Zebrafish dazl regulates cystogenesis upstream of the meiotic transition and germline stem cell specification and independent of meiotic checkpoints

Fertility and gamete reserves are maintained by asymmetric divisions of the germline stem cells to produce new stem cells or daughters that differentiate as gametes. Before entering meiosis, differentiating germ cells (GCs) of sexual animals typically undergo cystogenesis. This evolutionary conserved process involves synchronous and incomplete mitotic divisions of a germ cell daughter (cystoblast) to generate sister cells connected by stable intercellular bridges that facilitate exchange of materials to support a large synchronous population of gamete progenitors. Here we investigate cystogenesis in zebrafish and identified Deleted in azoospermia (Dazl), a conserved vertebrate RNA binding protein as a regulator of this process. Analysis of dazl mutants revealed an essential role for Dazl in regulating incomplete cytokinesis and germline cyst formation before the meiotic transition. Accordingly, dazl mutant GCs form defective ring canals, and ultimately remain as individual cells that fail to differentiate as meiocytes. In addition to promoting cystoblast divisions and meiotic entry, dazl function is required upstream of germline stem cell establishment and fertility. Summary Statement We show that zebrafish dazl is required for incomplete cytokinesis to generate germline cysts during cystogenesis, acts upstream of germline stem cell establishment, and is required for meiosis, and fertility.

to promoting cystoblast divisions and meiotic entry, dazl function is required upstream of germline stem cell establishment and fertility.

Introduction
In many organisms the germline is among the first cell types to be set aside during early development (Ginsburg, 1994;Illmensee and Mahowald, 1976;Illmensee et al., 1976;Wolf et al., 1983). Early germ cells, called primordial germ cells (PGCs), can be specified by maternal factors or by inductive signals (Farrell et al., 2018;Lawson and Hage, 1994;Marlow, 2015;Nieuwkoop and Sutasurya, 1979). Once specified, PGCs ignore somatic differentiation programs and migrate to the site where the gonad anlage forms (Braat et al., 1999a;Gross-Thebing et al., 2017;Nieuwkoop and Sutasurya, 1979;Strome and Updike, 2015). There the PGCs proliferate and eventually enter meiosis and differentiate to produce the gametes, sperm in males and oocytes in females. Although the earliest stages of PGC development in zebrafish have been extensively studied (Barton et al., 2016;Marlow, 2015;Paksa and Raz, 2015;Raz, 2003), the cellular events and molecular mechanisms involved in the transition from PGC to germline stem cell (GSC) and the earliest phases of gametogenesis are less well understood.

Germline cyst formation
A common and evolutionarily conserved feature of GCs is the asymmetric division of GSCs to produce a stem cell and a premeiotic daughter. These differentiating divisions have been classified as type-I or type-II divisions (Saito et al., 2007;Saito and Tanaka, 2009). Cells resulting from type-I divisions do not divide further but directly differentiate as meiotic cells and are observed in juvenile and adult teleost fish (medaka and in zebrafish) (Marlow and Mullins, 2008;Saito et al., 2007). Type-II divisions generate cystoblast cells that divide mitotically with incomplete cytokinesis to generate interconnected sisters in Drosophila (Cox and Spradling, 2003), Xenopus (Kloc et al., 2004) and medaka (Saito et al., 2007). The interconnections generated from incomplete cytokinesis form intercellular bridges (or ring canals) (Brown and King, 1964;de Cuevas et al., 1997;Fawcett et al., 1959;Greenbaum et al., 2011;Haglund et al., 2011;King, 1966, 1969;Koch et al., 1967;Lei and Spradling, 2016;Lin and Spradling, 1993;Mahowald, 1971;Marlow and Mullins, 2008;Pepling and Spradling, 1998;Robinson and Cooley, 1996;Spradling, 1993). In Drosophila ring canal formation involves regulation of the actin cytoskeleton, which localizes to the cleavage furrows and maintains midbody structures (Greenbaum et al., 2011). Arrest of the cytokinetic furrow and maintenance of the midbody involves a complex regulatory network that stabilizes the actomyosin meshwork to arrest abscission and maintain the contractile ring, forming ring canals instead of dividing (Greenbaum et al., 2011;Haglund et al., 2011;Hime et al., 1996;Robinson and Cooley, 1996). In mouse, interaction between the inactive serine-threonine kinase TEX14 and CEP55 regulates intercellular bridge stability in part by blocking abscission factors (Alix, Escrt complex) (Greenbaum et al., 2011;Greenbaum et al., 2006;Kim et al., 2015;Morita et al., 2007). Intercellular bridge and germline cyst formation is a conserved feature of germ cell biology and is crucial for fertility (e.g. (Greenbaum et al., 2006)). Intercellular bridges of germ cells are thought to fulfill several functions, including facilitating intercellular communication to produce numerous synchronous cells (de Cuevas et al., 1997;Lei and Spradling, 2013;Pepling and Spradling, 1998), coordinating critical stages such as meiotic entry (Robinson and Cooley, 1996;Stanley et al., 1972), maintenance of "functional ploidy" and gamete equivalence after meiotic divisions, and a mechanism for sensing and selection against abnormalities that would be detrimental to subsequent generations (Braun et al., 1989;LeGrand, 2001).

