Biology subjects

Spradling, A. C.

Publications and source records attributed to Spradling, A. C..

At least 19 recordsLinked to original sources

bioRxiv2026

The mouse oocyte Balbiani body reassembles in growing follicles to continue organelle selection and upon dispersal influences nuclear position

The Balbiani body (Bb), a universal oocyte structure enriched in mitochondria and organelles, forms during zygotene but grows and behaves differently in several animal groups. The mouse Bb forms during pre-follicular oogenesis, but whether it persists during and after the primordial follicle state is unknown, unlike the long lived Bbs in the oocytes of lower vertebrates. Here we report that the mouse Bb, which has been postulated to facilitate mitochondrial selection and organelle quality control, re-assembles in growing mouse follicles. The re-assembled Bb is organized around a single microtubule-organizing center (MTOC), surrounded by mitochondria, Golgi apparatus, endoplasmic reticulum, and endosomes, like the original Bb. Thus, mouse oocytes contain a Bb whose action is paused by quiescence, but otherwise more closely resembles the primordial follicle Bb and the Bb in zebrafish and Xenopus oocytes. The mouse Bb eventually disperses as its MTOC relocates from the perinuclear region to the oocyte cortex, driven by opposing microtubule and actin forces that also reposition the nucleus to the oocyte center. We suggest that the Bb re-assembles and persists during oocyte growth until its function in mitochondrial and organelle quality control is complete.

developmental biology↗

bioRxiv2025

The Drosophila ovary produces three follicle waves similar to those in mice

Ovarian follicles in most species are assumed to develop using a single pathway. However, in Drosophila pupae, lineage tracing 2,937 single-cell clones identified three follicle "waves" that follow distinct programs arrayed anterior to posterior in the germarium and developing ovarioles. 40 primordial germ cells (PGCs) become anterior germline stem cells (GSCs) that produce "wave 2" follicles throughout adulthood. 100 PGCs posterior to wave 2 develop into "wave 1.5" follicles that form the earliest laid eggs. Different follicle stem cells (FSCs) sequentially occupy the same two niches timed to provide both waves with specific follicle cells, consistent with programmed differences between waves 2 and 1.5. "Wave 1" PGCs located even further posterior proliferate, form cysts, interact with swarm cells, degenerate, break from the ovary 22-26 hours after puparium formation, and release lipid-enriched vacuoles. Some testis germ cells behave similarly. We speculate that wave 1 germ cells contribute directly or indirectly to the pharate adult ecdysone pulse that mediates adult development and sex-specific neural remodeling. Why Drosophila follicle waves generally resemble those clarified recently in mouse preadult ovaries merits additional study.

developmental biology↗

bioRxiv2025

The Drosophila ovarian terminal filament imports molecules needed to produce lipid droplets, the fusome, and functional germ cells

Oogenesis in Drosophila requires small molecules as biosynthetic precursors and regulators of follicle development. But how such molecules reach germline stem cells (GSCs), developing germline cysts and follicles in the germarium and early ovariole is poorly understood. The flat, stacked cells of the terminal filament (TF) form a specialized somatic structure positioned at the anterior end of ovarioles in virtually all insect ovaries, but physiological roles TFs play in adult ovaries remain little known. By briefly knocking down, specifically in the TF, exocyst components affecting vesicle trafficking, lipid importers such as LpR2, and organic anion importers such as Oatp30B, we found that the TF provides lipophilic molecules to GSCs and downstream germarium cells needed to maintain lipid droplets, and germ cell differentiation. When exocyst component Sec6 is knocked down, vesicles containing lipophilic cargos back up at the TF-germ cell junction, suggesting that endosomes move between the stacked TF cells and into cap cells by transcytosis. Some of these may be shuttled during the GSC cell cycle between the TF and newly forming fusome. Our studies suggest that TFs import lipophilic precursors and regulators to autonomously coordinate their ovarioles stem cell activity and cyst development.

developmental biology↗

bioRxiv2025

Early female germline development in Xenopus laevis: stem cells, nurse cells and germline cysts

