Biology subjects

Draper, B. W.

Publications and source records attributed to Draper, B. W..

11 recordsLinked to original sources

bioRxiv2025

Zebrafish lack a strong meiotic checkpoint response to defects in chromosome synapsis

The synaptonemal complex (SC) is a meiosis-specific structure that aligns homologous chromosomes and facilitates the repair of meiotic DNA double-strand breaks (DSBs). Defects in SC assembly or unrepaired DSBs trigger a prophase I checkpoint to prevent the formation of aneuploid gametes. The strength of these checkpoints varies among species and between sexes. Whether zebrafish (Danio rerio) have prophase I surveillance mechanisms that monitor chromosome synapsis and/or meiotic DSB repair has not been explored. To investigate how defects in SC formation affect gametogenesis in zebrafish, mutations in genes encoding two structural components of the SC, syce2 and sycp1 were examined. While syce2 and sycp1 fish exhibit defects in both synapsis and DSB repair, the two mutants show different reproductive outcomes. syce2 mutant females and males produce a significant percentage of normal progeny. In contrast, sycp1 mutant females produce fewer normal offspring, while sycp1 mutant males are infertile, with spermatocytes arrested at metaphase I. Notably, offspring from syce2 and sycp1 mutant mothers show extensive somatic mosaic aneuploidy, indicating that defects in the meiotic machinery can lead to genome instability during embryogenesis. Our findings suggest that a checkpoint monitoring the progression of synapsis may be weak or absent in the zebrafish, resembling the situation in yeast, plants, and Drosophila, rather than in mice and the nematode Caenorhabditis. elegans where robust mechanisms exists to silence unpaired chromosomes leading to meiocyte apoptosis. Author SummaryMeiosis is a specialized cell division that reduces chromosome numbers for the production of haploid gametes and is necessary for sexual reproduction. During meiosis prophase I, precise coordination is required for key events such as the formation and repair of DNA double-strand breaks and the synapsis of homologous chromosomes, which is mediated by the synaptonemal complex. Specialized checkpoint pathways monitor the proper execution of these events. Errors in either process can induce a checkpoint response leading to cell death or the production of aneuploid gametes. Here, we demonstrate that the checkpoint monitoring synapsis is weak--or potentially absent--in zebrafish, suggesting that a robust synapsis checkpoint is not conserved across all vertebrates. We discuss this finding in the context of the teleost-specific whole genome duplication and the widespread heterogametic switching (e.g., XY to ZW systems) within this diverse clade, which includes over 30,000 species.

cell biology↗

bioRxiv2024

Galvanotactic directionality of cell groups depends on group size

Motile cells migrate directionally in the electric field in a process known as galvanotaxis, important and under-investigated phenomenon in wound healing and development. We previously reported that individual fish keratocyte cells migrate to the cathode in electric fields, that inhibition of PI3 kinase reverses single cells to the anode, and that large cohesive groups of either unperturbed or PI3K-inhibited cells migrate to the cathode. Here we find that small uninhibited cell groups move to the cathode, while small groups of PI3K-inhibited cells move to the anode. Small groups move faster than large groups, and groups of unperturbed cells move faster than PI3K-inhibited cell groups of comparable sizes. Shapes and sizes of large groups change little when they start migrating, while size and shapes of small groups change significantly, lamellipodia disappear from the rear edges of these groups, and their shapes start to resemble giant single cells. Our results are consistent with the computational model, according to which cells inside and at the edge of the groups pool their propulsive forces to move but interpret directional signals differently. Namely, cells in the group interior are directed to the cathode independently of their chemical state. Meanwhile, the edge cells behave like individual cells: they are directed to the cathode/anode in uninhibited/PI3K-inhibited groups, respectively. As a result, all cells drive uninhibited groups to the cathode, while larger PI3K-inhibited groups are directed by cell majority in the group interior to the cathode, while majority of the edge cells in small groups win the tug-of-war driving these groups to the anode. Significance statementMotile cells migrate directionally in electric fields. This behavior - galvanotaxis - is important in many physiological phenomena. Individual fish keratocytes migrate to the cathode, while inhibition of PI3K reverses single cells to the anode. Uninhibited cell groups move to the cathode. Surprisingly, groups of PI3K-inhibited cells exhibit bidirectional behavior: larger/smaller groups move to the cathode/anode, respectively. A mechanical model suggests that inner and outer cells interpret directional signals differently, and that a tug-of-war between the outer and inner cells directs the cell groups. These results shed light on general principles of collective cell migration.

cell biology↗

bioRxiv2024

Rbpms2 promotes female fate upstream of the nutrient sensing Gator2 complex component, Mios

