RESUMO
During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. 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. Compared to tp53 single mutant controls, rad21l1-/- tp53-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Oogênese/fisiologia , Espermatogênese/fisiologia , Peixe-Zebra/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Pareamento Cromossômico , Feminino , Genes p53 , Gônadas/anatomia & histologia , Masculino , Mutação , Peixe-Zebra/fisiologia , CoesinasRESUMO
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.
Assuntos
Cromossomos/genética , Endodesoxirribonucleases/genética , Meiose/genética , Telômero/genética , Animais , Pareamento Cromossômico/genética , Quebras de DNA de Cadeia Dupla , Desenvolvimento Embrionário/genética , Feminino , Hibridização in Situ Fluorescente , Masculino , Prófase/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/patologia , Peixe-Zebra/genéticaRESUMO
The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Gônadas/crescimento & desenvolvimento , Morfogênese/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Animais , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Diferenciação Sexual/genética , Transdução de Sinais , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Peixe-Zebra/genéticaRESUMO
Although the zebrafish is a major model organism, how they determine sex is not well understood. In domesticated zebrafish, sex determination appears to be polygenic, being influenced by multiple genetic factors that may vary from strain to strain, and additionally can be influenced by environmental factors. However, the requirement of germ cells for female sex determination is well documented: animals that lack germ cells, or oocytes in particular, develop exclusively as males. Recently, it has been determined that oocytes are also required throughout the adult life of the animal to maintain the differentiated female state. How oocytes control sex differentiation and maintenance of the sexual phenotype is unknown. We therefore generated targeted mutations in genes for two oocyte produced signaling molecules, Bmp15 and Gdf9 and here report a novel role for Bmp15 in maintaining adult female sex differentiation in zebrafish. Females deficient in Bmp15 begin development normally but switch sex during the mid- to late- juvenile stage, and become fertile males. Additionally, by generating mutations in the aromatase cyp19a1a, we show that estrogen production is necessary for female development and that the function of Bmp15 in female sex maintenance is likely linked to the regulation of estrogen biosynthesis via promoting the development of estrogen-producing granulosa cells in the oocyte follicle.
Assuntos
Proteína Morfogenética Óssea 15/genética , Oócitos/metabolismo , Processos de Determinação Sexual/genética , Transdução de Sinais , Animais , Aromatase/genética , Aromatase/metabolismo , Proteína Morfogenética Óssea 15/metabolismo , Estrogênios/metabolismo , Feminino , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Masculino , Mutação , Fenótipo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is a key regulator of sex determination and/or gonadal sex differentiation across metazoan animals. This is unusual given that sex determination genes are typically not well conserved. The mechanisms by which zebrafish sex is determined have remained elusive due to the lack of sex chromosomes and the complex polygenic nature of sex determination in domesticated strains. To investigate the role of dmrt1 in zebrafish sex determination and gonad development, we isolated mutations disrupting this gene. We found that the majority of dmrt1 mutant fish develop as fertile females suggesting a complete male-to-female sex reversal in mutant animals that would have otherwise developed as males. A small percentage of mutant animals became males, but were sterile and displayed testicular dysgenesis. Therefore zebrafish dmrt1 functions in male sex determination and testis development. Mutant males had aberrant gonadal development at the onset of gonadal sex-differentiation, displaying reduced oocyte apoptosis followed by development of intersex gonads and failed testis morphogenesis and spermatogenesis. By contrast, female ovaries developed normally. We found that Dmrt1 is necessary for normal transcriptional regulation of the amh (anti-Müllerian hormone) and foxl2 (forkhead box L2) genes, which are thought to be important for male or female sexual development respectively. Interestingly, we identified one dmrt1 mutant allele that co-operates with a linked segregation distorter locus to generate an apparent XY sex determination mechanism. We conclude that dmrt1 is dispensable for ovary development but necessary for testis development in zebrafish, and that dmrt1 promotes male development by transcriptionally regulating male and female genes as has been described in other animals. Furthermore, the strong sex-ratio bias caused by dmrt1 reduction-of-function points to potential mechanisms through which sex chromosomes may evolve.
Assuntos
Desenvolvimento Sexual , Testículo/embriologia , Fatores de Transcrição/fisiologia , Peixe-Zebra/embriologia , Animais , Feminino , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/análise , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Cromossomos Sexuais , Processos de Determinação Sexual , Diferenciação Sexual , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/análiseRESUMO
Stationary symmetrical fish keratocyte cells break symmetry and become motile spontaneously but slowly. We found that applying electric field (EF) accelerates the polarization by an order of magnitude. While spontaneously polarized cells move persistently for hours, the EF-induced polarity is lost in a majority of cells when the EF is switched off. However, if the EF is applied for a long time and then switched off, the majority of cell move stably. Myosin inhibition abolishes spontaneous polarization, but does not slow down EF-induced polarization, and after the EF is turned off, motility does not stop; however, the cell movements are erratic. Our results suggest that the EF rapidly polarizes the cells, but that resulting polarization becomes stable slowly, and that the EF bypasses the requirement for myosin action in motility initiation.