In Drosophila, germline cysts arise through synchronous mitotic divisions and acquire a polarized architecture organized by the fusome, which guides oocyte specification and supports meiotic progression. Similar cyst structures exist in non-mammalian vertebrate ovaries, but their polarity and function have remained uncertain. Using single-cell RNA sequencing and high-resolution imaging, we reconstructed the germ cell differentiation trajectory in Xenopus laevis and uncovered striking parallels with invertebrate and mouse cyst development. We identified a distinct germline stem cell (GSC) population marked by piwil4, low translational activity, and expression of neuronal-specific and transposon-silencing genes. Downstream from GSCs, during cyst development, an asymmetric fusome-like structure (FLS) composed of stable microtubules forms a rosette-like connection between cystocytes and co-localizes with Golgi vesicles and ER, suggesting polarized trafficking. In contrast to previous claims, [~]90% of EdU-prelabeled cyst cells turned over rather than forming oocytes, consistent with a nurse cell fate. The striking parallels described here between cyst and fusome formation, polarization, cyst breakdown, and nurse cell turn over to produce relatively few oocytes, argue that amphibian cysts have important functions in female gametogenesis.

developmental biology↗

bioRxiv2025

Mouse pre-meiotic germline cysts contain fusome-like structure dependent on Dazl that mediates cyst fragmentation and oocyte development

Mouse female primordial germ cells (PGCs) undergo five synchronous, incomplete mitotic divisions, and send each resulting germline cyst into meiosis to fragment and produce 4-6 oocytes and 24-26 supportive nurse cells. However, no system of polarity has been found to specify mammalian oocytes, link them appropriately to nurse cells and enable them to acquire high quality organelles and cytoplasm. We report that mouse cysts develop an asymmetric Golgi, endoplasmic reticulum (ER) and microtubule-associated "fusome", similar to the oocyte-determining fusome in Drosophila cysts. The mouse fusome distributes asymmetrically among cyst cells and enriches in future oocytes with Pard3, and Golgi-endosomal UPR (unfolded protein response) proteins. Spindle remnants rich in stable acetylated microtubules, like those building the Drosophila and Xenopus fusomes, transiently link early mouse cyst cells for part of each cell cycle. A non-random gap in these microtubules predicts that initial cysts fragment into similar six-cell derivatives, providing a potential mechanism for producing uniform oocytes. Together with previous studies these results argue that a polarized fusome underlies the development of female gametes from the PGC to follicular oocyte stages in diverse animals including mammals.

developmental biology↗

bioRxiv2024

Early mouse ovarian follicles develop into a thecal cell network that generates androgens during peri-puberty

The earliest growing mouse follicles, wave 1, rapidly develop in the ovarian medulla, while the great majority, wave 2, are stored for later use as resting primordial follicles in the cortex. Wave 1 follicles are known to mostly undergo atresia, a fate sometimes associated with the persistence of steroidogenic theca cells, but this connection is poorly understood. We characterized wave 1 follicle biology using tissue clearing, lineage tracing and scRNAseq to clarify their contributions to offspring and to steroidogenesis. Wave 1 follicles lineage marked by E16.5 FoxL2 expression in granulosa cells, reach preantral stages containing theca cell layers by 2 weeks. Atresia begins about a week later, during which 80-100% of wave 1 follicles degrade their oocytes, turn over most granulosa cells, but retain theca cells which expand in number together with interstitial gland cells in the medulla. During puberty (5 weeks), these cells ultrastructurally resemble steroidogenic cells and highly express androgen biosynthetic genes. Unexpectedly, the FoxL2 lineage tag also marked about 400 primordial follicles, located near the medullar-cortical boundary, that become the earliest-activated wave 2 follicles. These "boundary" or "wave 1.5" follicles generate 70-100% of the earliest mature oocytes, while fewer than 26 wave 1 follicles with oocytes survive. Consistent with their largely distinct fates in steroid or oocyte production, granulosa cells of antral wave 1 and wave 2 follicles differentially express multiple genes, including Wnt4 and Igfbp5. SIGNIFICANCE STATEMENTWe studied the ovaries of juvenile mice 2-6 weeks (wk) of age using transcriptomics and cell lineage to characterize the earliest developing groups of follicles. Wave 1 follicles begin growing at birth in the ovarian medulla, but few produce mature oocytes. Instead, most undergo partial atresia and expand steroidogenic cells expressing androgenic genes at puberty. A newly identified primordial follicle subset located at the medulla-cortex boundary are the earliest to activate and grow. Their oocytes give rise to most early offspring, while no more than a few derive from surviving wave 1 follicles. Our results highlight the importance of spatially localized follicle subgroups and show that follicular waves aid reproduction in distinct ways, some of which involve substantial atresia and cellular remodeling.

