Summary Transcription factors related to the insect sex determination gene Doublesex (DMRT proteins) control sex determination and/or sexual differentiation in diverse metazoans, and are implicated in transitions between sex-determining mechanisms during vertebrate evolution [1]. In mice Dmrt1 is required for male gonadal differentiation in somatic cells and germ cells [2-4]. DMRT1 also maintains male gonadal sex: its loss, even in adults, can trigger sexual fate reprogramming in which male Sertoli cells transdifferentiate into their female equivalents – granulosa cells – and testicular tissue reorganizes to a more ovarian morphology [5]. Here we use a conditional Dmrt1 transgene to show that Dmrt1 is not only necessary but also sufficient to specify male cell identity in the mouse gonad. DMRT1 expression in the ovary silenced the female sex-maintenance gene Foxl2 and reprogrammed juvenile and adult granulosa cells into Sertoli-like cells, triggering formation of structures resembling male seminiferous tubules. DMRT1 can silence Foxl2 even in the absence of the testis-determining genes Sox8 and Sox9. mRNA profiling found that DMRT1 activates many testicular genes and downregulates ovarian genes and single cell RNA-seq in transdifferentiating cells identified dynamically expressed candidate mediators of this process. Strongly upregulated genes were highly enriched on chromosome X, consistent with sexually antagonistic functions. This study provides an in vivo example of single gene reprogramming of cell sexual identity. Our findings suggest a reconsideration of mechanisms involved in human disorders of sexual development (DSD) and empirically support evolutionary models where loss or gain of Dmrt1 function promotes establishment of new vertebrate sex determination systems.

SUMMARY Background The centrosome has a well-established role as a microtubule organizer during mitosis and cytokinesis. In addition, it facilitates the union of parental haploid genomes following fertilization by nucleating a microtubule aster along which the female pronucleus migrates towards the male pronucleus. Stable associations between the sperm aster and the pronuclei are essential during this directed movement. Results Our studies reveal that the zebrafish gene futile cycle (fue) is required in the zygote for male pronucleus-centrosome attachment and female pronuclear migration. We show that fue encodes a novel, maternally-provided long form of lymphoid-restricted membrane protein (lrmp), a vertebrate-specific gene of unknown function. Both maternal lrmp mRNA and protein are highly localized in the zygote, in a largely overlapping pattern at nuclear membranes, centrosomes, and spindles. Truncated Lrmp-EGFP fusion proteins identified subcellular targeting signals in the C-terminus of Lrmp, however endogenous mRNA localization is likely important to ensure strict spatial expression of the protein. Localization of both Lrmp protein and lrmp RNA is defective in fue mutant embryos, indicating that correct targeting of lrmp gene products is dependent on Lrmp function. Conclusions Lrmp is a conserved vertebrate gene whose maternally-inherited products are essential for nucleus-centrosome attachment and pronuclear congression during fertilization. Precise subcellular localization of lrmp products also suggests a requirement for strict spatiotemporal regulation of their function in the early embryo.

Summary Mammalian sex determination initiates in the fetal gonad with specification of bipotential precursor cells into male Sertoli cells or female granulosa cells. This choice was long presumed to be irreversible, but genetic analysis in the mouse recently revealed that sexual fates must be maintained throughout life. Somatic cells in the testis or ovary, even in adults, can be induced to transdifferentiate to their opposite-sex equivalents by loss of a single transcription factor, DMRT1 in the testis or FOXL2 in the ovary. Here we ask what mechanism DMRT1 prevents from triggering transdifferentiation. We find that DMRT1 blocks testicular retinoic acid (RA) signaling from activating genes normally involved in female sex determination and ovarian development and show that inappropriate activation of these genes can drive sexual transdifferentiation. By preventing activation of potential feminizing genes, DMRT1 allows Sertoli cells to participate in RA signaling, which is essential for reproduction, without being sexually reprogrammed.

Females homozygous for a mutation in cellular island (cei) produce embryos with defects in cytokinesis during early development. Analysis of the cytoskeletal events associated with furrow formation reveal that these defects include a general delay in furrow initiation as well as a complete failure to form furrow-associated structures in distal regions of the blastodisc. A linkage mapping-based candidate gene approach, including transgenic rescue, shows that cei encodes the zebrafish Aurora B kinase homologue. Genetic complementation analysis between the cei mutation and aurB zygotic lethal mutations corroborate gene assignment and reveal a complex nature of the maternal-effect cei allele, which appears to preferentially affect a function important for cytokinesis in the early blastomeres. Surprisingly, in cei mutant embryos a short yet otherwise normal furrow forms in the center of the blastodisc. Furrow formation is absent throughout the width of the blastodisc in cei mutant embryos additionally mutant for futile cycle, which lack a spindle apparatus, showing that the residual furrow signal present in cei mutants is derived from the mitotic spindle. Our analysis suggests that partially redundant signals derived from the spindle and astral apparatus mediate furrow formation in medial and distal regions of the early embryonic blastomeres, respectively, possibly as a spatial specialization to achieve furrow formation in these large cells. In addition, our data also suggest a role for Cei/AurB function in the reorganization of the furrow-associated microtubules in both early cleavage- and somite-stage embryos. In accordance with the requirement for cei/aurB in furrow induction in the early cleavage embryo, germ plasm recruitment to the forming furrow is also affected in embryos lacking normal cei/aurB function.

In animals, females deposit gene products into developing oocytes, which drive early cellular events in embryos immediately after fertilization. As maternal gene products are present before fertilization, the functional manipulation of maternal genes is often challenging to implement, requiring gene expression or targeting during oogenesis. Maternal expression can be achieved through transgenesis, but transgenic approaches are time consuming and subject to undesired epigenetic effects. Here, we have implemented in vitro culturing of experimentally manipulated immature oocytes to study maternal gene contribution to early embryonic development in the zebrafish. We demonstrate phenotypic rescue of a maternal-effect mutation by expressing wild-type product in cultured oocytes. We also generate loss-of-function phenotypes in embryos through either the expression of a dominant-negative transcript or injection of translation-blocking morpholino oligonucleotides. Finally, we demonstrate subcellular localization during the early cell divisions immediately after fertilization of an exogenously provided maternal product fused to a fluorescent protein. These manipulations extend the potential to carry out genetic and imaging studies of zebrafish maternal genes during the egg-to-embryo transition.