Biology subjects

Yamashita, Y. M.

Publications and source records attributed to Yamashita, Y. M..

At least 19 recordsLinked to original sources

bioRxiv2026

A genome quality control program for the haploid stage of spermatogenesis

Genome integrity is fundamental to cellular and organismal health, with the germline genome bearing the most profound impact. During spermatogenesis, post-meiotic germ cells, called spermatids, undergo a series of morphological changes over an extended period (a week in fruit flies, two weeks in humans) to yield mature sperm. Having undergone consecutive meiotic divisions, these spermatids lack homologous chromosomes or sister chromatids, critical templates for accurate DNA repair. How genome integrity of spermatids is ensured during this vulnerable yet prolonged period remains poorly understood. In this study, we show that the process of spermatogenesis is specifically compromised to eliminate damaged nuclei upon {gamma}-irradiation. We further find a non-canonical role of histone variant H2Av in the process of spermatid elimination. Our findings demonstrate the presence of a haploid-specific quality control program that ensures the integrity of the sperm genome transmitted to the next generation.

cell biology↗

bioRxiv2026

Developmental conversion of the nucleolus into an RNA Polymerase II transcriptional platform in Drosophila spermatocytes

The nucleolus is widely regarded as a specialized compartment for RNA polymerase I (Pol I)-driven ribosomal RNA transcription and ribosome biogenesis. Yet the presence of "atypical nucleoli", or nucleolus-like bodies (NLBs), which lack rRNA transcription despite containing canonical nucleolar components, has long been recognized, most notably during mammalian oogenesis and spermatogenesis. NLBs have been shown to have an essential function independent of rRNA transcription, but the nature of that function remained unclear. Here, we demonstrate that the nucleolus becomes an NLB during spermatocyte development in Drosophila melanogaster and, surprisingly, that this NLB serves as a platform for RNA polymerase II (Pol II)-mediated transcription. We find that the Y chromosome-linked fertility genes, which are heterochromatic in most cell types but highly expressed in spermatocytes, are transcribed at the spermatocyte NLB. We further show that the recruitment of active Pol II to the NLB requires known spermatocyte-specific transcriptional regulators. In their absence, the Y-linked fertility genes embedded within heterochromatin are not properly transcribed. Our findings reveal an active role for an NLB as a Pol II platform, and we propose that other NLBs may have similar functionality.

developmental biology↗

bioRxiv2025

D. melanogaster meiotic driver Stellate compromises sperm development by impeding nuclear envelope remodeling

Meiotic drive is a phenomenon that violates Mendels Law of Equal Segregation, leading to biased transmission of the meiotic driver to the offspring. D. melanogaster Stellate (Ste) is an X-linked meiotic driver that preferentially harms Y-chromosome-bearing spermatids, thereby favoring the transmission of the X chromosome to the next generation. We have recently shown that Ste protein segregates asymmetrically during meiosis I with a strong bias toward the Y-chromosome-inheriting side, leading to the eventual demise of the Y-chromosome-containing spermatids. However, the cellular mechanisms by which Ste protein interferes with spermatid development remain unknown. Here, we show that Ste-containing spermatids are delayed in the process of nuclear envelope remodeling, an essential process during sperm DNA compaction. We show that components of the nuclear lamina (such as Lamin Dm0, and the LEM domain proteins Otefin and Bocks) are rapidly removed during nuclear envelope remodeling during the early stages of normal spermatid development. However, Ste-containing spermatids retained these nuclear lamina proteins for a prolonged time. Their delayed removal is associated with defective formation of the dense complex, which is composed of a bundle of microtubules and serves as a structural support for sperm nuclear morphogenesis. Defective dense complex formation in Ste-containing spermatids led to defective sperm DNA compaction. Together, the present study reveals an unexpected cellular mechanism by which a meiotic driver, Ste, sabotages sperm development. Article summaryStellate is an X-chromosome-linked meiotic driver in D. melanogaster, which interferes with the process of spermatogenesis and causes preferential death of the Y-chromosome-containing spermatids. However, the cellular mechanisms by which Stellate interferes with spermatogenesis remain unknown. This study shows that Stellate-containing spermatids are defective in the process of nuclear envelope remodeling, an essential process during sperm DNA compaction. Defective nuclear envelope remodeling was associated with a failure to assemble the dense complex, a microtubule-rich structure that serves as structural support for sperm nuclear morphogenesis. Together, the study provides insights into a cellular strategy employed by a meiotic driver.