Dazl and fertility
The RNA binding protein (Rbp) Deleted in azoospermia-like (Dazl) is a member of the Deleted in azoospermia (DAZ) family composed of Daz, Daz-like (Dazl) and Boule (Fu et al., 2015). DAZ family members are exclusively expressed in the germline and contribute to various aspects of GC development in invertebrates and vertebrates (Fu et al., 2015).
Our study examines the earliest stages of gonadogenesis and provides evidence that Dazl is required for germline cyst formation and plays critical roles in GC amplification upstream of meiotic entry and establishment of GSCs, and thus is essential for fertility.
Here we describe zebrafish cystogenesis, a process during which PGCs transit from individual cells to GC clusters that undergo morphological changes characterized by complex cytoplasmic and nuclear rearrangements to form germline cysts. We show that GC numbers increase concomitantly with cyst formation. Following F-Actin distribution revealed that premeiotic cyst cells are interconnected by intercellular bridges or ring canals and contain aggregated or branched actin rich structures reminiscent of spectrosomes and fusomes (Cooley and Theurkauf, 1994;Deng and Lin, 1997;Lighthouse et al., 2008;Lin et al., 1994;Cooley, 1996, 1997;Tilney et al., 1996;Xue and Cooley, 1993;Yue and Spradling, 1992). Analysis of zinc finger nucleases and CRISPR-induced dazl mutant alleles revealed that zygotically transcribed dazl is essential for incomplete cytokinesis and germline cyst formation before meiosis begins.
In contrast to wild-type GC, dazl mutant GCs form defective ring canals, and ultimately remain as individual cells that fail to differentiate as meiocytes. In addition to promoting cystoblast divisions and meiotic entry, Dazl acts upstream of GSC establishment, and consequently dazl mutant fish develop exclusively as sterile males. Zygotic dazl is however dispensable for GC specification, germ granule formation, GC migration, and PGC divisions.
In addition, we show that the apoptotic pathway mediated by the cell cycle checkpoint kinase, chk2, is dispensable for early development, sex determination, gonadogenesis, and fertility in zebrafish. Although germline survival was prolonged in dazl mutants devoid of chk2, loss of dazl -/-GCs was not suppressed by tp53 or chk2 mutations, indicating that germline loss occurs via an independent mechanism. Our findings support a novel requirement for dazl in GCs for cystogenesis upstream of GSC establishment prior to the meiotic transition and provide evidence that type-II or amplifying divisions and cyst formation are critical for the mitotic to meiotic transition and fertility in zebrafish.