Assuntos
Escamas de Animais/metabolismo , Movimento Celular , Polaridade Celular , Estimulação Elétrica , Células Epiteliais/metabolismo , Miosinas/metabolismo , Escamas de Animais/citologia , Animais , Células Cultivadas , Ciclídeos , Fenótipo , Fatores de TempoRESUMO
BACKGROUND: Aberrant signaling between germ cells and somatic cells can lead to reproductive disease and depends on diffusible signals, including transforming growth factor-beta (TGFB) -family proteins. The TGFB-family protein Gsdf (gonadal soma derived factor) controls sex determination in some fish and is a candidate for mediating germ cell/soma signaling. RESULTS: Zebrafish expressed gsdf in somatic cells of bipotential gonads and expression continued in ovarian granulosa cells and testicular Sertoli cells. Homozygous gsdf knockout mutants delayed leaving the bipotential gonad state, but then became a male or a female. Mutant females ovulated a few oocytes, then became sterile, accumulating immature follicles. Female mutants stored excess lipid and down-regulated aromatase, gata4, insulin receptor, estrogen receptor, and genes for lipid metabolism, vitellogenin, and steroid biosynthesis. Mutant females contained less estrogen and more androgen than wild-types. Mutant males were fertile. Genomic analysis suggests that Gsdf, Bmp15, and Gdf9, originated as paralogs in vertebrate genome duplication events. CONCLUSIONS: In zebrafish, gsdf regulates ovarian follicle maturation and expression of genes for steroid biosynthesis, obesity, diabetes, and female fertility, leading to ovarian and extra-ovarian phenotypes that mimic human polycystic ovarian syndrome (PCOS), suggesting a role for a related TGFB signaling molecule in the etiology of PCOS. Developmental Dynamics 246:925-945, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Células-Tronco Adultas/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Fator de Crescimento Transformador beta/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/citologia , Humanos , Masculino , Síndrome do Ovário Policístico/etiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The visibility of monolayer graphene is dependent on its surrounding dielectric environment and the presence of any contamination associated with 2D layer transfer. Here, the optical contrast of residually contaminated monolayer graphene encased within a range of dielectric stacks characteristic of realistic devices is examined, highlighting the utility of optical microscopy for a graphene assessment, both during and after lithographic processing. Practically, chemical vapor deposited graphene is encapsulated in dielectric stacks of varying thicknesses of SiO2. Optical contrast is then measured and compared to predictions of a multilayer model. Experimentally measured contrast is in close agreement with simulation only when contamination is included.
RESUMO
Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain-sDMA interactions, piRNAs and piRNA targets.
Assuntos
Chaperonas Moleculares/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Elementos de DNA Transponíveis/genética , Feminino , Substâncias Macromoleculares , Masculino , Oócitos/metabolismo , Oócitos/ultraestrutura , Ovário/metabolismo , Mapeamento de Interação de Proteínas , Interferência de RNA , Proteínas de Ligação a RNA/química , Frações Subcelulares/metabolismo , Testículo/metabolismo , Transcrição Gênica , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
Female zebrafish have a prolific reproductive capacity, suggesting that a germline stem cell (GSC) population drives oocyte production. However, a zebrafish female GSC population has yet to be identified. Adult stem cells are defined by their ability to both self-renew and differentiate, and by their localization to a stem cell niche. We show here that mitotic and early meiotic germ cells are present in the adult ovary and that the zebrafish homolog of the conserved vertebrate GSC marker, nanos2, is expressed in a subset of pre-meiotic oogonia in the adult gonad. We propose that these nanos2(+) cells are GSCs. Importantly, we find that mitotic, nanos2(+), and early meiotic germ cells localize to the germinal zone, thus identifying this region as the probable ovarian GSC niche in zebrafish. nanos3, which encodes a conserved RNA-binding protein, is known to be required for the continued production of oocytes in the zebrafish. Although mammalian homologs of nanos3 are expressed in early spermatogonia, no study has defined the role of nanos3 in the regulation of vertebrate GSCs. Here we demonstrate that nanos3 function is required for the maintenance of GSCs, but not for their specification, and propose that nanos2 and nanos3 are partially redundant in this role.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Ovário/metabolismo , Células-Tronco/metabolismo , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Microscopia Confocal , Mitose/genética , Dados de Sequência Molecular , Mutação , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Filogenia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Espermatogônias/citologia , Espermatogônias/metabolismo , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/metabolismoRESUMO
Sex in zebrafish is not determined by a major chromosomal locus, but instead relies on a mechanism that is influenced by a germ cell-derived signal, as animals that lack germ cells, or specifically oocytes, develop as phenotypic males. These data suggest that during primary sex determination, an oocyte-derived signal acts on the bipotential somatic gonad to promote the female-specific program. However, it is not known if germ cells are required only during the primary sex-determining window, or if they are required throughout adult life to maintain the female sexual phenotype. Here, we show that while wild-type zebrafish do not switch sex as adults, germ cell-depleted adult females readily convert to a male phenotype. Notably, when oocytes are depleted, but germline stem cells remain, adult females sex-revert to sperm-producing males, indicating that a germ cell-derived signal acts on the somatic gonad to promote female development directly or indirectly by repressing male-specific gene expression. These results also confirm that signals from the somatic gonad in turn ensure that the sex appropriate gamete is produced.