evolutionary biology↗

bioRxiv2025

Defective splicing of Y-chromosome-linked gigantic genes underlies hybrid male sterility in Drosophila

The Y chromosome evolves rapidly, often differing dramatically even between closely related species. While such divergence has long been suspected to contribute to hybrid male sterility, leading to reproductive isolation and thus speciation, the underlying mechanisms remain elusive. Here, we identify a molecular basis linking Y chromosome divergence to reproductive isolation in Drosophila. We show that male hybrids between D. simulans and D. mauritiana fail to properly express key Y-linked fertility genes. These genes contain unusually large introns, exceeding megabases and show substantial sequence divergence between species. In the hybrids, these gigantic introns are misprocessed, resulting in widespread splicing defects, including aberrant "back-splicing" events that join later exons to earlier ones. Our findings suggest that sequence divergence within introns can disrupt essential gene expression through defective splicing, providing a mechanistic link between rapid Y chromosome evolution and hybrid sterility. This work highlights the underappreciated role of intronic divergence in speciation.

evolutionary biology↗

bioRxiv2025

Modular regulation of the stem cell transcriptome defines self-renewal, differentiation, and dedifferentiation

Adult stem cells maintain tissue homeostasis, yet are themselves vulnerable to loss. One common mechanism to replace lost stem cells is dedifferentiation, in which progeny revert to stem cell identity. It is a paradox how stem cells and progeny retain the same stem cell potential while exhibiting distinct current identities of self-renewal, differentiation, and dedifferentiation. Here, we show that the Drosophila male germline lineage solves this paradox via two parallel and complementary mechanisms to separate potential and identity. First, differentiating progeny maintain stem cell potency by inheriting perdurant stem cell mRNAs without actively transcribing them. Second, two known niche signals (Bmp and Jak-Stat) activate distinct sets of targets, defining three identities (self-renewal, differentiation, and dedifferentiation) based on the combination of their on/off states. Together, this study reveals how a pool of dedifferentiation-competent progeny is maintained to regenerate stem cells as needed without resulting in stem cell overproduction, and resolves the puzzle of why most stem cell systems require multiple independent niche signals. Significance StatementDedifferentiation is a mechanism by which differentiating cells revert back to stem cell identity to compensate for the loss of stem cells, allowing for long-term tissue homeostasis. It remains poorly understood how stem cells and progeny retain the same stem cell potential while exhibiting distinct current identities of self-renewal, differentiation, and dedifferentiation. This study reveals two parallel mechanisms to separate cells potential and identity. First, differentiating progeny maintain stem cell potency by inheriting perdurant stem cell mRNAs. Second, two known niche signals (Bmp and Jak-Stat) activate distinct sets of targets, defining three identities (self-renewal, differentiation, and dedifferentiation) based on the combination of their on/off states. This study reveals how a pool of dedifferentiation-competent progeny is maintained to regenerate stem cells.

developmental biology↗

bioRxiv2025

Testing the function of widespread transcription of the testis

Male germ cells have complex transcriptomes, with a large fraction of the genome being transcribed. This includes protein-coding genes (often not translated), non-coding DNA, and repetitive DNA, such as transposons and satellite DNA, which are normally silenced as heterochromatin. The significance of such widespread transcription remains unknown. Here, we show that a heterochromatin protein, HP2, is required for the transcription of AAGAG satellite DNA in Drosophila spermatocytes. HP2 depletion leads to abnormal retention of heterochromatin histone marks (H3K9me3) and spermatid death during sperm DNA packaging, leading to a model that transcription of AAGAG satellite DNA facilitates the remodeling of its heterochromatic nature in preparation for sperm DNA packaging. Strikingly, the severity of the spermatid death correlates with the amount of AAGAG satellite DNA carried by the spermatids, leading to preferential death of Y chromosome-containing spermatids over X-containing spermatids, and hence sex-ratio meiotic drive phenotype. We propose that widespread spermatocyte transcription may reflect the process of chromatin remodeling to allow sperm DNA packaging. We further propose that differential composition and amount of satellite DNA on chromosomes may underlie naturally occurring male meiotic drive.

