Biology subjects

Benner, L.

Publications and source records attributed to Benner, L..

8 recordsLinked to original sources

bioRxiv2026

A mdg4 Retrotransposon Screen for X-linked Female Sterile Alleles and its Relationship with the Transcription Factor OVO

In the germline, the mdg4 retrotransposon integrates in close proximity to the location of OVO DNA binding motifs, suggesting that insertion bias is driven by the OVO transcription factor. A classical genetic example of this is the reversion of the dominant female-sterile allele, ovoD1, by the transposition of mdg4 into the ovo promoter where OVO protein binds. We wanted to take advantage of this relationship and determine if we could recover female sterile alleles along the X chromosome due to mdg4 insertion, with the hypothesis that these would be genes that OVO binds and transcriptionally regulates in the germline. We mobilized the mdg4 retrotransposon with the use of mutants for the lncRNA gene flamenco (flam) and recovered 17 recessive female sterile alleles out of a total of 1,192 chromosomes screened. We identified 11 complementation groups, for which a mdg4 insertion was responsible for female sterility in 7 groups. Notably, a complementation group consisting of 6 alleles was found to be the result of a Doc transposable element insertion into the gene Grip91 and is potentially evidence for a Doc insertional hotspot in the genome. Our screen also uncovered that 7/17 recessive female sterile chromosomes contained multiple transposable element insertions indicating that flam- females derepress numerous transposable elements that can lead to multiple transposon insertions along a single chromosome, as has been suggested previously. Altogether, we found that mdg4 did have an insertion bias into OVO bound regions of the genome that can result in female sterility, however, this was the case for a minority of the female sterile alleles recovered with this method. Article SummaryThe retrotransposon mdg4 preferentially inserts near binding sites of the female germline transcription factor OVO in Drosophila melanogaster, most notably at the ovo locus itself. We leveraged this relationship to screen for X-linked recessive female-sterile mutations generated by mdg4 mobilization in flamenco mutant females. From 1,192 chromosomes, we recovered 17 female-sterile alleles across 11 complementation groups. mdg4 insertions were significantly enriched in OVO-bound regions but accounted for only a subset of sterility phenotypes, revealing substantial background mutagenesis by other transposable elements. These results refine the OVO-mdg4 relationship and highlight both the promise and limitations of transposon-based genetic screens.

genetics↗

bioRxiv2025

Programmed meiotic errors facilitate dichotomous sperm production in the silkworm, Bombyx mori

The goal of meiosis is typically to produce haploid gametes (eggs or sperm). Failure to do so is catastrophic for fertility and offspring health. However, Lepidopteran (moths and butterflies) males produce two morphs of sperm: nucleated (eupyrene) sperm which fertilize the egg, and anucleated (apyrene) sperm, both of which are essential for fertilization. The meiotic differences in the two types of spermatogenesis have not been well characterized, and our knowledge of the molecular differences between eupyrene and apyrene spermatogenesis are extremely limited in all systems. The only factor identified as being required for apyrene spermatogenesis is Sex-lethal (Sxl). Here, we show through cytological analysis of early meiotic events that there are several key differences in the genesis of apyrene sperm and eupyrene sperm. Specifically, apyrene spermatocytes fail to condense and pair their chromosomes during meiotic prophase I. In addition, telomeres do not attach to the nuclear envelope. Due to these differences, full-length synaptonemal complex does not form. RNA sequencing of both eupyrene- and apyrene-producing testes revealed distinct changes in transcriptional programs, including down-regulation of a myriad of meiotic genes and cell cycle checkpoint factors during apyrene meiosis. By comparing wild-type and Sxl-knockout apyrene testes, we found that Sxl is not required for the changes in the expression of the meiotic genes but instead plays a role in checkpoint inactivation to allow this error-prone meiosis to proceed. Together, our findings reveal significant insights into two converging molecular pathways that promote the formation of dimorphic sperm in Lepidoptera.

genetics↗

bioRxiv2024

Diverse somatic Transformer and sex chromosome karyotype pathways regulate gene expression in Drosophila gonad development