Materials and methods
Fish strains dazl ae57 and dazl ae34 mutant fish strains were generated using Crispr-Cas9 mutagenesis as in . dazl Δ7 was generated using zinc fingers nucleases (Ekker, 2008;Foley et al., 2009b) as detailed below. Complementation tests were performed by intercrossing carriers of each dazl mutant allele. To generate double mutants, dazl ae57 was crossed with tp53 M214K or the chk2 sa20350 allele, generated in The Sanger Institute's Zebrafish Mutation Project (Kettleborough et al., 2013) and obtained through ZIRC.
Genomic DNA and cDNA from chk2 sa20350 mutant tissues were sequenced to verify the genomic mutation and determine the transcript produced from the mutant allele (primers in Supplemental Table 1). To visualize the GCs, mutations were crossed into the ziwi:GFP GC reporter line (Leu and Draper, 2010). All procedures and experimental protocols were performed in accordance with NIH guidelines and were approved by the Einstein (protocol #20140502) and Icahn School of Medicine at Mount Sinai Institutional (ISMMS) Animal Care and Use Committees (IACUC #2017-0114).
Mutagenesis dazl ae34 and dazl ae57 allele were generated by CRISPR-Cas9 mediated mutagenesis based on . dazl gRNAs targeting exon 6 were designed using the CHOPCHOP webtool (Montague et al., 2014) (Supplemental Table1). Briefly, the gene specific target and the constant oligonucleotides were annealed, and the fragment was filled in using T4 DNA polymerase. Next, the fragment was transcribed and cleaned up to yield sgRNA using the MEGAscript SP6 kit (Life technology, Ambion). 1 nl of 12.5 ng/µl of gRNA and 1 nl of Cas9 protein (300 ng/ul) (Jinek et al., 2012) along with phenol red (Sigma Aldrich) were co-injected at one-cell stage. At 24 hpf, uninjected and injected embryos (n=8 each) were assayed by PCR amplification (primers in Supplemental table 1) followed by T7 endonuclease digest (Hwang et al., 2013), and those with new banding patterns were sequenced to confirm mutagenesis. Injected embryos were raised to adulthood, and individuals carrying mutations were identified by extracting gDNA from their progeny and fin tissue and assaying as above. Smaller bands compared to the WT allele, indicative of de novo mutations, were extracted from the gel, cloned into a PCR4 TOPO vector (Sigma), and sequenced in both directions to determine the mutated sequence. Fish harboring dazl ae34 or dazl ae57 mutations were outcrossed to AB fish. All mutations were verified by sequencing both genomic DNA and cDNA from mutant animals. Total RNA was extracted from pooled embryos from heterozygote intercrosses or AB strain WT (n=20-30) using Trizol (Life Technologies, 15596). cDNA was prepared with SuperScript III/IV Reverse Transcription Kit (Life Technologies, 18080-051). RT-PCR was performed to amplify the dazl coding region using Easy-A High Fidelity Taq polymerase (600400, Agilent) (primers in Supplemental Table 1). The PCR fragments were TOPO cloned into pCR8/GW/TOPO (K250020, Invitrogen) and sequenced (Macrogen). Sequences were analyzed using Sequencher or MacVector software.
The dazl D7 allele, a 7nt deletion resulting in a frame shift and subsequent premature stop codon at amino acid 54, was generated using Zinc finger nucleases (ZFNs) (Foley et al., 2009a). The dazl genomic region was PCR amplified and sequenced. ZFNs targeting the region in exon two just upstream of the RRM were purchased from Sigma and injected (500pg) at one-cell-stage into wild-type embryos. Buffer (Becker and Hart, 1999). Then, permeabilized in 2% Triton X-100. Following the primary antibody solution, Alexa fluor 568 phalloidin was added. Samples were mounted in vectashield with DAPI and images were acquired using a Zeiss Axio Observer inverted microscope equipped with ApotomeII and a CCD camera, a Zeiss Zoom dissecting scope equipped with ApotomeII, or a Leica SP5 DMI at the Microscopy CoRE at the IMSSM.

Germ cell, cyst and oocyte-like cell quantification.
Vasa protein was used as a marker to identify and count individual GCs. Z-series stacks of gonads at each stage were obtained using a Zeiss Axio Observer inverted microscope equipped with ApotomeII and a CCD camera, or Zeiss Zoom dissecting scope equipped with ApotomeII, or a Leica SP5 DMI at the Microscopy CoRE at the ISMMS. Cells were manually counted by analyzing each slice within each Z-stack for Vasa positivity and nuclear morphology (DAPI) defining each category during the cystogenesis process and the number of cysts per cell. Quantification of spectrosomes was performed by counting each actin-rich structure through the Z-stack. Cell area and volume was measured after a manual segmentation of the cell in each plane through the Z-stack. All the above experiments were performed using ZEN pro (Zeiss), Leica Application Suite (LAS) or ImageJ/FIJI, Imaris (Oxford instruments).

Statistical analysis
Statistical differences were assessed using Prism software and paired Student's t-test.