developmental biology↗

bioRxiv2023

Chromatin and gene expression changes during female Drosophila germline stem cell development illuminate the biology of highly potent stem cells

Highly potent animal stem cells either self renew or launch complex differentiation programs, using mechanisms that are only partly understood. Drosophila female germline stem cells (GSC) perpetuate without change over evolutionary time and generate cystoblast daughters that develop into nurse cells and oocytes. Cystoblasts initiate differentiation by generating a transient syncytial state, the germline cyst, and by increasing pericentromeric H3K9me3 modification, actions likely to suppress transposable element activity. Relatively open GSC chromatin is further restricted by Polycomb repression of testis or somatic cell-expressed genes briefly active in early female germ cells. Subsequently, Neijre/CBP and Myc help upregulate growth and reprogram GSC metabolism by altering mitochondrial transmembrane transport, gluconeogenesis and other processes. In all these respects GSC differentiation resembles development of the totipotent zygote. We propose that the totipotent stem cell state was shaped by the need to resist transposon activity over evolutionary time scales.

developmental biology↗

bioRxiv2022

Chromosome-scale scaffolding of the fungus gnat genome (Diptera: Bradysia coprophila)

BackgroundThe fungus gnat, Bradysia (Sciara) coprophila, has compelling chromosome biology. Paternal chromosomes are eliminated during spermatogenesis whereas both maternal X sister chromatids are retained. Embryos start with three copies of the X chromosome, but 1-2 copies are eliminated from somatic cells as part of sex determination, and one is eliminated in the germline to restore diploidy. These developmentally normal events present opportunities to study chromosome movements that are unusual in other systems. To support such studies, we previously generated a highly contiguous optical-map-scaffolded long-read assembly (Bcop_v1) of the male somatic genome. However, the scaffolds were not chromosome-scale, the majority of the assembly lacked chromosome assignments, and the order and orientation of the contigs along chromosomes remained unknown. FindingsMale pupae Hi-C data was used to correct, order, and orient the contigs from Bcop_v1 into chromosome-scale scaffolds, producing the updated assembly, Bcop_v2. Several orthogonal analyses allowed us to (i) identify the corresponding chromosome for each scaffold, (ii) orient them with respect to polytene maps, and (iii) determine that they were highly concordant with the chromosomes they represent. Gene annotations produced for Bcop_v1 were lifted over to Bcop_v2. Chromosomal repeat distributions highlight a potential telomeric sequence. Finally, the Hi-C data shed new light on three "fold-back regions" seen to physically interact in images of polytene X chromosomes. ConclusionsStudies of the unusual chromosome movements in Bradysia coprophila will benefit from the updated assembly (Bcop_v2) where each somatic chromosome is represented by a single scaffold.

genomics↗

bioRxiv2021

Drosophila renal stem cells enhance fitness by delayed remodeling of adult Malpighian tubules

Drosophila renal stem cells (RSCs) contradict the common expectation that stem cells maintain tissue homeostasis. RSCs are abundant, quiescent and confined to the peri-ureter region of the kidney-like Malpighian tubules (MTs). Although derived during pupation like intestinal stem cells, RSCs initially remodel the larval MTs only near the intestinal junction. However, following adult injury to the ureter by xanthine stones, RSCs remodel the damaged region in a similar manner. Thus, RSCs represent stem cells encoding a developmental redesign. The remodeled tubules have a larger luminal diameter and shorter brush border, changes linked to enhanced stone resistance. However, RSC-mediated modifications also raise salt sensitivity and reduce fecundity. Our results suggest that RSCs arose by arresting developmental progenitors to preserve larval physiology until a time in adulthood when it becomes advantageous to complete development by RSC activation. One-Sentence SummaryActivated Drosophila renal stem cells rebuild the adult Malphigian tubules using a less efficient but more stone-resistant design.

