Biology subjects

Libuda, D. E.

Publications and source records attributed to Libuda, D. E..

13 recordsLinked to original sources

bioRxiv2026

Genomic sequence and structural variations accumulating between laboratory lineages of wild type C. elegans

Laboratory cultivation subjects model organisms to selective pressure and genetic drift that can result in the accumulation of many genomic and phenotypic differences over time. The nematode Caenorhabditis elegans has been used for research since the 1970s, and studies comparing the N2 Bristol and CB4856 Hawaiian isolates provided foundational knowledge about metazoan genome evolution. Most comparative genomics studies have used these isolates because their long-term geographical isolation promoted a high degree of genomic divergence within the species. Further, there is growing evidence of phenotypic differences between laboratory lineages of each wild type isolate after repeated independent lab cultivation of these strains. To examine the genomic divergence between different laboratory lineages the Bristol and Hawaiian backgrounds, we first generated de novo genome assemblies of two Bristol and two Hawaiian lineages from Illumina and PacBio sequencing reads. Following genome assembly, we quantified Single Nucleotide Polymorphisms (SNPs), short insertion/deletions (indels), and genomic structural variants (SVs). Between laboratory lineages of the Bristol isolate, we identified 25,432 SNPs, 5,202 indels, and 441 SVs. When aligning laboratory lineages of the Hawaiian isolate, we identified 4,518 SNPs, 1,188 indels, and 387 SVs. For both sets of comparisons, we find that SNPs and indels are broadly enriched in introns and depleted from coding sequences. In contrast to SNPs and indels, we find that genomic SVs are enriched in intergenic sequences. Taken together, our analyses reveal the accumulation of genomic divergence between lineages of Bristol and Hawaiian C. elegans from independent lab cultivation, and how these variants may underpin emergent phenotypic differences observed in the two most popularly used C. elegans wild type isolates. Author SummaryLaboratory model organisms, like natural populations, are subject to evolutionary pressures and genomic changes during prolonged laboratory cultivation. In this study we comprehensively quantify SNPs, indels, and SVs between independent lab cultivations of the C. elegans lineages of the Bristol and Hawaiian isolates.

genetics↗

bioRxiv2026

Caenorhabditis elegans form sexually dimorphic and dynamic germ granules throughout meiotic prophase I progression

In sexually reproducing organisms, germ cells faithfully transmit both the genome and epigenetic information across generations through the formation of haploid gametes, such as eggs and sperm. Small RNA pathways tune gene expression in a sex-specific manner during germ cell development to facilitate both proper germ cell formation and transgenerational inheritance of epigenetic information. In Caenorhabditis elegans, components of small RNA pathways localize to germ granules, liquid-like membraneless organelles within the cytoplasm of developing germ cells. During oogenesis, germ granules form hierarchal sub-compartments that may be required for proper germ cell development and epigenetic inheritance. However, germ granule structure during spermatogenesis remains largely undescribed. Here we determine that the germ granule structural components PGL-1 and ZNFX-1 display sexually dimorphic foci morphology and size during meiotic prophase I progression. Further, we quantitate the sexually dimorphic sub-compartmentalization of these two proteins within the germ granule, determining that while PGL-1 and ZNFX-1 do associate during germ cell development, the extent of overlap varies between sexes and throughout meiotic progression. Additionally, we identify WAGO-4, a Argonaute protein central to gene regulation by small RNA pathways, as a sexually dimorphic component of the germ granule during germ cell development. Together, our studies reveal that the overall structure of the germ granule, as well as an Argonaute protein housed inside, are sexually dimorphic, which may underpin sex-specific regulation by small RNA pathways during germ cell development. Author SummarySmall RNA pathways are critical for the regulation and passage of genomic and epigenetic information to the next generation. Components of these pathways are housed in germ granules during egg and sperm development. Previous work examining germ granule structure in Caenorhabditis elegans focused on oocytes and the late stages of meiosis I. Here, we comprehensively characterize the localization of two structural and one functional component of the germ granule throughout meiotic progression of both developing egg and sperm cells. We identify that both biophysical properties and germ granule configurations are dependent upon meiotic stage and sex.

developmental biology↗

bioRxiv2026

High-resolution global recombination mapping in C. elegans reveals sexual dimorphisms shaped by meiotic chromosomal features and structures

