Conserved meiotic mechanisms in the cnidarian Clytia hemisphaerica revealed by Spo11 knockout

During meiosis, each duplicated chromosome pairs and recombines with its unique homolog to ensure the shuffling of genetic information across generations. Functional studies in classical model organisms have revealed a surprising diversity in the chronology and interdependency of the earliest meiotic steps such as chromosome movements, pairing, association via Synaptonemal Complex formation (synapsis), recombination and the formation of chiasmata. A key player is Spo11, an evolutionarily conserved topoisomerase-related transesterase that initiates meiotic recombination via the catalysis of programmed DNA double stranded breaks (DSBs). While DSBs are required for pairing and synapsis in budding yeast and mouse, alternative pathways are employed during female meiosis of the fruit fly and nematode Caenorhabditis elegans. Here, to provide a comparative perspective on meiotic regulation from a distinct animal clade, we chart gametogenesis in Clytia hemisphaerica jellyfish and examine the role of Spo11 using CRISPR-Cas9 mutants, generated clonally from F0 polyp colonies. Spo11 mutant females fail to assemble synaptonemal complexes and chiasmata, such that homologous chromosome pairs disperse during oocyte growth. Subsequent meiotic divisions are abnormal but produce viable progeny. Clytia thus shares an ancient eukaryotic dependence of synapsis and chromosome segregation on Spo11-generated DSBs. It provides a valuable additional experimental model for dissecting meiotic mechanisms during animal gametogenesis, and for building a comparative framework for distinguishing evolutionarily conserved versus flexible features of meiosis.

RNAi-Related Dicer and Argonaute Proteins Play Critical Roles for Meiocyte Formation, Chromosome-Axes Lengths and Crossover Patterning in the Fungus Sordaria macrospora

RNA interference (RNAi) is a cellular process involving small RNAs that target and regulate complementary RNA transcripts. This phenomenon has well-characterized roles in regulating gene and transposon expression. In addition, Dicer and Argonaute proteins, which are key players of RNAi, also have functions unrelated to gene repression. We show here that in the filamentous Ascomycete Sordaria macrospora, genes encoding the two Dicer (Dcl1 and Dcl2) and the two Argonaute (Sms2 and Qde2) proteins are dispensable for vegetative growth. However, we identified roles for all four proteins in the sexual cycle. Dcl1 and Sms2 are essential for timely and successful ascus/meiocyte formation. During meiosis per se, Dcl1, Dcl2, and Qde2 modulate, more or less severely, chromosome axis length and crossover numbers, patterning and interference. Additionally, Sms2 is necessary both for correct synaptonemal complex formation and loading of the pro-crossover E3 ligase-protein Hei10. Moreover, meiocyte formation, and thus meiotic induction, is completely blocked in the dcl1 dcl2 and dcl1 sms2 null double mutants. These results indicate complex roles of the RNAi machinery during major steps of the meiotic process with newly uncovered roles for chromosomes-axis length modulation and crossover patterning regulation.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here we report that testis-specific RAD1 disruption resulted in impaired DSB repair, germ cell depletion and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss also caused defects in homolog synapsis, ATR signaling and meiotic sex chromosome inactivation. Comprehensive testis phosphoproteomics revealed that RAD1 and ATR coordinately regulate numerous proteins involved in DSB repair, meiotic silencing, synaptonemal complex formation, and cohesion. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

Ciliary control of meiotic chromosomal pairing mechanics and germ cell morphogenesis

Meiosis is a cellular program essential for the production of haploid gametes. A hallmark of meiosis is chromosomal pairing via synaptonemal complexes, and a major focus traditionally has been to understand synaptonemal complex formation. However, chromosomal pairing also depends on cytoplasmic counterparts that tether and rotate telomeres on the nuclear envelope, shuffling chromosomes and mechanically driving their homology searches1–8. Rotating telomeres slide on perinuclear microtubules and are ultimately pulled towards the centrosome7,9,10, forming the “zygotene chromosomal bouquet configuration”11. The bouquet is universally conserved and is essential for pairing and fertility1–8,12. However, despite its discovery in 190011, how the cytoplasmic counterparts of bouquet formation are mechanically regulated has remained enigmatic. Here, by studying zebrafish oogenesis, we report and comprehensively characterize the “zygotene cilium” - a previously unrecognized cilium in oocytes. We show that the zygotene cilium specifically connects to the bouquet centrosome and constitutes a cable system of the cytoplasmic bouquet machinery. Farther, zygotene cilia extend throughout the germline cyst, a conserved cellular organization of germ cells. By analyzing multiple ciliary mutants, we demonstrate that the zygotene cilium is essential for chromosomal pairing, germ cell morphogenesis, ovarian development and fertility. We further show that the zygotene cilium is conserved in both male meiosis in zebrafish, as well as in mammalian oogenesis. Our work uncovers the novel concept of a cilium as a critical player in meiosis and sheds new light on reproduction phenotypes in ciliopathies. Furthermore, most cells in metazoans are ciliated and exhibit specific nuclear dynamics. We propose a cellular paradigm that cilia can control chromosomal dynamics.