The somatic sex determination gene transformer (tra) is required for the highly sexually dimorphic development of most somatic cells, including those of the gonads. In addition, somatic tra is required for the germline development even though it is not required for sex determination within germ cells. Germ cell autonomous gene expression is also necessary for their sex determination. To understand the interplay between these signals, we compared the phenotype and gene expression of larval wild-type gonads and the sex-transformed tra gonads. XX larval ovaries transformed into testes were dramatically smaller than wild-type, with significant reductions in germ cell number, likely due to altered geometry of the stem cell niche. Additionally, there was a defect in progression into spermatocyte stages. XY larval testes transformed into ovaries had excessive germ cells, possibly due to the earlier onset of cell division. We suggest that germ cells are neither fully female nor male following somatic sex transformation, with certain pathways characteristic of each sex expressed in tra mutants. We found multiple patterns of somatic and germline gene expression control exclusively due to tra, exclusively due to sex chromosome karyotype, but usually due to a combination of these factors showing tra and sex chromosome karyotype pathways regulate gene expression during Drosophila gonad development.

developmental biology↗

bioRxiv2024

Muskelin acts as a substrate receptor of the highly regulated Drosophila CTLH E3 ligase during the maternal-to-zygotic transition

The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. We have previously shown that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch (TRAL) are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies. In particular, Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting degradation of ME31B and its cofactors to the MZT are unknown. Here, we show that the developmental specificity of the CTLH complex is mediated by multi-pronged regulation, including transcriptional control by the transcription factor OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which we demonstrate acts as a substrate adaptor for the Drosophila CTLH complex. Although conserved, Muskelin has structural roles in other species, suggesting a surprising functional plasticity. Finally, we find that Muskelin has few targets beyond the three known RNA binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, seemingly with the goal of regulating three important RNA binding proteins.

developmental biology↗

bioRxiv2023

OVO Positively Regulates Essential Maternal Pathways by Binding Near the Transcriptional Start Sites in the Drosophila Female Germline

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVOs role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA Polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

developmental biology↗

bioRxiv2023

Female germline expression of OVO transcription factor bridges Drosophila generations

OVO is required for karyotypically female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovoD1allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo utilizes a number of unannotated splice variations in the extended exon and a minor population of ovo-B transcripts utilizes an alternative splice. This indicates that classical ovo alleles such as ovoD1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, we deleted the ovo-A and ovo-B promoters showing that only ovo-B is required for female germ cell viability and there is an early and polyphasic developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, we endogenously tagged ovo and found nuclear OVO in all differentiating female germ cells throughout oogenesis in adults. We also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next. Article Summaryovo has long been considered to be at the top of the female germline sex determination pathway. We utilized updated genetic methods to determine OVO expression, localization, and requirement in the embryonic and adult germline. Our results indicate that OVO is always present, and likely required, in the Drosophila female germline.

genetics↗

bioRxiv2020

Functional tagging of endogenous proteins and rapid selection of cell pools (Rapid generation of endogenously tagged piwi in ovarian somatic sheath cells.)

The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We present a concise workflow to establish modified cells, characterize the edited allele and probe the function of the tagged protein.

cell biology↗

bioRxiv2018

Drosophila small ovary encodes a zinc-finger repressor required for ovarian differentiation

Repression is essential for coordinated cell type-specific gene regulation and controlling the expression of transposons. In the Drosophila ovary, stem cell regeneration and differentiation requires controlled gene expression, with derepression leading to tissue degeneration and ovarian tumors. Likewise, the ovary is acutely sensitive to deleterious consequences of transposon derepression. The small ovary (sov) locus was identified in a female sterile screen, and mutants show dramatic ovarian morphogenesis defects. We mapped the locus to the uncharacterized gene CG14438, which encodes a zinc-finger protein that colocalizes with the essential Heterochromatin Protein 1 (HP1a). We demonstrate that Sov functions to repress inappropriate cell signaling, silence transposons, and suppress position-effect variegation in the eye, suggesting a central role in heterochromatin stabilization.

developmental biology↗