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Infertile ovotestis (mixture of ovary and testis) often occurs in intersex individuals under certain pathological and physiological conditions. However, how ovotestis is formed remains largely unknown. Here, we report the first comprehensive single-cell developmental atlas of the model ovotestis. We provide an overview of cell identities and a roadmap of germline, niche, and stem cell development in ovotestis by cell lineage reconstruction and a uniform manifold approximation and projection. We identify common progenitors of germline stem cells with two states, which reveal their bipotential nature to differentiate into both spermatogonial stem cells and female germline stem cells. Moreover, we found that ovotestis infertility was caused by degradation of female germline cells via liquid–liquid phase separation of the proteasomes in the nucleus, and impaired histone-to-protamine replacement in spermatid differentiation. Notably, signaling pathways in gonadal niche cells and their interaction with germlines synergistically determined distinct cell fate of both male and female germlines. Overall, we reveal a cellular fate map of germline and niche cell development that shapes cell differentiation direction of ovotestis, and provide novel insights into ovotestis development.

Spermatogenesis is an essential process for producing sperm cells. Reproductive strategy is successfully evolved for a species to adapt to a certain ecological system. However, roles of newly evolved genes in testis autophagy remain unclear. In this study, we found that a newly evolved gene srag (Sox9-regulated autophagy gene) plays an important role in promoting autophagy in testis in the lineage of the teleost Monopterus albus. The gene integrated into an interaction network through a two-way strategy of evolution, via Sox9-binding in its promoter and interaction with Becn1 in the coding region. Its promoter region evolved a cis element for binding of Sox9, a transcription factor for male sex determination. Both in vitro and in vivo analyses demonstrated that transcription factor Sox9 could bind to and activate the srag promoter. Its coding region acquired ability to interact with key autophagy initiation factor Becn1 via the conserved C-terminal, indicating that srag integrated into preexisting autophagy network. Moreover, we determined that Srag enhanced autophagy by interacting with Becn1. Notably, srag transgenic zebrafish revealed that Srag exerted the same function by enhancing autophagy through the Srag–Becn1 pathway. Thus, the new gene srag regulated autophagy in testis by integrated into preexisting autophagy network.

Photoreceptor degeneration and damages often lead to blindness, and the underlying molecular mechanisms are largely unknown. There is also a lot of missing information for establishing the role of macroautophagy/autophagy in the retinopathy. We recently generated knockout mouse lines of the essential gene Tubgcp4 (tubulin, gamma complex associated protein 4) and revealed an interplay between essential genes and autophagy regulation. Complete knockout of Tubgcp4 in mice results in early embryonic lethality due to abnormal spindle assembly, whereas heterozygotes are viable through dosage compensation from one wild-type allele, suggesting a dosage effect of the essential gene. However, haploinsufficiency of TUBGCP4 impairs assembly of TUBG/γ-tubulin ring complexes and disturbs autophagy homeostasis of the retina, with pathological phenotypes of photoreceptor degeneration and a decrease of electroretinography responses. TUBGCP4 can inhibit autophagy by competing with ATG3 to interact with ATG7, thus interfering with lipidation of LC3B. Taken together, these findings demonstrate dosage effect of the essential gene Tubgcp4 for viability of mutant mice, and suggest key roles of TUBGCP4 in embryo development and retinal homeostasis by autophagy regulation. Abbreviations: ATG3: autophagy related 3; ATG7: autophagy related 7; CRISPR: clustered regularly interspaced short palindromic repeats; ERG: electroretinography; HCQ: hydroxychloroquine; LC3B: microtubule-associated protein 1 light chain 3 beta; NFE2L2: nuclear factor, erythroid 2 like 2; ONL: outer nuclear layer; PPARGC1A: peroxisome proliferator-activated receptor gamma coactivator-1 alpha; RB1CC1: RB1 inducible coiled-coil 1; SQSTM1: sequestosome 1; TUBGCP: tubulin, gamma complex associated protein; TUBGRC/γ: TuRCs gamma-tubulin ring complexes.

BackgroundZebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors.ResultsHere, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS).ConclusionsOverall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

Additional file 1. Additional figures and tables.

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. How autophagosomal vesicle forms in a spatio-temporally regulated manner remains elusive. Our recent study revealed that small GTPase, RAB37 (RAB37, member RAS oncogene family), functions as a key organizer of autophagosomal membrane biogenesis. RAB37 interacts with ATG5 (autophagy related 5) and promotes autophagosome formation by modulating ATG12–ATG5-ATG16L1 complex assembly. These findings provide new insights into autophagy regulation.

Photoreceptor degeneration and damages often lead to blindness, and the underlying molecular mechanisms are largely unknown. There is also a lot of missing information for establishing the role of macroautophagy/autophagy in the retinopathy. We recently generated knockout mouse lines of the essential gene Tubgcp4 (tubulin, gamma complex associated protein 4) and revealed an interplay between essential genes and autophagy regulation. Complete knockout of Tubgcp4 in mice results in early embryonic lethality due to abnormal spindle assembly, whereas heterozygotes are viable through dosage compensation from one wild-type allele, suggesting a dosage effect of the essential gene. However, haploinsufficiency of TUBGCP4 impairs assembly of TUBG/γ-tubulin ring complexes and disturbs autophagy homeostasis of the retina, with pathological phenotypes of photoreceptor degeneration and a decrease of electroretinography responses. TUBGCP4 can inhibit autophagy by competing with ATG3 to interact with ATG7, thus interfering with lipidation of LC3B. Taken together, these findings demonstrate dosage effect of the essential gene Tubgcp4 for viability of mutant mice, and suggest key roles of TUBGCP4 in embryo development and retinal homeostasis by autophagy regulation. Abbreviations: ATG3: autophagy related 3; ATG7: autophagy related 7; CRISPR: clustered regularly interspaced short palindromic repeats; ERG: electroretinography; HCQ: hydroxychloroquine; LC3B: microtubule-associated protein 1 light chain 3 beta; NFE2L2: nuclear factor, erythroid 2 like 2; ONL: outer nuclear layer; PPARGC1A: peroxisome proliferator-activated receptor gamma coactivator-1 alpha; RB1CC1: RB1 inducible coiled-coil 1; SQSTM1: sequestosome 1; TUBGCP: tubulin, gamma complex associated protein; TUBGRC/γ: TuRCs gamma-tubulin ring complexes

Germline stem cells are essential in the generation of both male and female gametes. In mammals, the male testis produces sperm throughout the entire lifetime, facilitated by testicular germline stem cells. Oocyte renewal ceases in postnatal or adult life in mammalian females, suggesting that germline stem cells are absent from the mammalian ovary. However, studies in mice, rats, and humans have recently provided evidence for ovarian female germline stem cells (FGSCs). A better understanding of the role of FGSCs in ovaries could help improve fertility treatments. Here, we developed a rapid and efficient method for isolating FGSCs from ovaries of neonatal mice. Notably, our FGSC isolation method could efficiently isolate on average 15 cell "strings" per ovary from mice at 1-3 days postpartum. FGSCs isolated from neonatal mice displayed the string-forming cell configuration at mitosis (i.e. a "stringing" FGSC (sFGSC) phenotype) and a disperse phenotype in postnatal mice. We also found that sFGSCs undergo vigorous mitosis especially at 1-3 days postpartum. After cell division, the sFGSC membranes tended to be connected to form sFGSCs. Moreover, F-actin filaments exhibited a cell-cortex distribution in sFGSCs, and E-cadherin converged in cell-cell connection regions, resulting in the string-forming morphology. Our new method provides a platform for isolating FGSCs from the neonatal ovary, and our findings indicate that FGCSs exhibit string-forming features in neonatal mice. The sFGSCs represent a valuable resource for analysis of ovary function and an in vitro model for future clinical use to address ovarian dysfunction.