Crossover recombination events during meiosis repair double-strand DNA breaks and ensure accurate chromosome segregation in most organisms. For many species, the genomic distribution of crossovers is nonrandom and sexually dimorphic. While many species evolved kilobase-scale "hotspots" for crossover formation, the Caenorhabditis elegans genome lacks hotspots, and crossovers are enriched across megabase-scale domains. Further, genetic and cytological studies indicate the crossover frequency in C. elegans spermatogenesis is higher relative to oogenesis in many but not all genetic intervals. To determine the genomic features that contribute to the sexually dimorphic recombination landscape in the absence of hot spots, we defined and analyzed the recombination landscape across the whole genome in C. elegans using whole-genome sequencing and high-resolution recombination mapping in single worms bearing recombinant chromosomes from individual sperm and oocytes. We find that the spatial distribution of crossovers is sexually dimorphic on chromosomes I, II, and III, and that the global rate of double-crossover events is 4.7-fold higher in spermatocytes. Additionally, we find that pairing and synapsis may contribute to the sexually dimorphic crossover landscape. In comparison to the spermatocyte crossover landscape, a higher proportion of oocyte crossovers are formed in the domains directly adjacent to the pairing centers of each chromosome. Further, reducing the genetic dosage of the synaptonemal complex central region protein SYP-2, which is a meiotic chromosome structural protein required for homologous chromosome synapsis, reshapes the oocyte crossover landscape to resemble observations in wild-type spermatocytes. Finally, we found that spermatocyte crossovers are partially enriched in H3K36me3-marked euchromatic regions, while many oocyte crossovers are enriched in H3K27me3-marked heterochromatic regions. Taken together, our studies reveal how synaptonemal complex component dosage and local chromatin states influence crossover placement and the sex-specific regulation of meiotic recombination. Author SummaryProduction of viable eggs and sperm depends on accurate chromosome segregation during meiosis. Segregation of parental copies of homologous chromosomes requires the reciprocal exchange and physical linkage of DNA that arises through crossover recombination. Increasing evidence indicates the existence of sexual dimorphisms during meiotic recombination. In this study, we generated and analyzed high-resolution recombination maps specific to spermatogenesis and oogenesis in the nematode C. elegans, which reveals sex-specific crossover distributions and a higher rate of crossing over in sperm cells. Further, we indicate how specific chromosomal features and structures differentially affect the crossover landscape in eggs versus sperm. Our work highlights how, in a system absent of pre-defined "hotspots" for recombination, local chromatin structures, chromosomal pairing domains, and the abundance of synaptonemal complex proteins are potential drivers for establishing the observable sex differences in crossover recombination.

genetics↗

bioRxiv2025

WAGO-1 is a sexually dimorphic Argonaute protein required for proper germ granule structure and gametogenesis

Germ cell proliferation and proper genome inheritance are critical for maintaining fertility through generations. To promote proper germ cell development, small RNA pathways employ Argonaute proteins (AGOs) to modulate gene expression and protect against deleterious genomic elements while not silencing against self. Here we identify sexual dimorphisms in localization and function of protein structural features of the Argonaute WAGO-1 that affects sex-specific gene regulation during C. elegans germ cell development. During meiotic prophase I progression, we find that germ granule structural proteins and the PIWI AGO, PRG-1, display dynamic and distinct localization patterns between egg and sperm development which coincide with differential WAGO-1 localization and biophysical properties. Sexually dimorphic functions of specific WAGO-1 protein structural domains underpin these differences. Disruption or modification to the N-terminus intrinsically disordered region (IDR) of WAGO-1 leads to loss of PGL-1 phase separation only during spermatogenesis. Further, we find that these germ granule disruptions are likely due to prolonged association of the IDR with the RNA-binding pocket of WAGO-1. In addition, deletion of the MID and part of the PIWI domains causes male-specific sterility and disruption to WAGO-1 localization with PGL-1 during oogenesis. Finally, we demonstrate that these disruptions to WAGO-1 protein structure dynamically change the mRNA and sRNA landscape of adult males and hermaphrodites, in which the AGOs ALG-3/4 and VSRA-1 are misregulated. Together, these data suggest that WAGO-1 differentially regulates genes during oogenesis versus spermatogenesis, and that these differences in gene regulation may be due to the sex-specific configuration and biophysical properties of WAGO-1 within the germ granule.