Functional characterization of Caenorhabditis elegans cbs-2 gene during meiosis

Cystathionine β-synthase (CBS) is a eukaryotic enzyme that maintains the cellular homocysteine homeostasis and catalyzes the conversion of homocysteine to L-cystathionine and Hydrogen sulfide, via the trans-sulfuration pathway. In Caenorhabditis elegans, two cbs genes are present: cbs-1 functions similarly as to human CBS, and cbs-2, whose roles are instead unknown. In the present study we performed a phenotypic characterization of the cbs-2 mutant. The null cbs-2 mutant is viable, fertile and shows the wild-type complement of six bivalents in most oocyte nuclei, which is indicative of a correct formation of crossover recombination. In absence of synaptonemal complex formation (syp-2 mutant), loss of cbs-2 leads to chromosome fragmentation, suggesting that cbs-2 is essential during inter-sister repair. Interestingly, although proficient in the activation of the DNA damage checkpoint after exposure to genotoxic stress, the cbs-2 mutant is defective in DNA damage-induced apoptosis in meiotic germ cells. These results suggest possible functions for CBS-2 in meiosis, distinct from a role in the trans-sulfuration pathway. We propose that the C. elegans CBS-2 protein is required for both inter-sister repair and execution of DNA damage-induced apoptosis.

Chromosome-autonomous feedback downregulates meiotic DSB competence upon synaptonemal complex formation

The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in Saccharomyces cerevisiae mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog-engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants ecm11 and gmc2 experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis.

Multivalent weak interactions between assembly units drive synaptonemal complex formation

The synaptonemal complex (SC) is an ordered but highly dynamic structure assembled between homologous chromosomes to control interhomologous crossover formation, ensuring accurate meiotic chromosome segregation. However, the mechanisms regulating SC assembly and dynamics remain unclear. Here, we identified two new SC components, SYP-5 and SYP-6, in Caenorhabditis elegans that have distinct expression patterns and form distinct SC assembly units with other SYPs through stable interactions. SYP-5 and SYP-6 exhibit diverse in vivo SC regulatory functions and distinct phase separation properties in cells. Charge-interacting elements (CIEs) are enriched in SC intrinsically disordered regions (IDRs), and IDR deletion or CIE removal confirmed a requirement for these elements in SC regulation. Our data support the theory that multivalent weak interactions between the SC units drive SC formation and that CIEs confer multivalency to the assembly units.

A novel allele of ASY3 promotes meiotic stability in autotetraploid Arabidopsis lyrata

In this study we performed a genotype-phenotype association analysis of meiotic stability in ten autotetraploid Arabidopsis lyrata and A. lyrata/A. arenosa hybrid populations collected from the Wachau region and East Austrian Forealps. The aim was to determine the effect of eight meiosis genes under extreme selection upon adaptation to whole genome duplication. Individual plants were genotyped by high-throughput sequencing of the eight meiosis genes (ASY1, ASY3, PDS5b, PRD3, REC8, SMC3, ZYP1a/b) implicated in synaptonemal complex formation and phenotyped by assessing meiotic metaphase I chromosome configurations. Our results reveal that meiotic stability varied greatly (20-100%) between individual tetraploid plants and was associated with segregation of a novel allele orthologous to the budding yeast RED1 chromosome axis protein, Asynapsis3 (ASY3), derived from A. lyrata. The adaptive ASY3 protein possesses a putative in-frame tandem duplication (TD) of a serine-rich region upstream of the coiled-coil domain that has arisen at sites of DNA microhomology. The frequency of multivalents observed in plants homozygous for the ASY3 TD haplotype was significantly lower than plants heterozygous for TD/ND (non-duplicated) ASY3 haplotypes. Chiasma distribution was significantly altered in the stable plants compared to the unstable plants with a shift from proximal and interstitial to predominantly distal locations. The number of HEI10 foci at pachtyene that mark class I crossovers was significantly reduced in meiotic nuclei from ASY3 TD homozygous plants compared to ASY3 ND/TD heterozygotes, indicating an adaptive consequence of the ASY3 TD allele. From the ten populations, fifty-eight alleles of these 8 meiosis genes were identified, demonstrating dynamic population variability at these loci which nevertheless exhibit signatures of strong hard selective sweeps. Widespread chimerism between alleles originating from A. lyrata/A. arenosa and diploid/tetraploids indicates that this group of rapidly evolving genes provide precise adaptive control over meiotic recombination in the tetraploids, the very process that gave rise to them.Author summaryWhole genome duplication can promote adaptability, but is a dramatic mutation usually resulting in meiotic catastrophe and genome instability. Here we focus on a case of coordinated stabilization of meiotic recombination in ten autotetraploid Arabidopsis lyrata and A. lyrata/A. arenosa hybrid populations from the Wachau region and East Austrian Forealps. We fuse population genomic data with a genotype-phenotype association study, concentrating on the effects of eight meiosis genes (ASY1, ASY3, PDS5b, PRD3, REC8, SMC3, ZYP1a/b) implicated in synaptonemal complex formation in the tetraploids under extreme selection. Our analysis demonstrates that a novel allele of the meiotic chromosome axis protein Asynapsis3 that contains an in-frame duplication of a serine-rich region is the major determinant of male meiotic stability. This adaptive restabilisation appears to be achieved by a reduction in the number of meiotic crossovers as well as a shift in their positioning towards the chromosome ends. Of the eight genes, fifty-eight alleles were identified, indicating dynamic population variability at these loci under extreme selection. In addition, widespread allelic chimerism between alleles originating from A. lyrata/A. arenosa and diploid/tetraploids indicates that this group of rapidly evolving genes provide precise adaptive control over meiotic recombination in the tetraploids, the very process that gave rise to them.