CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

. Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix. Developmental Biology, Elsevier, 2017, 432 (2) AbstractInformation on the mechanisms orchestrating sexual differentiation of the bipotential gonads into testes or ovaries in amphibians is limited. The aim of this study was to investigate the development of Xenopus laevis gonad, to identify the earliest signs of sexual differentiation, and to describe mechanisms driving these processes. We used light and electron microscopy, immunofluorescence and cell tracing. In order to identify the earliest signs of sexual differentiation the sex of each tadpole was determined using genotyping with the sex markers. Our analysis revealed a series of events participating in the gonadal development, including cell proliferation, migration, cell adhesion, stroma penetration, and basal lamina formation. We found that during the period of sexual differentiation the sites of intensive cell proliferation and migration differ between male and female gonads. In the differentiating ovaries the germ cells remain associated with the gonadal surface epithelium (cortex) and a sterile medulla forms in the ovarian centre, whereas in the differentiating testes the germ cells detach from the surface epithelium, disperse, and the cortex and medulla fuse.Cell junctions that are more abundant in the ovarian cortex possibly can favor the persistence of germ cells in the cortex. Also the stroma penetrates the female and male gonads differently.These finding indicate that the crosstalk between the stroma and the coelomic epitheliumderived cells is crucial for development of male and female gonad.

Xenopus laevis is an amphibian (frog) species widely used in developmental biology and genetics. To unravel the molecular machinery regulating sex differentiation of Xenopus gonads, we analyzed for the first time the transcriptome of developing amphibian gonads covering sex determination period. We applied microarray at four developmental stages: (i) NF50 (undifferentiated gonad during sex determination), (ii) NF53 (the onset of sexual differentiation of the gonads), (iii) NF56 (sexual differentiation of the gonads), and (iv) NF62 (developmental progression of differentiated gonads). Our analysis showed that during the NF50, the genetic female (ZW) gonads expressed more sex-specific genes than genetic male (ZZ) gonads, which suggests that a robust genetic program is realized during female sex determination in Xenopus . However, a contrasting expression pattern was observed at later stages (NF56 and NF62), when the ZW gonads expressed less sex-specific genes than ZZ gonads, i.e., more genes may be involved in further development of the male gonads (ZZ). We identified sexual dimorphism in the expression of several functional groups of genes, including signaling factors, proteases, protease inhibitors, transcription factors, extracellular matrix components, extracellular matrix enzymes, cell adhesion molecules, and epithelium-specific intermediate filaments. In addition, our analysis detected a sexually dimorphic expression of many uncharacterized genes of unknown function, which should be studied further to reveal their identity and if/how they regulate gonad development, sex determination, and sexual differentiation. Comparison between genes sex-specifically expressed in developing gonads of Xenopus and available transcriptome data from zebrafish, two reptile species, chicken, and mouse revealed significant differences in the genetic control of sex determination and gonad development. This shows that the genetic control of gonad development is evolutionarily malleable. Electronic supplementary material The online version of this article (10.1007/s00427-019-00630-y) contains supplementary material, which is available to authorized users.