Assuntos
Regulação da Expressão Gênica/fisiologia , Células Germinativas/metabolismo , Fenótipo , Processos de Determinação Sexual/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Células Germinativas/citologia , Técnicas Histológicas , Imuno-Histoquímica , Hibridização In Situ , Masculino , Metronidazol , Modelos Biológicos , Dados de Sequência Molecular , Plasmídeos/genética , Células-Tronco/citologiaRESUMO
Reproductive success relies on proper establishment and maintenance of biological sex. In many animals, including mammals, the primary gonad is initially ovary biased. 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.
Assuntos
Oócitos , Oogênese , Proteínas de Ligação a RNA , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Feminino , Masculino , Regulação da Expressão Gênica no Desenvolvimento , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Nutrientes/metabolismo , Oócitos/metabolismo , Oogênese/genética , Ovário/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Testículo/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
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.
RESUMO
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.
RESUMO
The T-box transcription factor Eomesodermin (Eomes) has been implicated in patterning and morphogenesis in frog, fish and mouse. In zebrafish, one of the two Eomes homologs, Eomesa, has been implicated in dorsal-ventral patterning, epiboly and endoderm specification in experiments employing over-expression, dominant-negative constructs and antisense morpholino oligonucleotides. Here we report for the first time the identification and characterization of an Eomesa mutant generated by TILLING. We find that Eomesa has a strictly maternal role in the initiation of epiboly, which involves doming of the yolk cell up into the overlying blastoderm. By contrast, epiboly progression is normal, demonstrating for the first time that epiboly initiation is genetically separable from progression. The yolk cell microtubules, which are required for epiboly, are defective in maternal-zygotic eomesa mutant embryos. In addition, the deep cells of the blastoderm are more tightly packed and exhibit more bleb-like protrusions than cells in control embryos. We postulate that the doming delay may be the consequence both of overly stabilized yolk cell microtubules and defects in the adhesive properties or motility of deep cells. We also show that Eomesa is required for normal expression of the endoderm markers sox32, bon and og9x; however it is not essential for endoderm formation.
Assuntos
Padronização Corporal/fisiologia , Movimento Celular/fisiologia , Gástrula/embriologia , Proteínas com Domínio T/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Western Blotting , Adesão Celular/fisiologia , DNA Complementar/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Microtúbulos/fisiologia , Dados de Sequência Molecular , Fatores de Transcrição SOX/metabolismo , Análise de Sequência de DNA , Proteínas com Domínio T/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Both male and female zebrafish have a population of germline stem cells that produce gametes throughout the life of the fish. These cells localize to specific regions in the gonads and can be identified because they uniquely express the nanos2 gene, which encodes a conserved regulator of translation. A method is presented here for identifying germline stem cells in the ovary and testis using a combined protocol for whole-mount fluorescent RNA in situ hybridization to detect nanos2 mRNA and immunofluorescence to detect the pan-germ cell marker Vasa.
Assuntos
Células Germinativas , Peixe-Zebra , Animais , Feminino , Masculino , Peixe-Zebra/genética , Gônadas , Testículo , Células-TroncoRESUMO
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. BMP15's 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.
RESUMO
BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.
Assuntos
Insuficiência Ovariana Primária , Peixe-Zebra , Humanos , Animais , Masculino , Feminino , Ativação de Macrófagos/genética , Oócitos/fisiologia , Insuficiência Ovariana Primária/genéticaRESUMO
Recent research has elucidated mechanochemical pathways of single cell polarization, but much less is known about collective motility initiation in adhesive cell groups. We used galvanotactic assays of zebrafish keratocyte cell groups, pharmacological perturbations, electric field switches, particle imaging velocimetry, and cell tracking to show that large cell groups initiate motility in minutes toward the cathode. Interestingly, while PI3K-inhibited single cells are biased toward the anode, inhibiting PI3K does not affect the cathode-directed cell group migration. We observed that control groups had the fastest cathode-migrating cell at the front, while the front cells in PI3K-inhibited groups were the slowest. Both control and PI3K-inhibited groups rapidly repolarized when the electric field direction was reversed, and the group migration continued after the electric field was switched off. Inhibiting myosin disrupted the cohesiveness of keratocyte groups and abolished the collective directionality and ability to switch direction when the electric field is reversed. Our data are consistent with a model according to which cells in the group sense the electric field individually and mechanical integration of the cells results in coherent group motility.