Biology subjects

Hawley, R. S.

Publications and source records attributed to Hawley, R. S..

10 recordsLinked to original sources

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↗

bioRxiv2025

Using evolution as a tool: Replacing corolla in Drosophila melanogaster with its Drosophila mauritiana ortholog creates a novel hypomorphic allele

In Drosophila melanogaster females, as in most organisms, the segregation of meiotic chromosomes depends on the proper distribution of crossovers along paired maternal and paternal chromosomes. In most cases, crossovers require the synaptonemal complex (SC), a conserved multi-protein structure that forms between homologous chromosomes in meiotic prophase I. Recent studies leveraging hypomorphic alleles suggest that the SC plays a more direct role in the distribution of crossover events. However, identifying additional hypomorphic mutations that avoid catastrophic phenotypes by partially disrupting the SC has been challenging. Here, to create a new hypomorphic allele of the D. melanogaster SC gene corolla, we used CRISPR/Cas9 to replace it with the coding sequence of its Drosophila mauritiana ortholog, yielding corollamau. Since the amino acid sequence of SC proteins is rapidly diverging while maintaining the general tripartite structure of the SC, we hypothesized that this replacement would enable the assembly of the SC but show defects in crossover distribution. Indeed, at 25 {degrees}C corollamau homozygous females exhibited full-length SC with defects in SC maintenance and crossover formation, resulting in moderate levels of chromosome missegregation. At 18 {degrees}C, SC maintenance was rescued, and recombination rates were improved, although they remained significantly lower than observed in wild type. Importantly, these phenotypes are less severe than observed in corolla null mutant flies, suggesting corollamau is a hypomorphic allele. Unexpectedly, in homozygotes we also observed unique polycomplexes composed of the SC proteins Corolla and Corona but lacking the transverse filament protein C(3)G. Overall, we report a novel hypomorphic allele of corolla that will enable future studies on the role of the SC in crossover distribution. Further, the unique polycomplexes found in mutant flies may provide new insights into SC protein-protein interactions and SC architecture. Author SummaryIn many species, the success of sexual reproduction relies on a protein structure called the synaptonemal complex (SC). The SC forms between the maternal and paternal copies of chromosomes and functions to ensure crossing over. Most prior studies have used SC mutants that have grave defects, preventing the study of nuances in SC function. Here, we replace one of the SC genes in Drosophila melanogaster with the ortholog of a close relative, creating a new allele that displays a partial loss-of-function phenotype. At the standard rearing temperature, flies homozygous for this allele exhibit SC maintenance defects, a reduced number of crossover events, and aberrant chromosome segregation. In flies reared at a lower temperature, SC maintenance is rescued but the defects in recombination and chromosome segregation persist. We also found a unique SC protein aggregate in these flies. Altogether, this new mutant reflects a novel approach to study the structure and function of the SC.

genetics↗

bioRxiv2025

The recombination landscape of Drosophila melanogaster can be repatterned by a single gene

Meiotic recombination plays an important role in ensuring proper chromosome segregation during meiosis I through the creation of chiasmata that connect homologous chromosomes. Recombination plays an additional role in evolution by creating new allelic combinations. Organisms display species-specific crossover patterns, but how these patterns are established is poorly understood. Drosophila mauritana displays a differing recombination pattern compared to Drosophila melanogaster, with D. mauritiana experiencing a reduced centromere effect, the suppression of recombination emanating from the centromeres. To evaluate the contribution of the synaptonemal complex (SC) C(3)G protein to these recombination rate differences, the D. melanogaster allele was replaced with D. mauritiana c(3)G coding sequence. We found that the D. mauritiana C(3)G could interact with the D. melanogaster SC machinery to build full length tripartite SC and chromosomes segregated accurately, indicating sufficient crossovers were generated. However, the placement of crossovers was altered, displaying an increase in frequency of the centromere-proximal euchromatin indicating a decrease in the centromere effect similar to that observed in D. mauritiana. Recovery of chromatids with more than one crossover was also increased, likely due to the larger chromosome span now available for crossovers. As replacement of a single gene mediated a strong shift of one species crossover pattern towards another species, it indicates a small number of discrete factors may have major influence on species-specific crossover patterning. Additionally, it demonstrates the SC, a structure known to be required for crossover formation in many species, is likely one of these discrete factors. Lay AbstractMeiotic crossovers are important for ensuring proper chromosome segregation and generating genetic diversity. Different species display unique crossover patterns but the mechanisms that establish these patterns are poorly understood. The synaptonemal complex (SC) is built between meiotic chromosomes and promotes crossover formation. Replacement of the SC gene c(3)G in the fruit fly Drosophila melanogaster with Drosophila mauritiana c(3)G resulted in full-length SC assembly and proper chromosome segregation, but the D. melanogaster crossover pattern was shifted to appear more similar to D. mauritiana. This demonstrates that crossover patterning can be largely influenced by minor changes in the makeup of the SC.