Formation of germline cysts
Following their arrival at the gonad, PGCs proliferate before initiating meiosis to form a bipotential gonad comprised of oocyte-like cells (OLCs) (Leu and Draper, 2010;Tong et al., 2010;Tzung et al., 2015;. Timing of entry into meiosis and OLC abundance are thought to contribute to sexual fate determination, such that more GCs promote female fate (Ye et al., 2019). Conversely, low numbers of germ cells and OLCs drive male development (Dai et al., 2015;Orban et al., 2009;Tzung et al., 2015). Despite these correlations highlighting the importance of proper development of OLCs, little is known about the actual mechanisms and cell biology regulating GC numbers and meiotic entry in zebrafish. Type-II divisions of GSCs have been observed in juvenile and adult teleosts (medaka and in zebrafish) and are thought to be amplifying divisions (Beer and Draper, 2013;Marlow and Mullins, 2008;Saito et al., 2007). In zebrafish this process likely occurs between 5-14 days when PGCs proliferate before initiating meiosis (Leerberg et al., 2017;Leu and Draper, 2010;Tong et al., 2010;Tzung et al., 2015;; however, how cysts form has not been described yet. To investigate cystogenesis, we labeled wild-type GCs with anti-Vasa antibody between 7 and 14 days (d) (Figure 1). At 7d to 10d, Vasa positive cells were detected in wild-type as individual GCs or clustered GCs ( Figure 1A, G). Individual GCs were dispersed within the gonad, while clustered GCs formed closely associated groups of cells ( Figure 1B,G). At this stage, the GC nuclei had condensed DNA as revealed by DAPI with no visible nucleolus, and the nuclear cytoplasm interface was highly convoluted with a high nucleus to cytoplasm ratio ( Figure 1C,G). The first somatic gonadal cells have been reported to be adjacent to the GCs at around 5d (Braat et al., 1999b;Draper et al., 2007;Leerberg et al., 2017). Similarly, we observed a few presumptive gonadal somatic cells surrounding the Vasa antibody labeled GC during this period ( Figure 1). Following clustering, GCs underwent a morphological transition, such that individual GCs became difficult to discern within a Vasa positive mass ( Figure 1C). This step was characterized by complex cytoplasmic and nuclear rearrangements. The most prominent feature was the appearance of a compact gonad such that individual GC boundaries were not apparent within the irregular cytoplasmic compartments devoid of Vasa and DAPI ( Figure   1C). During this period, DAPI signal was reduced and Vasa distribution shifted from the perinuclear aggregates typical of PGCs to diffusely cytoplasmic, suggesting that these morphological changes may correspond to mitotic divisions (Tong et al., 2010). The simultaneous obscuring of individual cell boundaries as the multiple domains devoid of Vasa emerge suggests that the phenomena is synchronous ( Figure 1C). Because this phenomenon likely corresponds to amplification of GC numbers that precedes emergence of germline cysts; hereafter, we describe these Vasa positive masses as cystogenic cells and this morphogenetic process as cystogenesis. The amplification step is characterized by cystogenic cells with a compact round nucleus and patent perinuclear Vasa granules circumscribed by scant cytoplasm (Figure 1  To further examine cyst architecture, determine cyst size, and investigate whether early cysts were interconnected by cytoplasmic bridges, we labeled GCs with Vasa antibody and F-Actin with Phalloidin to visualize cell membranes and the cytoskeleton (Supplemental Figure 4). At 10d wild-type gonads (Figure 2A Rodriguez- Mari et al., 2013;Tzung et al., 2015); therefore, we conclude that these are premeiotic GCs cysts.

dazl mutants
Although Dazl has not been shown to be required for cyst formation, we reasoned that Dazl was a compelling candidate regulator of this conserved process, since in mouse and human fetal ovary it interacts with Tex14, a regulator of ring canal formation (Greenbaum et al., 2006;Reynolds et al., 2005;Rosario et al., 2017;Zagore et al., 2018). In zebrafish dazl is expressed at all stages of GC development, but its function at this particular stage of germline development is unknown (Hashimoto et al., 2004;Howley and Ho, 2000;Kosaka et al., 2007;Maegawa et al., 1999). To test the hypothesis that dazl regulates germline cyst formation, we generated three mutant alleles disrupting zebrafish dazl using zinc finger nucleases (Foley et al., 2009a) and Crispr-Cas9 . We recovered dazl Δ7 using zinc finger nucleases to target the first exon of dazl ( Figure 3A).
Sequencing of the genomic and mutant cDNA revealed a frameshift mutation that caused a premature stop codon within dazl that removes all functional domains ( Figure 3B, C).
Using Crispr-Cas9 methods to target exon 6 of dazl, we recovered two additional alleles.