Reproductive success relies on proper establishment and maintenance of biological sex. In many animals, including mammals, the primary gonad is initially ovary in character. We previously showed the RNA binding protein (RNAbp), Rbpms2, is required for ovary fate in zebrafish. Here, we identified Rbpms2 targets in oocytes (Rbpms2-bound oocyte RNAs; rboRNAs). We identify Rbpms2 as a translational regulator of rboRNAs, which include testis factors and ribosome biogenesis factors. Further, genetic analyses indicate that Rbpms2 promotes nucleolar amplification via the mTorc1 signaling pathway, specifically through the mTorc1-activating Gap activity towards Rags 2 (Gator2) component, Missing oocyte (Mios). Cumulatively, our findings indicate that early gonocytes are in a dual poised, bipotential state in which Rbpms2 acts as a binary fate-switch. Specifically, Rbpms2 represses testis factors and promotes oocyte factors to promote oocyte progression through an essential Gator2-mediated checkpoint, thereby integrating regulation of sexual differentiation factors and nutritional availability pathways in zebrafish oogenesis.

developmental biology↗

bioRxiv2023

Macrophage activation drives ovarian failure and masculization

In humans, premature ovarian insufficiency (POI) is caused by autoimmunity and genetic factors, such as mutation of BMP15, a key ovarian determining gene. The cellular mechanisms associated with ovarian failure caused by BMP15 mutation and immune contributions to the disorder are not understood. BMP15s role in ovarian follicle development is conserved in vertebrates, including zebrafish. Using zebrafish, we established a causal link between macrophage activation and ovarian failure. We identified a germline-somatic gonadal cell-macrophage axis underlying ovarian atresia. Germline loss of Bmp15 triggers this axis that single-cell RNA sequencing and genetic analyses indicate involves activation of ovarian somatic cells that express conserved macrophage-activating ligands. Genetic ablation of macrophages blocks premature oocyte loss. Thus, the axis identified here represents potential therapeutic targets to preserve female fertility. One-Sentence SummarySex reversal due to Bmp15 deficiency requires macrophage activation by Csf1a, which is expressed by specialized pre-follicle cells in zebrafish.

developmental biology↗

bioRxiv2021

Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary

Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage ([≥]5 dpf). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA sequencing studies which identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells and interstitial stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, for select examples, we used gene knockout experiments to determine the role of newly identified genes. Our results reveal novel insights into ovarian development and function and the sequencing information will provide a valuable resource for future studies.

developmental biology↗

bioRxiv2020

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

Meiosis produces haploid gametes that will give rise to the next diploid generation. Chromosome segregation errors occurring at one or both meiotic divisions result in aneuploidy, which can lead to miscarriages or birth defects in humans. During meiosis I, ring-shaped cohesin complexes play important roles to aid in the proper segregation of homologous chromosomes. While REC8 is a specialized meiosis-specific cohesin that functions to hold sister chromatids together, the role of its vertebrate-specific paralog, RAD21L, is poorly understood. Here we tested if Rad21l1, the zebrafish homolog of human and mouse RAD21L, is required for meiotic chromosome dynamics during oogenesis and spermatogenesis. We found that Rad21l1 is an abundant component of meiotic chromosomes where it localizes to both the chromosome axes and the transverse filament of the synaptonemal complex (SC). Knocking out rad21l1 causes nearly the entire mutant population to develop as fertile males, suggesting the mutation triggers a sex reversal from female to male due to a failure in oocyte production. The rad21l1-/- mutant males display normal fertility at sexual maturity. Sex reversal was partially suppressed in the absence of tp53, suggesting that the rad21l1-/- mutation causes defects leading to a Tp53 dependent response, specifically in females. The rad21l1-/-;tp53-/- double mutant females produced elevated rates of decomposing eggs and deformed offspring compared to tp53-/- controls. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of Spo11 does not suppress the sex reversal phenotype. Overall, our data highlight an exceptional sexually dimorphic phenotype caused by knocking out a meiotic-specific cohesin subunit. We propose that Rad21l1 is required for maintaining the integrity of meiotic chromatin architecture during oogenesis. Author SummaryA prominent symptom of age-linked reproductive decline in women is the increased rate of miscarriage and birth defects due to aneuploidy. Aneuploidy can arise when chromosomes fail to segregate properly during meiosis, the process of creating haploid gametes from a diploid germ cell. Oocyte progression normally arrests prior to anaphase I, after homologous chromosomes have formed crossovers, but before ovulation, which triggers the first round of segregation. This prolonged arrest makes oocytes especially vulnerable to degradation of meiotic chromosome structure and homolog connections over time. Cohesin complexes play a major role in maintaining the meiotic chromosome architecture. Here we assess the role of the vertebrate-specific Rad21l1 cohesin subunit in zebrafish. We find that while males appear mostly unaffected by loss of Rad21l1, oocyte production is massively compromised, leading to sex reversion to males. Sex reversion can be partially prevented in the absence of Tp53, demonstrating that loss or Rad21l1 leads to a Tp53-dependent response in oocytes. Strikingly, double mutant rad21l1 tp53 females produce large numbers of poor quality eggs and malformed offspring. This demonstrates a cohesin-linked vulnerability in female meiosis not present in males and sheds light on a potential mechanism associated with the decline in female reproductive health.