genetics↗

bioRxiv2024

Patterns of crossover distribution in Drosophila mauritiana necessitate a re-thinking of the centromere effect on crossing over

We present a SNP-based crossover map for Drosophila mauritiana. Using females derived by crossing two different strains of D. mauritiana, we analyzed crossing over on all five major chromosome arms. Analysis of 105 male progeny allowed us to identify 327 crossover chromatids bearing single, double, or triple crossover events, representing 398 separate crossover events. We mapped these crossover sites along these five chromosome arms using a genome sequence map that includes the euchromatin-heterochromatin boundary. Confirming previous studies, we show that the overall crossover frequency in D. mauritiana is higher than is seen in D. melanogaster. Much of the increase in exchange frequency in D. mauritiana is due to a greatly diminished centromere effect. Using larval neuroblast metaphases from D. mauritiana -D. melanogaster hybrids we show that the lengths of the pericentromeric heterochromatin do not differ substantially between the two species, and thus cannot explain the observed differences in crossover distribution. Using a new and robust maximum likelihood estimation tool for obtaining Weinstein tetrad distributions, we observed an increase in bivalents with two or more crossovers when compared to D. melanogaster. This increase in crossing over along the arms of D. mauritiana likely reflects an expansion of the crossover-available euchromatin caused by the reduction in the centromere effect. The pattern of crossing over in D. mauritiana conflicts with the commonly accepted view of centromeres as polar suppressors of exchange (whose intensity is buffered by sequence non-specific heterochromatin) and demonstrates the importance of expanding such studies into other species of Drosophila. Article SummaryIn meiosis, homolog segregation is usually ensured by crossovers. The number and distribution of crossovers is in part regulated by cis-acting factors such as the cis- acting centromere effect, a polar suppression of exchange emanating from the vicinity of the centromere. We use SNP-based crossover mapping to show that in Drosophila mauritiana, the centromere effect is greatly reduced on four of the five major chromosome arms. We conclude that the centromere effect differs between Drosophila mauritiana and Drosophila melanogaster and that the ability to attenuate the centromere effect is not a general property of heterochromatin.

genetics↗

bioRxiv2020

Highly contiguous assemblies of 101 drosophilid genomes

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long read sequencing allow high quality genome assemblies for tens or even hundreds of species to be generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of high-quality assemblies for 101 lines of 95 drosophilid species encompassing 14 species groups and 35 sub-groups with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. These assemblies, along with detailed wet lab protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution within this key group.

evolutionary biology↗

bioRxiv2018

Molecular analysis of Drosophila melanogaster B chromosomes reveals their origin, composition, and structure

The number of chromosomes carried by an individual species is one of its defining characteristics. Some species, however, can also carry supernumerary chromosomes referred to as B chromosomes. B chromosomes were recently identified in a laboratory stock of Drosophila melanogaster--an established model organism with a wealth of genetic and genomic resources--enabling us to subject them to extensive molecular analysis. We isolated the B chromosomes by pulsed-field gel electrophoresis and determined their composition through next-generation sequencing. Although these B chromosomes carry no known euchromatic sequence, they are rich in transposable elements and long arrays of short nucleotide repeats, the most abundant being the uncharacterized AAGAT satellite repeat. Fluorescent in-situ hybridization on metaphase chromosome spreads revealed this repeat is located on Chromosome 4, strongly suggesting the origin of the B chromosomes is Chromosome 4. Cytological and quantitative comparisons of signal intensity between Chromosome 4 and the B chromosomes supports the hypothesis that the structure of the B chromosome is an isochromosome. We also report the identification of a new B chromosome variant in a related laboratory stock. This B chromosome has a similar repeat signature as the original but is smaller and much less prevalent. We examined additional stocks with similar genotypes and did not find B chromosomes, but did find these stocks lacked the AAGAT satellite repeat. Our molecular characterization of D. melanogaster B chromosomes is the first step towards understanding how supernumerary chromosomes arise from essential chromosomes and what may be necessary for their stable inheritance.

genetics↗

bioRxiv2018

A nuclear hormone receptor and lipid metabolism axis are required for the maintenance and regeneration of reproductive organs