Zygotic dazl is dispensable for PGC specification and migration
First, we determined whether GCs were specified in dazl mutants by examining the GC marker nanos3 (Koprunner et al., 2001) at shield stage and found no differences in PGC number, position or morphology between dazl mutants and siblings ( Figure 4A; n=45 embryos; 12 dazl +/+ ; 22 dazl ae57/+ ; 11 dazl ae57/ae57 ). Having confirmed that GCs were specified, we examined two GC markers, nanos3 (Koprunner et al., 2001) (Figure 4B) and Vasa, and quantified GC number and position at 30hpf. GCs were present in all progeny of dazl heterozygous parents at this stage ( Figure 4C-J). Although the number of GCs in individuals varied, no significant differences in GC number were found at this stage ( Figure 4J). Moreover, Vasa labeled germ granules of PGCs were indistinguishable between dazl mutants and siblings ( Figure 4D, F, H insets; n=28; 5 dazl +/+ ; 17 dazl ae57/+ ; 6 dazl ae57/ae57 ). Defective GC migration can lead to death of GCs or loss of germline identity (Draper et al., 2007;Gross-Thebing et al., 2017;Weidinger et al., 2003), and dazl has been implicated in PGC migration in Xenopus (Houston and King, 2000). Therefore, we examined Vasa labeled PGC position in dazl mutants. Vasa positive PGCs were present and indistinguishable between dazl ae57 and dazl D7 mutants and siblings. Although the occasional stray germ cell was observed, there were no significant differences in PGC migration between dazl mutants and siblings ( Figure 4B-H). Moreover, we observed no differences between dazl ae57/ae57 , dazl D7/D7 ; or dazl ae57/D7 mutants in any of the assays performed in this study. Therefore, we conclude that if dazl is required for GC specification, PGC granule formation, or germ cell viability, maternal dazl must be sufficient at these stages.

dazl is required for cystogenesis
To determine if dazl was required for cystogenesis we examined Vasa and actin labeled Taken together these results indicate that initial transition and early amplification phases do not require dazl.
Next, we examined dazl mutants at 12d, when cysts emerge in wildtype. In contrast to the interconnected Vasa positive cyst cells in wild-type and dazl heterozygotes ( Figure   5E Figure 6). In contrast, dazl mutant cell area and volume did not decrease, suggesting failed division (Supplemental Figure 6).
Because somatic gonadal cells encapsulating the GCs were intact, and because dazl expression is GC specific (Hashimoto et al., 2004;Maegawa et al., 1999), Dazl likely acts within the GCs to promote cystogenesis.
Failure to generate a cyst in dazl mutants could be due to abnormal cyst architecture or cytokinesis such as failure to arrest the cytokinetic furrow, particularly given that Dazl interacts with cytokinesis and ring canal factors (Reynolds et al., 2005;Rosario et al., 2019;Rosario et al., 2017;Zagore et al., 2018). Actin is a prominent marker of intercellular connections and subcellular structures of GC cysts in other species, including the spectrosome of GSCs, and the branched fusome that connects germline cyst cells (de Cuevas et al., 1997;Kloc et al., 2004;Lin and Spradling, 1995;Snapp et al., 2004;Spradling et al., 1997). Close inspection of actin (labeled with Phalloidin) in gonads with wild-type genotypes at 8 and 10d revealed an actin rich density within GCs, potentially a spectrosome ( Figure 6A-A'; C-C'). These aggregates were also present in dazl mutant GCs at 8 and 10d ( Figure 6B-B'; D-D'). By 12d, when cysts are abundant in wild-type, actin was present in branched structures reminiscent of the fusome of other species (Hime et al., 1996;Kloc et al., 2004;Warn et al., 1985) ( Figure 6E-E'). In contrast, at this stage in dazl mutants the spectrosome-like aggregates persist in Vasa+ cells ( Figure 6F-F'). By 14dpf these actin structures resolved and were no longer detected in wild-type cysts ( Figure 6G-G', I). However, the actin densities persisted and appeared to have duplicated as two actin aggregates were present in dazl mutant GCs as opposed to the single aggregate observed at 8 and 10d in dazl mutants (compare Figure 6H-H' and 6B-B', J), possibly reflecting failed division or cell fusion.
The main structures connecting cells within cysts are the ring canals, thought to be the products of arrested cytokinesis (Haglund et al., 2011;Hime et al., 1996;Robinson and Cooley, 1996). At 10d ring canals were present between cyst cells in wild-type ( Figure   7A-A") and in dazl mutants ( Figure 7B Figure 7D-D"; F-F"). Taken all together, we conclude that dazl is essential for incomplete cytokinesis/type-II divisions and germline cyst formation.