developmental biology↗

bioRxiv2024

Co-transcriptional splicing facilitates transcription of gigantic genes

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.

developmental biology↗

bioRxiv2024

Insulin signaling regulates R2 retrotransposon expression to orchestrate transgenerational rDNA copy number maintenance

Preserving a large number of essential yet highly unstable ribosomal DNA (rDNA) repeats is critical for the germline to perpetuate the genome through generations. Spontaneous rDNA loss must be countered by rDNA copy number (CN) expansion. Germline rDNA CN expansion is best understood in Drosophila melanogaster, which relies on unequal sister chromatid exchange (USCE) initiated by DNA breaks at rDNA. The rDNA-specific retrotransposon R2 responsible for USCE-inducing DNA breaks is typically expressed only when rDNA CN is low to minimize the danger of DNA breaks; however, the underlying mechanism of R2 regulation remains unclear. Here we identify the insulin receptor (InR) as a major repressor of R2 expression, limiting unnecessary R2 activity. Through single-cell RNA sequencing we find that male germline stem cells (GSCs), the major cell type that undergoes rDNA CN expansion, have reduced InR expression when rDNA CN is low. Reduced InR activity in turn leads to R2 expression and CN expansion. We further find that dietary manipulation alters R2 expression and rDNA CN expansion activity. This work reveals that the insulin pathway integrates rDNA CN surveying with environmental sensing, revealing a potential mechanism by which diet exerts heritable changes to genomic content.

cell biology↗

bioRxiv2024

RNA polymerase II-mediated rDNA transcription mediates rDNA copy number expansion in Drosophila

Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNAs repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as rDNA magnification, has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary.

genetics↗

bioRxiv2022

Deregulation of Y-linked protamine-like genes in sex chromosome-biased spermatid demise

Meiotic drive is a phenomenon wherein a genetic element achieves a higher rate of transmission than dictated by Mendelian segregation (1-3). One proposed mechanism for meiotic drivers to achieve biased transmission is by sabotaging essential processes of gametogenesis (e.g. spermatogenesis), leading to demise of gametes that contain their opponents (1). Studies in D. simulans have recently found that a set of meiotic driver genes contain a sequence homologous to protamines (4, 5), critical proteins that package sperm chromatin (6-8). However, the underlying mechanisms of drive and the relevance of protamine-like sequences in meiotic drive remain unknown. While studying the function of Modulo, the homolog of Nucleolin in Drosophila melanogaster (9, 10), we unexpectedly discovered Y-linked protamine genes function as a meiotic driver: we found that modulo mutants known sterility is caused by deregulation of the autosomal protamine-like gene (Mst77F) and its Y chromosome-linked homolog (Mst77Y). Modulo regulates these genes at the step of polyadenylation of the transcripts. We show that Mst77Y likely acts as a dominant-negative form of Mst77F, interfering with the process of histone-to-protamine transition, leading to nuclear decompaction. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear decompaction and results in the biased demise of X chromosome-bearing sperm. We propose that dominant-negative protamine variants may be a common strategy found in male meiotic drive and may explain known rapid divergence of protamine genes. Significance statementProtamines are small, highly positively charged proteins that are required for packaging DNA to produce mature sperm with highly-condensed nuclei capable of fertilization. Even small changes in the dosage of protamines in humans is associated with infertility. Yet, despite their essential function, protamines are rapidly evolving. It has been speculated that protamines rapid divergence may be explained by their potential participation in genomic conflict. Our work implicates the involvement of Y chromosome-linked multicopy protamine-like genes in meiotic drive in Drosophila melanogaster. Our results suggest that dominant negative protamines can sabotage the process of nuclear compaction during spermiogenesis, revealing a potential cellular mechanism of sperm killing in meiotic drive.