genetics↗

bioRxiv2020

Reduced Fgf signaling during early zebrafish development causes ciliopathy-associated phenotypes

FGF signaling is used reiteratively during development and elicits several different responses, such as cell proliferation, differentiation, or migration. We parsed the complex FGF intracellular response by creating triple homozygous mutants in the Pea3 subgroup of ETS transcription factors, designated 3etv mutants. The Pea3 proteins Etv4 and Etv5 are expressed in areas of FGF activity; however, their role in FGF signal transduction as either positive or negative modulators of FGF activity was unclear. Using 3etv mutants, we found these genes act redundantly and have phenotypes consistent with known FGF defects in inner ear, pectoral fin, and posterior mesoderm development. Additionally, we uncovered a novel role for the FGF/Pea3 pathway during body axis straightening. 3etv larvae develop a curly-tail up (CTU) phenotype that we linked to mis-regulation of the polycystin and urotensin pathways, which have opposing actions to ensure a straight body orientation along the dorsal-ventral axis. We find that the Etv4/5 transcription factors act as positive regulators of FGF signaling and propose a model where Etv4/5 are required for cilia function downstream of Fgf8a. Summary StatementPea3 transcription factor triple mutants reveal a role for FGF signaling in balancing polycystin and urotensin signaling to achieve a straight body axis.

developmental biology↗

bioRxiv2019

CIC-DUX4 expression drives the development of small round cell sarcoma in transgenic zebrafish: a new model revealing a role for ETV4 in CIC-mediated sarcomagenesis

CIC-DUX4 sarcoma is a rare subtype of sarcoma characterized by a devastating prognosis and resistance to conventional therapeutic strategies. So far, only few models of the disease have been reported, and its biological mechanisms remain to be elucidated. We established mosaic transgenic zebrafish expressing the human CIC-DUX4 fusion under the control of the {beta}-actin promoter. CIC-DUX4 transgenic fish rapidly developed aggressive soft tissue tumors with a high penetrance. RNAseq profiling revealed that fish tumors shared major common targets with human tumors and cell lines, including the overexpression of the Pea3 transcription factors, etv4 and etv5. Tumor development was strongly impaired in etv4-deficient zebrafish, implicating Etv4 as a critical effector of CIC-DUX4-mediated oncogenesis. Altogether, we report here the first in vivo model of CIC-DUX4 sarcoma in zebrafish, which will represent a major tool for future preclinical research.

cancer biology↗

bioRxiv2018

Fibroblast growth factor receptors function redundantly during zebrafish embryonic development

Fibroblast growth factor (Fgf) signaling regulates many processes during development. In many cases, one tissue layer secretes an Fgf ligand that binds and activates an Fgf receptor (Fgfr) expressed by a neighboring tissue. Although many Fgf ligands have known requirements in development, less is known about the requirements for the receptors. We have generated null mutations in each of the five fgfr genes in zebrafish. Considering the many requirements for Fgf signaling throughout development and that null mutations in the mouse Fgfr1 and Fgfr2 genes are embryonic lethal, it was surprising that all zebrafish homozygous mutants are viable and fertile, with no discernable embryonic defect. Instead, we have discovered surprising complexity of the Fgf pathway, where multiple receptors are involved in coordinating developmental processes. For example, mutations in the ligand fgf8a cause loss of the midbrain-hindbrain boundary, whereas in the fgfr mutants, this phenotype is only seen in embryos that are triple mutant for fgfr1a;fgfr1b;fgfr2, but not in any single and double mutant combinations. We show that this apparent fgfr redundancy is also seen during the development of several other tissues, including posterior mesoderm, pectoral fins, viscerocranium, and neurocranium. These data therefore begin to define the Fgfrs that function with a particular Fgf ligand to regulate early development in zebrafish.\n\nSummary statementAnalysis of fibroblast growth factor receptor mutants in zebrafish shows that functional redundancy assures robustness of a core developmental signaling pathway.

developmental biology↗

bioRxiv2018

The telomere bouquet is a hub where meiotic double-strand breaks, synapsis, and stable homolog juxtaposition are coordinated in the zebrafish, Danio rerio