Type-II/cystogenic divisions are required for fertility
As discussed, two modes of GSC divisions, type-I/direct differentiating and type-II/cystogenic, have been described (Marlow and Mullins, 2008;Nakamura et al., 2010;Saito et al., 2007). In mouse, the ring canals that connect cyst cells are dispensable for fertility in females but are required in males (Greenbaum et al., 2006). In contrast, defects in ring canal formation in Drosophila disrupt fertility of both sexes (Hime et al., 1996;Yue and Spradling, 1992). Type-II divisions fail in dazl mutants. To determine if cystogenic divisions are required for fertility in zebrafish, dazl mutants were raised to adulthood. As expected based on the germline specific expression of dazl (Kosaka et al., 2007;Maegawa et al., 1999), no overt morphological defects were observed among dazl -/-.
To confirm that infertility of dazl mutants was specific for dazl mutation and to assess mutant allele strengths we performed complementation tests. While heterozygosity for each allele caused no phenotypes or fertility deficits ( Figure 8A, B, D', D'-E, E' n=9), dazl ae57/D7 mutants were exclusively sterile males ( Figure 8C, F, F' n=2).
Examination of Vasa-labeled GCs (Leerberg et al., 2017) and DNA with DAPI, revealed oocytes ( Figure 8G) or sperm ( Figure 2H) in wild-type siblings at 2 months of age, and lack of Vasa+ GCs in dazl ae57/D7 mutants ( Figure 9I; n=2). In contrast, dazl ae57/ae34 mutants were fertile males or females, albeit with a male bias (n= 5 females, n=10 males). Based on these observations, we conclude that dazl mediated cystogenesis/type-II division is essential for GSC establishment and fertility in zebrafish. Moreover, dazl ae57 and dazl D7 are strong loss of function alleles, but dazl ae34 retains sufficient function to support normal germline development.
Because zebrafish dazl mutant GCs are not detected after 18d, we investigated whether blocking cell death by tp53 checkpoint activation could suppress germline loss in dazl mutants. To do so, we crossed the tp53 mutation into the dazl ae57 background to generate double mutants. Analysis of dissected adult (>6 months) gonads revealed ovary or testis in tp53 mutants that were heterozygous for dazl ae57 (n=9). In contrast, all dazl ae57/57 whether tp53 heterozygous (n=4) or tp53 -/-(n=5) lacked GCs and were sterile males. To investigate whether tp53 mutation could prolong GC viability in dazl mutants, we analyzed Vasa-labeled GCs at 40dpf. As expected, OLCs or oocytes were present in tp53 mutants that were heterozygous for dazl ae57 (Supplemental Figure 6; n=6). In contrast, no GCs were detected at 40d in dazl ae57/57 gonads regardless of tp53 genotypes (n=6 heterozygous; n=4 tp53 -/-) (Supplemental Figure 7). Therefore, we conclude that GC loss in dazl mutants is Tp53 independent.
In mouse, some infertility phenotypes associated with germline cell death can be suppressed by chek2 mutation, which also acts via tp63 (Bolcun-Filas et al., 2014).
Zebrafish have a single chek2 gene on chromosome 5. The zebrafish chek2 sa20350 mutant allele was recovered in the Sanger mutation screen; however, the mutant phenotype has not been reported (Kettleborough et al., 2013). We obtained the chk2 sa20350 nonsense allele (Kettleborough et al., 2013), which we confirmed by sequencing genomic and cDNA from mutants harboring a C to A mutation that creates a premature stop codon (Q-stop) to yield a truncated Chek2 protein lacking the protein kinase domain ( Figure 9A; Supplemental Figure 8) and developed a DCAPs genotyping assay ( Figure 9B). Mutation of chek2 does not disrupt meiosis or fertility in Drosophila (Abdu et al., 2002) or mouse (Bolcun-Filas et al., 2014). Similarly, we found that chek2 mutation caused no overt phenotypes, and did not interfere with viability, sexual differentiation or fertility in zebrafish as both male and female adult chek2 mutants were fertile (n=9 females and n=13 males).
Therefore, we conclude that chek2 is not required for germline development or fertility in zebrafish.
Taken together these results indicate that dazl is required for cyst development and maintenance of the germline by a mechanism that acts independent of chk2 and Tp53 checkpoints.