developmental biology↗

bioRxiv2023

De novo genome assemblies reveal structural variations between laboratory and natural isolates of C. elegans

Genomic structural variations (SVs) and transposable elements (TEs) can be significant contributors to genome evolution, altered gene expression, and risk of genetic diseases. Recent advancements in long-read sequencing have greatly improved the quality of de novo genome assemblies and enhanced the detection of sequence variants at the scale of hundreds or thousands of bases. Comparisons between two diverged wild isolates of Caenorhabditis elegans, the Bristol and Hawaiian strains, have been widely utilized in the analysis of small genetic variations. Genetic drift, including SVs and rearrangements of repeated sequences such as TEs, can occur over time from long-term maintenance of wild type isolates within the laboratory. To comprehensively detect both large and small structural variations as well as TEs due to genetic drift, we generated de novo genome assemblies and annotations for each strain from our lab collection using both long- and short-read sequencing and compared our assemblies and annotations with that of other lab wild type strains. Within our lab assemblies, we annotate over 3.1Mb of sequence divergence between the Bristol and Hawaiian isolates: 337,584 SNPs, 94,503 small insertion-deletions (<50bp), and 4,334 structural variations (>50bp). Further, we define the location and movement of specific DNA TEs between N2 Bristol and CB4856 Hawaiian wild type isolates. Specifically, we find the N2 Bristol genome has 20.6% more TEs from the Tc1/mariner family than the CB4856 Hawaiian genome. Moreover, we identified Zator elements as the most abundant and mobile TE family in the genome. Using specific TE sequences with unique SNPs, we also identify 38 TEs that moved intrachromosomally and 9 TEs that moved interchromosomally between the N2 Bristol and CB4856 Hawaiian genomes. By comparing the de novo genome assembly of our lab collection Bristol isolate to the VC2010 Bristol assembly, we also reveal that lab lineages display over 2 Mb of total variation: 1,162 SNPs, 1,528 indels, and 897 SVs with 95% of the variation due to SVs. Overall, our work demonstrates the unique contribution of SVs and TEs to variation and genetic drift between wild type laboratory strains assumed to be isogenic despite growing evidence of genetic drift and phenotypic variation. Author SummaryFor multiple model organisms, propagation of wild type strains in independent labs can lead to multiple phenotypic differences over time. To assess recombination, map mutations, and understand genomic changes during speciation, Caenorhabditis elegans researchers primarily use the wild type isolates Bristol and Hawaiian. Here, we map structural variations, transposable elements, and sequence divergence between the Bristol and Hawaiian natural isolates and between genomes of different lab lineages of these same strains.

genomics↗

bioRxiv2022

Sexual dimorphic regulation of recombination by the synaptonemal complex

In sexually reproducing organisms, germ cells faithfully transmit the genome to the next generation by forming haploid gametes, such as eggs and sperm. Although most meiotic proteins are conserved between eggs and sperm, many aspects of meiosis are sexually dimorphic. The mechanisms regulating recombination display sex-specific differences in multiple organisms such that the same proteins in each sex are being utilized in different ways to produce sexually dimorphic outcomes. The synaptonemal complex (SC), a large ladder-like structure that forms between homologous chromosomes, is essential for regulating meiotic chromosome organization and promoting recombination. To assess whether sex-specific differences in the SC underpin sexually dimorphic aspects of meiosis, we examined two Caenorhabditis elegans SC central region proteins, SYP-2 and SYP-3, in oogenesis and spermatogenesis and uncovered sex-specific roles for the SYPs in regulating meiotic recombination. We find that SC composition is regulated by sex-specific mechanisms throughout meiotic prophase I. During pachytene, both oocytes and spermatocytes differentially regulate the stability of SYP-2 and SYP-3 within an assembled SC, with increased SYP-2 dynamics in spermatocytes and increased SYP-3 dynamics in oocytes. Further, we uncover that the relative amount of SYP-2 and SYP-3 within the SC is independently regulated in both a sex-specific and a recombination-dependent manner. Specifically, we find that SYP-2 regulates the early steps of recombination in both sexes, while SYP-3 controls the timing and positioning of crossover recombination events across the genomic landscape in only oocytes. Taken together, we demonstrate dosage-dependent regulation of individual SC components with sex-specific functions in recombination. These sexual dimorphic features of the SC provide insights into how spermatogenesis and oogenesis adapted similar chromosome structures to differentially regulate and execute recombination.