Understanding how stem cells and their progeny maintain and regenerate reproductive organs is of fundamental importance. The freshwater planarian Schmidtea mediterranea provides an attractive system to study these processes because its hermaphroditic reproductive system (RS) arises post-embryonically and when lost can be fully and functionally regenerated from the proliferation and regulation of experimentally accessible stem and progenitor cells. By controlling the function of a nuclear hormone receptor gene (nhr-1), we established conditions in which to study the formation, maintenance and regeneration of both germline and somatic tissues of the planarian RS. We found that nhr-1(RNAi) not only resulted in the gradual degeneration and complete loss of the adult hermaphroditic RS, but also in the significant downregulation of a large cohort of genes associated with lipid metabolism. One of these, Smed-acs-1, a homologue of Acyl-CoA synthetase, was indispensable for the development, maintenance and regeneration of the RS, but not for the homeostasis or regeneration of other somatic tissues. Remarkably, supplementing nhr-1(RNAi) animals with either bacterial Acyl-CoA synthetase or the lipid metabolite Acetyl-CoA rescued the phenotype restoring the maintenance and function of the hermaphroditic RS. Our findings uncovered a likely evolutionarily conserved role for nuclear hormone receptors and lipid metabolism in the regulation of stem and progenitor cells required for the long-term maintenance and regeneration of animal reproductive organs, tissues and cells.

developmental biology↗

bioRxiv2018

Synaptonemal complex architecture facilitates the chromosome-specific regulation of recombination in Drosophila

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double strand breaks (DSBs) into crossovers or gene conversions. The removal of any central region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct three in-frame deletions within the predicted coiled-coil region of the C(3)G protein. These three deletion mutants disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, allowing us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently than the autosomes.

genetics↗

bioRxiv2018

High-quality genome assemblies of 15 Drosophila species generated using Nanopore sequencing

The Drosophila genus is a unique group containing a wide range of species that occupy diverse ecosystems. In addition to the most widely studied species, Drosophila melanogaster, many other members in this genus also possess a well-developed set of genetic tools. Indeed, high-quality genomes exist for several species within the genus, facilitating studies of the function and evolution of cis-regulatory regions and proteins by allowing comparisons across at least 50 million years of evolution. Yet, the available genomes still fail to capture much of the substantial genetic diversity within the Drosophila genus. We have therefore tested protocols to rapidly and inexpensively sequence and assemble the genome from any Drosophila species using single-molecule sequencing technology from Oxford Nanopore. Here, we use this technology to present high-quality genome assemblies of 15 Drosophila species: 10 of the 12 originally sequenced Drosophila species (ananassae, erecta, mojavensis, persimilis, pseudoobscura, sechellia, simulans, virilis, willistoni, and yakuba), four additional species that had previously reported assemblies (biarmipes, bipectinata, eugracilis, and mauritiana), and one novel assembly (triauraria). Genomes were generated from an average of 29x depth-of-coverage data that after assembly resulted in an average contig N50 of 4.4 Mb. Subsequent alignment of contigs from the published reference genomes demonstrates that our assemblies could be used to close over 60% of the gaps present in the currently published reference genomes. Importantly, the materials and reagents cost for each genome was approximately $1,000 (USD). This study demonstrates the power and cost-effectiveness of long-read sequencing for genome assembly in Drosophila and provides a framework for the affordable sequencing and assembly of additional Drosophila genomes.

genomics↗

bioRxiv2018

Rapid low-cost assembly of the Drosophila melanogaster reference genome using low-coverage, long-read sequencing

Accurate and comprehensive characterization of genetic variation is essential for deciphering the genetic basis of diseases and other phenotypes. A vast amount of genetic variation stems from large-scale sequence changes arising from the duplication, deletion, inversion, and translocation of sequences. In the past 10 years, high-throughput short reads have greatly expanded our ability to assay sequence variation due to single nucleotide polymorphisms. However, a recent de novo assembly of a second Drosophila melanogaster reference genome has revealed that short read genotyping methods miss hundreds of structural variants, including those affecting phenotypes. While genomes assembled using high-coverage long reads can achieve high levels of contiguity and completeness, concerns about cost, errors, and low yield have limited widespread adoption of such sequencing approaches. Here we resequenced the reference strain of D. melanogaster (ISO1) on a single Oxford Nanopore MinION flow cell run for 24 hours. Using only reads longer than 1 kb or with at least 30x coverage, we assembled a highly contiguous de novo genome. The addition of inexpensive paired reads and subsequent scaffolding using an optical map technology achieved an assembly with completeness and contiguity comparable to the D. melanogaster reference assembly. Comparison of our assembly to the reference assembly of ISO1 uncovered a number of structural variants (SVs), including novel LTR transposable element insertions and duplications affecting genes with developmental, behavioral, and metabolic functions. Collectively, these SVs provide a snapshot of the dynamics of genome evolution. Furthermore, our assembly and comparison to the D. melanogaster reference genome demonstrates that high-quality de novo assembly of reference genomes and comprehensive variant discovery using such assemblies are now possible by a single lab for under $1,000 (USD).

genomics↗