Discussion
Our study examines the earliest stages of gonadogenesis and provides evidence that the conserved RBP, Dazl, is required for germline cyst formation and plays critical roles in germ cell amplification, and acts upstream of meiosis and establishment of germline stem cells to promote fertility. Immunoaffinity screens for Dazl targets have identified regulators of incomplete cytokinesis (Kim et al., 2015;Reynolds et al., 2005;Rosario et al., 2019;Rosario et al., 2017;Zagore et al., 2018). This work provides genetic evidence that Dazl is required to form germline cysts interconnected by ring canals. Specifically, dazl mutant GCs initiate cyst formation, but ring canals collapse, such that mutant cells ultimately remain as individuals that fail to differentiate as meiocytes. In addition to promoting type-II cystoblast divisions we show dazl acts upstream of meiotic entry and GSC establishment. Finally, we show that Dazl and type-II cystogenic divisions are essential for fertility as GCs are lost from dazl mutants by a mechanism independent of meiotic checkpoint regulators.

Conserved dazl functions in gametogenesis and the PGC to meiotic transition
In cultured cells, dazl promotes meiotic entry (Chen et al., 2014;Haston et al., 2009a;Jung et al., 2017;Yu et al., 2009). Similarly, in mouse dazl is required for "licensing" or acquisition of meiotic competence and to generate gametes; dazl mutant GCs remain PGC like and fail to develop as male or female in response to masculinizing or feminizing signals (Gill et al., 2011;Hu et al., 2015b). In medaka, dazl is also required for gametogenesis and GC development (Li et al., 2016;Xu et al., 2007) but is not required for sexually dimorphic gonad development in response to somatic cues (Nishimura et al., 2018). Here we show that zygotic dazl is required for gametogenesis and meiotic progression in zebrafish. However, in contrast to mouse dazl mutant gonads which fail to sexually differentiate, and medaka dazl mutants, which develop as either sex (Nishimura et al., 2018), zebrafish dazl mutants develop exclusively as males. In medaka, recovery of male or female dazl mutants with only early PGCs provided evidence that PGC-like cells could support development of both sexes (Nishimura et al., 2018). Although, PGCs were initially present in zebrafish dazl mutants, no females were recovered. Thus, in zebrafish PGCs devoid of dazl are not sufficient to support cystogenesis, establishment of GSCs, or a fertile gonad. In agreement with this notion, recent work in mouse and pig indicates that commitment to germline fate only occurs after the PGCs reach the gonad and requires dazl (Nicholls et al., 2019).

A role for dazl in germline cyst formation
In many organisms, proliferation of GCs before meiosis is often accompanied by synchronous, incomplete divisions that form a germline cyst. During cyst formation, GSCs form cystoblasts by mitotic divisions (Leu and Draper, 2010;Pepling and Spradling, 1998). Here we characterized cystogenesis in juvenile zebrafish gonads and demonstrate a requirement for Dazl in germline cyst formation. This process begins with congregation of individual migratory GCs that then undergo synchronous division to generate premeiotic cysts. Notably, as we observe in zebrafish dazl mutants, dazl -/-GCs of mouse (Chen et al., 2014;Gill et al., 2011;Hu et al., 2015a;Lin et al., 2008) and medaka (Nishimura et al., 2018) remain as individual cells, which were described as PGC-like in mice and medaka (Chen et al., 2014;Gill et al., 2011;Nishimura et al., 2018). Here we show that PGCs lacking dazl begin the cystogenic process, including formation of ring canals. However, without Dazl, ring canals collapse signifying failure to mature bridges or completion of cytokinesis. In wild-type, the spectrosome-like actin aggregates progress to branched fusome-like structures that ultimately resolve in late cysts. It is unclear why these structures seem to disappear once the cysts form. Labeling and lineage tracing will be required to determine if cyst breakdown occurs as has been shown in mice, or if instead cysts are stable and there is selection as occurs in Drosophila (de Cuevas et al., 1997;Lei and Spradling, 2013;Pepling and Spradling, 1998). In contrast, actin remains in spectrosome-like aggregates, which are subsequently duplicated in dazl mutants.
These duplicated structures potentially indicate cell fusion or more likely failed cytokinesis because cell size does not change in dazl mutant GCs in contrast to wild-type GCs which become smaller.
Interestingly, during cystogenesis the characteristic perinuclear Vasa granules of PGCs (Knaut et al., 2000) are transiently lost and are later reestablished in premeiotic cystocytes. Significantly, zygotic Dazl is not required for Vasa translation, but Dazl protein or successful cyst formation is required to reestablish perinuclear Vasa aggregates. In zebrafish all premeiotic GCs express Vasa, but a subset also express nanos2; those that express both are thought to be the GSCs (Beer and Draper, 2013;Cao et al., 2019;Draper, 2017). Based on the observation that all GCs seem to enter a cyst state in wildtype from which Vasa+ premeiotic cells and a limited number of nanos2+ GSCs emerge, and the finding that GCs of both populations are lost in dazl mutants, it is tempting to speculate that the germline cyst not only serves to amplify premeiotic GC numbers but also plays a role in specification of the GSCs. Very recent work in mouse and pig similarly implicates dazl in commitment to germline fates (Nicholls et al., 2019), suggesting that whether PGCs, the precursors of the germline are specified by maternal inheritance of germ plasm or are induced later, dazl plays and evolutionarily conserved role in commitment or specification of the germline after they reach the gonad anlage.