Meiosis is a cellular program that generates haploid gametes for sexual reproduction. While chromosome events that contribute to reducing ploidy (homologous chromosome pairing, synapsis, and recombination) are well conserved, their execution varies across species and even between sexes of the same species. The telomere bouquet is a conserved feature of meiosis that was first described nearly a century ago, yet its role is still debated. Here we took advantage of the prominent telomere bouquet in zebrafish, Danio rerio, and super-resolution microscopy to show that axis morphogenesis, synapsis, and the formation of double-strand breaks (DSBs) all take place within the immediate vicinity of telomeres. We established a coherent timeline of events and tested the dependence of each event on the formation of Spo11-induced DSBs. First, we found that the axis protein Sycp3 loads adjacent to telomeres and extends inward, suggesting a specific feature common to all telomeres seeds the development of the axis. Second, we found that newly formed axes near telomeres engage in presynaptic co-alignment by a mechanism that depends on DSBs, even when stable juxtaposition of homologous chromosomes at interstitial regions is not yet evident. Third, we were surprised to discover that ~30% of telomeres in early prophase I engage in associations between two or more chromosome ends and these interactions decrease in later stages. Finally, while pairing and synapsis were disrupted in both spo11 males and females, their reproductive phenotypes were starkly different; spo11 mutant males failed to produce sperm while females produced offspring with severe developmental defects. Our results support zebrafish as an important vertebrate model for meiosis with implications for differences in fertility and genetically derived birth defects in males and females.\n\nAuthor SummaryInherent to reproduction is the transmission of genetic information from one generation to the next. In sexually reproducing organisms, each parent contributes an equal amount of genetic information, packaged in chromosomes, to the offspring. Diploid organisms, like humans, have two copies of every chromosome, while their haploid gametes (e.g. eggs and sperm) have only one. This reduction in ploidy depends on the segregation of chromosomes during meiosis, resulting in gametes with one copy of each chromosome. Missegregation of the chromosomes in the parents leads to abnormal chromosome numbers in the offspring, which is usually lethal or has detrimental developmental effects. While it has been known for over a century that homologous chromosomes pair and recombine to facilitate proper segregation, how homologs find their partners has remained elusive. A structure that has been central to the discussion of homolog pairing is the bouquet, or the dynamic clustering of telomeres during early stages of meiosis. Here we use zebrafish to show that the telomere bouquet is the site where key events leading to homologous chromosome pairing are coordinated. Furthermore, we show that deletion of spo11, a gene required for proper recombination in most studied organisms, resulted in very different effects in males and females where males were sterile while females produced deformed progeny.

genetics↗

bioRxiv2018

wnt4a Promotes Female Development and Reproductive Duct Elongation in Zebrafish

In laboratory strains of zebrafish, sex determination occurs in the absence of a typical sex chromosome and it is not known what regulates the proportion of animals that develop as male or female. Many sex determination and differentiation genes that act downstream of a sex chromosome are well conserved among vertebrates, but studies that test their contribution to this process have mostly been limited to mammalian models. In mammals, WNT4 is a signaling ligand that is essential for ovary and Mullerian duct development, where it function, in part, to antagonize the male-promoting FGF9 signal. Wnt4 is highly conserved in non-mammalian vertebrates, but it is not known if Wnt4 plays a role in sex determination and/or the differentiation of sex organs outside of mammals. This is an especially interesting question in teleost, such as zebrafish, because they lack an Fgf9 ortholog. Here we show that wnt4a is the ortholog of mammalian Wnt4, and that wnt4b was present in the last common ancestor of humans and zebrafish, but was lost in mammals. We found that wnt4a is expressed in the somatic cells of juvenile gonads during the time sex determination likely occurs. We show that wnt4a loss-of-function mutants develop predominantly as males and conclude that wnt4a activity promotes female sex determination in zebrafish. Additionally, both male and female wnt4a mutants are sterile because their reproductive ducts do not connect to the vent, where wnt4a is normally expressed. Yet when dissected from homozygous wnt4a mutant gonads, both sperm and eggs can produce fertile offspring. Together these results strongly argue that Wnt4a is a conserved regulator of female sex determination and reproductive duct development in non-mammalian vertebrates.\n\nSUMMARYWnt4 is a key regulator of ovary development in mammals, but it is not known if it plays a similar role in other vertebrates. Here we show that zebrafish wnt4a is the ortholog of mammalian Wnt4. We show that wnt4a is expressed in zebrafish somatic gonad cells during the time sex determination likely occurs. Through analysis of wnt4a mutants, we show that Wnt4a promotes female sex determination and the development of the male and female reproductive. We conclude that Wnt4/Wnt4a is likely a conserved regulator of ovarian and reproductive duct development in all vertebrates

developmental biology↗