developmental biology↗

bioRxiv2022

BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during C. elegans meiosis

The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Here we show that BRCA1/BRC-1 and the SMC-5/6 complex limit intersister crossover recombination as well as error-prone repair pathways during meiotic prophase I. Using genetic and cytological methods to monitor repair of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5/6 repress intersister crossover recombination events, with meiotic cells becoming more dependent upon these proteins to repair DSBs in late meiotic prophase I. Sequencing of conversion tracts from homolog-independent DSB repair events indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 also specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal that functional BRC-1 enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). Taken together, our study illuminates the coordinate interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.

genetics↗

bioRxiv2022

Aging and sperm signals alter DNA break formation and repair in the C. elegans germline

Female reproductive aging is associated with decreased oocyte quality and fertility. The nematode Caenorhabditis elegans is a powerful system for understanding the biology of aging and exhibits age-related reproductive defects that are analogous to those observed in many mammals, including dysregulation of DNA repair. C. elegans germline function is influenced simultaneously by both reproductive aging and signals triggered by limited supplies of sperm, which are depleted over chronological time. To delineate the causes of DNA repair defects in aged C. elegans germlines, we assessed both DNA double strand break (DSB) induction and repair during meiotic prophase progression in aged germlines which were depleted of self-sperm, mated, or never exposed to sperm. We find that germline DSB induction is dramatically reduced only in hermaphrodites which have exhausted their endogenous sperm, suggesting that a signal due specifically to sperm depletion downregulates DSB formation. We also find that DSB repair is delayed in aged germlines regardless of whether hermaphrodites had either a reduction in sperm supply or an inability to endogenously produce sperm. These results demonstrate that in contrast to DSB induction, DSB repair defects are a feature of C. elegans reproductive aging independent of sperm presence. Finally, we demonstrate that the ubiquitin E2 ligase variant UEV-2 is required for efficient DSB repair specifically in young germlines, implicating UEV-2 in the regulation of DNA repair during reproductive aging. In summary, our study demonstrates that DNA repair defects are a feature of C. elegans reproductive aging and uncovers parallel mechanisms regulating efficient DSB formation in the germline. Author SummaryAging leads to a decline in the quality of the female reproductive cells, known as oocytes. Oocytes subjected to reproductive aging experience an increase in both infertility and aneuploidies that cause miscarriages and birth defects. The nematode Caenorhabditis elegans is a classic model system used to determine the mechanisms of aging. Old C. elegans oocytes accrue many defects which may contribute to their reduced quality, including dysregulation of DNA repair. C. elegans fertility and germline function is also regulated oocyte-independently by sperm-dependent signals. To determine how aging and sperm may independently impact DNA repair in aging C. elegans oocytes, we control oocyte aging and sperm presence independently to evaluate their effects on DNA break formation and repair. We find that running out of sperm reduces the levels of DNA breaks which are produced, but the efficiency of DNA repair declines during aging independent of sperm effects. We also identify a protein which specifically promotes DNA repair in the oocytes of young animals, suggesting that this protein may regulate DNA repair in the germline during aging. Taken together, our research defines aging-specific and aging-independent mechanisms which regulate the genome integrity of oocytes.

genetics↗

bioRxiv2021

Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion

The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin inducible degron (AID) system to immobilize both hermaphrodites and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans. ARTICLE SUMMARYC. elegans is a powerful model system for visualizing biological processes in live cells. In addition to the challenge of suppressing the worm movement for live imaging, most immobilization techniques only work with hermaphrodites. Here, we describe a new genetic immobilization tool that conditionally immobilizes both worm sexes for live imaging studies. Additionally, we demonstrate that this tool can be used for live imaging the C. elegans germline without causing large defects to germline progression or fertility in either sex.

cell biology↗

bioRxiv2020

Automated and customizable quantitative image analysis of whole C. elegans germlines

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-throughput image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: 1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and, 2) assessment of these individual nuclei based on their position within the germline. We demonstrate the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at the single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields utilizing the C. elegans germline as a model system.