Chk2 mediated apoptosis is dispensable for sexual differentiation and fertility
Apoptosis is a feature of zebrafish gonads differentiating as testis (Rodriguez-Mari et al., 2010;Uchida et al., 2002;, but the pathways involved in regulating cell death during this process are not understood. Because zebrafish mutants disrupting tp53 can differentiate as either sex, and mutation of tp53 suppresses oocyte cell death associated with DNA damage, germline apoptosis is thought to be regulated by both tp53 pathway independent and dependent checkpoints (Bolcun-Filas et al., 2014;Rodriguez-Mari et al., 2010). Here we tested the possibility that the meiotic checkpoint kinase chk2, which, in response to DNA damage or aneuploidy, activates both tp53 and p63 could account for the tp53-independent activity (Abdu et al., 2002;Bolcun-Filas et al., 2014;Sperka et al., 2012). Our observation that mutation of zebrafish chk2, as previously observed for Drosophila and mouse chk2 (Abdu et al., 2002;Bolcun-Filas et al., 2014) and zebrafish tp53 (Bolcun-Filas et al., 2014;Rodriguez-Mari et al., 2010), does not alter viability, sex-specific differentiation, or fertility indicates that normal gonad development occurs independent of Chk2-mediated pathways. Therefore, the pathway that regulates cell death associated with testis differentiation in zebrafish remains to be determined. Based on its ability to suppress death of dazl mutant germ cells in mouse mutants, Bax emerges as a compelling candidate (Nicholls et al., 2019).

Loss of the dazl mutant germline occurs independent of chk2 and p53
Mutation of chek2 does not disrupt meiosis or fertility in Drosophila (Abdu et al., 2002) or mouse (Bolcun-Filas et al., 2014). Similarly, we determined that mutation of zebrafish chek2 does not interfere with meiosis or fertility in zebrafish. Because p53 and chk2, though not essential for normal development can suppress GC loss in other contexts, we expected that simultaneous mutation of chk2 or p53 mutation might similarly suppress germline loss in dazl mutants much as tp53 mutation suppresses oocyte loss and sex reversal of brca, zar1, fancl mutants (Miao et al., 2017;Rodriguez-Mari et al., 2010;Shive et al., 2010). In contrast, although loss of chk2, but not p53, could prolong survival of abnormal dazl mutant GCs, these cells did not progress further in meiosis and eventually were not maintained, resulting in sterility. This is similar to failure of tp53 mutation to suppress GC loss and sterility of zebrafish vasa mutants (Hartung et al., 2014). Like dazl mutant GCs, vasa, a Dazl target (Haston et al., 2009b;Kee et al., 2009;Li et al., 2019;Reynolds et al., 2005) and zili mutant GCs become vacuolated (Houwing et al., 2008). In contrast, brca, zar1, and fancl mutant oocytes do not and reach later meiotic stages (Miao et al., 2017;Rodriguez-Mari et al., 2010;Shive et al., 2010). Failure of tp53/chk2 to suppress GC loss in dazl -/is consistent with dazl function in a premeiotic GC program that acts before and independent of these meiotic checkpoint pathway regulators.