measuring-embryonic-viability-and-brood-size-in-caenorhabditis-elegans

Measuring Embryonic Viability and Brood Size in <em>Caenorhabditis elegans</em>

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

University of Delaware

Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in  to identify putative genes that interact with  during meiosis. Using a temperature-sensitive allele of , , we identified eleven suppressors of -induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.

Identification of Suppressors of  Embryonic Lethality in .

The C-terminus of SPE-11 is required for proper embryonic development in .

To investigate the dynamic localization of Topoisomerase II in live  we have generated a C-terminally GFP-tagged version of TOP-2 at the endogenous locus. We found that TOP-2::GFP localizes in a similar pattern to the previously published TOP-2::3XFLAG strain and does not disrupt the meiotic chromosome segregation functions of this enzyme.

Endogenous localization of TOP-2 in  using a C-terminal GFP-tag.

We have generated Tyrosyl-DNA phosphodiesterase 2 (TDPT-1)  strains where CRISPR/Cas9 was used to endogenously tag the protein at either the C- or N-terminus and validated the functionality of the resulting tagged TDPT-1 proteins. We have found that both the N-terminally tagged (  and C-terminally tagged (  ) worm TDPT-1 does not affect embryonic viability compared to wild type. Using the N-terminally tagged  strain we show, for the first time, that TDPT-1 is expressed in nuclei of the germ line and the soma. Moreover, we validate the expression of TDPT-1 at the protein level using the C-terminally tagged (  ) strain.

Characterization of N- and C-terminal endogenously tagged Tyrosyl-DNA phosphodiesterase 2 (TDPT-1)  strains.

During meiotic prophase I, accurate segregation of homologous chromosomes requires the establishment of chromosomes with a meiosis-specific architecture. The sister chromatid cohesin complex and the enzyme Topoisomerase II (TOP-2) are important components of meiotic chromosome architecture, but the relationship of these proteins in the context of meiotic chromosome segregation is poorly defined. Here, we analyzed the role of TOP-2 in the timely release of the sister chromatid cohesin subunit REC-8 during spermatogenesis and oogenesis of Caenorhabditis elegans. We show that there is a different requirement for TOP-2 in meiosis of spermatogenesis and oogenesis. The loss-of-function mutation top-2(it7) results in premature REC-8 removal in spermatogenesis, but not oogenesis. This correlates with a failure to maintain the HORMA-domain proteins HTP-1 and HTP-2 (HTP-1/2) on chromosome axes at diakinesis and mislocalization of the downstream components that control REC-8 release including Aurora B kinase. In oogenesis, top-2(it7) causes a delay in the localization of Aurora B to oocyte chromosomes but can be rescued through premature activation of the maturation promoting factor via knockdown of the inhibitor kinase WEE-1.3. The delay in Aurora B localization is associated with an increase in the length of diakinesis bivalents and wee-1.3 RNAi mediated rescue of Aurora B localization in top-2(it7) is associated with a decrease in diakinesis bivalent length. Our results imply that the sex-specific effects of TOP-2 on REC-8 release are due to differences in the temporal regulation of meiosis and chromosome structure in late prophase I in spermatogenesis and oogenesis.

TOP-2 is differentially required for the proper maintenance of the cohesin subunit REC-8 on meiotic chromosomes in Caenorhabditis elegans spermatogenesis and oogenesis.

Article	Year
Identification of Suppressors of Embryonic Lethality in . G3 (Bethesda, Md.)\| PubMed ID: 32086248	2020
The C-terminus of SPE-11 is required for proper embryonic development in . microPublication biology\| PubMed ID: 32550511	2020
Endogenous localization of TOP-2 in using a C-terminal GFP-tag. microPublication biology\| PubMed ID: 34095779	2021
Characterization of N- and C-terminal endogenously tagged Tyrosyl-DNA phosphodiesterase 2 (TDPT-1) strains. microPublication biology\| PubMed ID: 35622466	2022
TOP-2 is differentially required for the proper maintenance of the cohesin subunit REC-8 on meiotic chromosomes in Caenorhabditis elegans spermatogenesis and oogenesis. Genetics\| PubMed ID: 35951744	2022

Aimee Jaramillo-Lambert

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