cell biology↗

bioRxiv2020

Sister chromatid repair maintains genomic integrity during meiosis in Caenorhabditis elegans

During meiosis, the maintenance of genome integrity is critical for generating viable haploid gametes [1]. In meiotic prophase I, double-strand DNA breaks (DSBs) are induced and a subset of these DSBs are repaired as interhomolog crossovers to ensure proper chromosome segregation. DSBs in excess of the permitted number of crossovers must be repaired by other pathways to ensure genome integrity [2]. To determine if the sister chromatid is engaged for meiotic DSB repair during oogenesis, we developed an assay to detect sister chromatid repair events at a defined DSB site during Caenorhabditis elegans meiosis. Using this assay, we directly demonstrate that the sister chromatid is available as a meiotic repair template for both crossover and noncrossover recombination, with noncrossovers being the predominant recombination outcome. We additionally find that the sister chromatid is the exclusive recombination partner for DSBs during late meiotic prophase I. Analysis of noncrossover conversion tract sequences reveals that DSBs are processed similarly throughout prophase I and recombination intermediates remain central around the DSB site. Further, we demonstrate that the SMC-5/6 complex is required for long conversion tracts in early prophase I and intersister crossovers during late meiotic prophase I; whereas, the XPF-1 nuclease is required only in late prophase to promote sister chromatid repair. In response to exogenous DNA damage at different stages of meiosis, we find that mutants for SMC-5/6 and XPF-1 have differential effects on progeny viability. Overall, we propose that SMC-5/6 both processes recombination intermediates and promotes sister chromatid repair within meiotic prophase I, while XPF-1 is required as an intersister resolvase only in late prophase I.

genetics↗

bioRxiv2020

Excess crossovers impede faithful meiotic chromosome segregation in C. elegans

During meiosis, at least one crossover must form between each pair of homologous chromosomes to ensure their proper partitioning. However, most organisms limit the number of crossovers by a phenomenon called crossover interference; why this occurs is not well understood. Here we investigate the functional consequences of extra crossovers in Caenorhabditis elegans. Using a fusion chromosome that exhibits a high frequency of supernumerary crossovers, we find that essential chromosomal structures are mispatterned, subjecting chromosomes to improper spindle forces and leading to congression and segregation defects. Moreover, we uncover mechanisms that counteract these errors; anaphase I chromosome bridges were often able to resolve in a LEM-3 nuclease dependent manner, and tethers between homologs that persisted were frequently resolved during Meiosis II by a second mechanism. This study thus provides evidence that excess crossovers impact chromosome patterning and segregation, and also sheds light on how these errors are corrected.

cell biology↗

bioRxiv2019

Synaptonemal complex proteins direct and constrain the localization of crossover-promoting proteins during Caenorhabditis elegans meiosis

Crossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. During Caenorhabditis elegans meiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs, but the roles of these SYP proteins in promoting CO formation are poorly understood. Here, we investigate the relationships between the SYP proteins and conserved CO-promoting factors by examining the immunolocalization of these factors in meiotic mutants where SYP proteins are absent, reduced, or mis-localized. Although COs do not form in syp null mutants, CO-promoting proteins COSA-1, MSH-5, and ZHP-3 nevertheless become co-localized at a variable number of DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, CO-promoting proteins consistently track with SYP-1 localization. Moreover, CO-promoting proteins usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where SC forms between sister-chromatids, suggesting that CO regulation occurs within these structures. Further, we find that sister chromatids in the meiotic cohesin mutant rec-8 require both CO-promoting proteins and the SC to remain connected. Taken together, our findings support a model in which SYP proteins promote CO formation by directing and constraining the localization of CO-promoting factors to ensure that CO maturation occurs only between properly aligned homologous chromosomes.\n\nArticle SummaryErrors during meiosis are the leading cause of birth defects and miscarriages in humans. Thus, the coordinated control of meiosis events is critical for the faithful inheritance of the genome each generation. The synaptonemal complex (SC) is a meiosis-specific structure that assembles between homologs chromosomes and is critical for the establishment and regulation of crossovers, which ensure the accurate segregation of the homologous chromosomes at meiosis I. Here we show that the SC proteins function to regulate crossovers by directing and constraining the localization of proteins involved in promoting the formation of crossovers.

genetics↗