genetics↗

bioRxiv2022

Drosophila Males Use 5'-to-3' Phased Biogenesis to Make Stellate-silencing piRNAs that Lack Homology to Maternally Deposited piRNA Guides

PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In animals with a maternally specified germline, e.g. Drosophila melanogaster, maternally deposited piRNAs initiate piRNA biogenesis in the progeny. Normal fertility in D. melanogaster males requires repression of tandemly repeated Stellate genes by piRNAs from Suppressor of Stellate [Su(Ste)]. Because the Su(Ste) loci are on the Y chromosome, Su(Ste) piRNAs are not deposited in oocytes. How the male germline produces Su(Ste) piRNAs in the absence of maternally deposited Su(Ste) piRNAs is unknown. Here, we show that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis triggered by maternally deposited 1360/Hoppel transposon piRNAs. Strikingly, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers the developmentally programmed mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.

developmental biology↗

bioRxiv2022

Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study (Li et al., 2022). With over 44,000 nuclei and 6,000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.

developmental biology↗

bioRxiv2022

Sex-specific topological differences in germline cell lineage trees

From insects to mammals, oocytes ad sperm develop within germline cysts comprising cells connected by intercellular bridges (ICBs). In numerous insects, formation of the cyst is accompanied by growth of the fusome - a membranous that permeates the cyst. Fusome composition and function are best understood in Drosophila melanogaster: during oogenesis, the fusome dictates cyst topology and size and facilitates oocyte selection, while during spermatogenesis, the fusome synchronizes the cysts response to DNA damage. Despite its myriad and sex-specific roles during insect gametogenesis, fusome growth and inheritance in females and its structure and connectivity in males have remained challenging to investigate. Here, we take advantage of advances in high resolution confocal microscopy and computational image processing tools to reconstruct the topology, growth, and distribution of the fusome in both sexes. Our findings inform a theoretical model for fusome assembly and inheritance during oogenesis, shedding light on symmetry-breaking processes that lead to oocyte selection. In males, we find that cell divisions can deviate from the maximally branched pattern observed in females, leading to greater topological variability. Our work consolidates existing disjoint experimental observations and contributes a readily generalizable computational approach for quantitative studies of gametogenesis within and across species. Author summaryThe ubiquity of germline cysts across animals and accelerating advances in microscopy call for quantitative and highly resolved studies of their developmental dynamics. Here we use Drosophila melanogaster gametogenesis as a model system, alongside a supervised learning algorithm to study a shared organelle that arises during sperm and oocyte development - the fusome. The fusome is a highly specialized membranous organelle that permeates the cyst in both sexes. Our three-dimensional (3D) reconstructions of the fusome and quantitative measurements at successive stages of cyst development during oogenesis shed light on the evolution of cell fate asymmetry within the germline cyst in females, where the cyst gives rise to a single oocyte. In males, where each cell of the cyst goes on to form sperm, the fusome fragments and exhbits topologies that deviate from the stereotypic maximally branched topology found in females. Our findings can be interpreted in the context of the divergent outcomes of gametogenesis in both sexes and highlight the centrality of quantitative measurements in evaluating hypotheses in biological sciences.

developmental biology↗

bioRxiv2021

The retrotransposon R2 maintains Drosophila ribosomal DNA repeats

Ribosomal DNA (rDNA) loci contain the hundreds of tandemly repeated copies of ribosomal RNA genes needed to support cellular viability. This repetitiveness makes it highly susceptible to copy number (CN) loss, threatening multi-generational maintenance of rDNA. How this threat is counteracted to avoid extinction of the lineage has remained unclear. Here, we show that the rDNA-specific retrotransposon R2 is essential for rDNA CN maintenance in the Drosophila male germline, despite the perceived disruptive nature of transposable elements. Depletion of R2 led to defective rDNA CN maintenance, causing a decline in fecundity over generations and eventual extinction. This study reveals that active retrotransposons can provide a benefit to their hosts, contrary to their reputation as genomic parasites, which may contribute to their widespread success throughout taxa. One Sentence SummaryThe retrotransposon R2 initiates restoration of ribosomal DNA copies to trans-generationally maintain essential locus.