developmental biology↗

bioRxiv2021

Mouse oocytes develop in cysts with the help of nurse cells

Mammalian oocytes develop initially in cysts containing many more germ cells than the primordial oocytes they generate. We identified abundant nurse cells with reduced unique molecular identifiers (UMI)/cell from ovaries aged E14.5 to P1. Low UMI nurse cells are found in cysts and express the same major meiotic genes as pro-oocytes of the same stage, suggesting they are oocyte sisters that are signaled to transfer cytoplasm at different times and only subsequently diverge. Oocyte vs nurse cell selection occurs in cysts with a robust microtubule cytoskeleton, that closely interact with somatic cells and that develop a dense actin cytoskeleton around nurse cell nuclei that are held back from cytoplasmic transfer. Mouse and Drosophila nurse cells undergo programmed cell death by acidification from adjacent somatic pre-granulosa cells that express V-ATPases and cathepsin proteins. Disrupting acidification in cultured mouse ovaries blocked nurse cell turnover. About 200 genes are induced in mouse dictyate oocytes as previously reported, including Tuba1c and Tubb2b, genes that we find contribute to Balbiani body formation. Thus, mouse oocytes are specified within germline cysts and develop with the assistance of nurse cells using highly conserved mechanisms.

developmental biology↗

bioRxiv2021

A gut commensal niche regulates stable association of a multispecies microbiota

The intestines of animals are typically colonized by a complex, relatively stable microbiota that influences health and fitness, but the underlying mechanisms of colonization remain poorly understood. As a typical animal, the fruit fly, Drosophila melanogaster, is associated with a consistent set of commensal bacterial species, yet the reason for this consistency is unknown. Here, we use gnotobiotic flies, microscopy, and microbial pulse-chase protocols to show that a commensal niche exists within the proventriculus region of the Drosophila foregut that selectively binds bacteria with exquisite strain-level specificity. Primary colonizers saturate the niche and exclude secondary colonizers of the same strain, but initial colonization by Lactobacillus physically remodels the niche to favor secondary colonization by Acetobacter. Our results provide a mechanistic framework for understanding the establishment and stability of an intestinal microbiome. One-Sentence SummaryA strain-specific set of bacteria inhabits a defined spatial region of the Drosophila gut that forms a commensal niche.

microbiology↗

bioRxiv2020

Fmr1 translationally activates stress-sensitive mRNAs encoding large proteins in oocytes and neurons

Mutations in Fmr1, encoding the RNA binding protein FMRP, are leading causes of intellectual disability, autism, and female infertility, but FMRPs mechanism of action is controversial. In contrast to its previously postulated function as a translation repressor acting by stalling elongation, we recently found that FMRP activates translation initiation of large proteins in Drosophila oocytes up to [~]2-fold. We report here that FMRPs function as a translational activator is conserved in the mammalian brain. Reanalysis of mouse cortex ribosome profiling data shows that translation of large proteins in Fmr1 mutants is down-regulated 2.0-1.2-fold; ribosome stalling appears not to influence FMRP target protein translation in either cortex or oocyte tissue. Consistent with an activator function, most FMRP targets are associated with clinical syndromes when reduced, but not when over-expressed. Fmr1-dependent translation of one target, the N-end rule E3 ligase Poe/UBR4, occurs in microscopically visible ribonucleoprotein particles. These "Poe particles" require FMRP for their formation, are distinct from P bodies, and depend on actively elongating ribosomes, as indicated by their dissolution following a brief puromycin treatment. N-end rule-mediated proteolysis via Poe/UBR4 restrains cell growth and limits MAPK signaling in nervous tissue. Thus, loss of FMRP reduces production of an important growth repressor.

neuroscience↗

bioRxiv2020

Single-molecule sequencing of long DNA molecules allows high contiguity de novo genome assembly for the fungus fly, Sciara coprophila

The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in aiding these studies. We approached assembling the Sciara genome using multiple sequencing technologies: PacBio, Oxford Nanopore MinION, and Illumina. To find an optimal assembly using these datasets, we generated 44 Illumina assemblies using 7 short-read assemblers and 50 long-read assemblies of PacBio and MinION sequence data using 6 long-read assemblers. We ranked assemblies using a battery of reference-free metrics, and scaffolded a subset of the highest-ranking assemblies using BioNano Genomics optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. Moreover, we anchored nearly half of the Sciara genome sequence into chromosomes. Finally, we used the signal level of both the PacBio and Oxford Nanopore data to explore the presence or absence of DNA modifications in the Sciara genome since DNA modifications may play a role in imprinting in Sciara, as they do in mammals. These data serve as the foundation for future research by the growing community studying the unique features of this emerging model system.

genomics↗

bioRxiv2020

Differentiating Drosophila female germ cells initiate Polycomb silencing by altering PRC2 sampling

Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. We show that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing and how it is established. GSCs resemble pluripotent mammalian embryonic cells in lacking silenced chromatin, but most GSC daughters, like typical somatic cells, induce Polycomb silencing as they differentiate into nurse cells. Developmentally controlled changes in the levels of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate differentiation. In germline stem cells, abundant Pcl inhibits silencing by slowing PRC2 and diverting it from PRE sequences. During differentiation, core PRC2 represses inactive loci while Scm and residual Pcl cooperate to enrich PRC2 and silence traditional Polycomb domains. We propose that PRC2-interacting proteins regulate the transition from a variable to stable transcription state during differentiation by altering the rate that PRC2 samples regulatory sequences.

developmental biology↗

bioRxiv2019

Escort-like somatic cells mediate early mouse fetal ovarian development but surface-derived Lgr5+ cells support primordial follicles

Ovarian murine somatic cells are essential to form first wave medullar follicles and second wave primordial follicles. Using single cell RNA sequencing we characterized the transcriptomes of both somatic and germline ovarian cells during fetal and early neonatal development. Wnt4-expressing somatic cells we term "escort-like cells (ELCs)" interact with incoming germ cells and early developing cysts of both sexes. In the medullar region, ELCs differentiate into the granulosa cells of fast-growing first wave follicles. In contrast, after E12.5, Lgr5+ pre-granulosa cells ingress from the ovarian surface epithelium and replace cortical escort-like cells. These surface-derived cells become the main population of granulosa cells supporting primordial follicles, and differ in transcription from ELC derivatives. Reflecting their different cellular origins, ablation of Lgr5+ cells at E16.5 using Lgr5-DTR-EGFP eliminates second wave follicles, but first wave follicles continue to develop normally and support fertility. Our findings provide striking evidence that somatic cell behavior supporting germline cyst development in mice and Drosophila has been evolutionarily conserved.

developmental biology↗

bioRxiv2019

An abundant quiescent stem cell population in Drosophila Malpighian tubules protects principal cells from kidney stones

Adult Drosophila Malpighian tubules have low rates of cell turnover but are vulnerable to damage caused by stones, like their mammalian counterparts, kidneys. We show that Drosophila renal stem cells (RSCs) comprise a unique, unipotent regenerative compartment. RSCs respond only to loss of nearby principal cells (PCs), cells critical for maintaining ionic balance. Perhaps due to the large size of PCs they are outnumbered by RSCs, which replace each lost cell with multiple PCs of lower ploidy. RSCs share a developmental origin with highly active intestinal stem cells (ISCs), and like ISCs generate daughters by asymmetric Notch signaling, yet RSCs remain quiescent in the absence of damage. Nevertheless, the capacity for RSC-mediated repair extends the lifespan of flies carrying kidney stones. We propose that abundant, RSC-like stem cells exist in other tissues with low rates of turnover where they may have been mistaken for differentiated tissue cells.

developmental biology↗

bioRxiv2019

Altered germline cyst and oocyte differentiation in Tex14 mutant mice reveal a new mechanism underlying female reproductive life-span

In adult mammalian females, primordial follicles that form in the fetal/neonatal ovary are the only source to sustain adult ovarian function. Our previous studies revealed that during oocyte differentiation and primordial follicle formation in mouse fetal ovaries, primary oocytes form via gaining cytoplasm and organelles from sister germ cells that are connected to them by intercellular bridges within germline cysts. To better understand the role of intercellular bridges in oocyte differentiation, we analyzed mutant females lacking testis-expressed 14 (Tex14), a gene involved in cytokinesis and bridge formation. In Tex14-/- fetal ovaries, germ cells divide to form a reduced number of cysts in which sister germ cells are still connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wildtype cysts, Tex14-/- cysts fragment at a higher frequency and produce a greatly reduced number of primary oocytes with highly precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14+/- germline cysts are less fragmented and generate primary oocytes that are smaller than wild type. Interestingly, enlarged Tex14-/- primary oocytes are much more stable than wild type oocytes and more efficiently sustain folliculogenesis, whereas undersized Tex14+/- primary oocytes turn over at an accelerated rate. Our observations directly link the nature of fetal germ cell connectivity to cytoplasmic enrichment during oocyte differentiation and to oocyte developmental potential in the adult ovary. Our results imply that the duration of adult ovarian function is strongly influenced by the number of primary oocytes acquiring highly enriched cytoplasm during oocyte differentiation in fetal ovaries, rather than just by the size of the primordial follicle pool.

developmental biology↗

bioRxiv2019

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with [~]1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

genomics↗