb insertion (Sk-2(INS1)) and part of a >220-kb inversion (Sk-2(INV1)). These are the first two chromosome rearrangements to be formally identified in a Neurospora Sk element, providing evidence that they are at least partially responsible for Sk-based recombination suppression. Additionally, the proximity of these chromosome rearrangements to rfk-1 (a critical component of the spore-killing mechanism) suggests that they have played a key role in the evolution of meiotic drive in Neurospora.