cell biology↗

bioRxiv2021

Defective satellite DNA clustering into chromocenters underlies hybrid incompatibility in Drosophila

Although rapid evolution of pericentromeric satellite DNA repeats is theorized to promote hybrid incompatibility (HI)(1-4), how divergent repeats affect hybrid cells remains poorly understood. Recently, we demonstrated that sequence-specific DNA-binding proteins cluster satellite DNA from multiple chromosomes into chromocenters, thereby bundling chromosomes to maintain the entire genome in a single nucleus(5, 6). Here we show that ineffective clustering of divergent satellite DNA in the cells of Drosophila hybrids results in chromocenter disruption, associated micronuclei formation and tissue atrophy. We further demonstrate that previously identified HI factors trigger chromocenter disruption and micronuclei in hybrids, linking their function to a conserved cellular process. Together, we propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation.

cell biology↗

bioRxiv2021

me31B regulates stem cell homeostasis by preventing excess dedifferentiation in the Drosophila male germline.

Tissue-specific stem cells maintain tissue homeostasis by providing a continuous supply of differentiated cells throughout the life of organisms. Differentiated/differentiating cells can revert back to a stem cell identity via dedifferentiation to help maintain the stem cell pool beyond the lifetime of individual stem cells. Although dedifferentiation is important to maintain the stem cell population, it is speculated to underlie tumorigenesis. Therefore, this process must be tightly controlled. Here we show that a translational regulator me31B plays a critical role in preventing excess dedifferentiation in the Drosophila male germline: in the absence of me31B, spermatogonia (SGs) dedifferentiate into germline stem cells (GSCs) at a dramatically elevated frequency. Our results show that the excess dedifferentiation is likely due to misregulation of nos, a key regulator of germ cell identity and GSC maintenance. Taken together, our data reveal negative regulation of dedifferentiation to balance stem cell maintenance with differentiation.

developmental biology↗

bioRxiv2020

Localized mRNA translation mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis

Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g. Plasmodium microgametocytes and human and Drosophila sperm), very little is known about cytoplasmic cilia assembly. Here we show that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the localized translation of these axonemal dyneins and their incorporation into the axoneme directly from the cytoplasm. We found that this RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. We propose that cytoplasmic cilia require the local translation of key protein constituents such that these proteins are incorporated efficiently into the axoneme. Author SummaryCytoplasmic cilia, which are found in human and Drosophila sperm, are unique in that the axoneme is exposed to the cytoplasm. The authors show that a novel RNP granule containing axonemal dynein mRNAs facilitates localized translation of these axonemal proteins, facilitating cytoplasmic cilia formation.

developmental biology↗

bioRxiv2018

The modular mechanism of chromocenter formation in Drosophila

A central principle underlying the ubiquity and abundance of pericentromeric satellite DNA repeats in eukaryotes has remained poorly understood. In our previous study (Jagannathan et al., 2018), we proposed that the interchromosomal clustering of satellite DNAs into nuclear structures known as chromocenters ensures encapsulation of all chromosomes into a single nucleus. Chromocenter disruption led to micronuclei formation, resulting in cell death. Here we show that chromocenter formation is mediated by a modular network, where interactions between two sequence-specific satellite DNA-binding proteins, D1 and Prod, bound to their cognate satellite DNAs, bring the full complement of chromosomes into the chromocenter. D1 prod double mutants die during embryogenesis, exhibiting enhanced phenotypes associated with chromocenter disruption, revealing the universal importance of satellite DNAs and chromocenters. Taken together, we propose that interactions between chromocenter modules, consisting of satellite DNA binding proteins and their cognate satellite DNA, package the Drosophila genome within a single nucleus.

molecular biology↗