Quantitative imaging reveals real-time Pou5f3-Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish.

Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3-Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3-Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3-Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis.

Transplantation of adult fibroblast nuclei into the central region of metaphase II eggs resulted in mid-blastula transition embryos.

Recently, a novel technical method to perform somatic nuclear transplantation (NT) in zebrafish using nonactivated eggs as recipients without the need to detect the micropyle was developed in our lab. However, the use of spermatozoa as an activating agent prevented to know whether the inserted nucleus compromised embryonic and early larval developmental ability. The aim of the present work was to test the developmental ability of the embryos reconstructed by transplanting adult fibroblast nuclei into the central region of the metaphase II egg but subsequently activated by only water. In addition, because an oocyte aging facilitates the activation in mammalian oocytes, this work also pursued to test whether the use of limited-aged eggs (2 h) as recipients improved the activation process in zebrafish NT. The adult somatic nucleus located in the central region of the nonactivated egg resulted in the 12% of mid-blastula transition embryos versus the 20% when the transplant is in the animal pole (p >or= 0.05). This suggests that the central region of the nonactivated metaphase II eggs can be a suitable place for nucleus deposition in NT in zebrafish. These results reinforce the possibility to use nonactivated metaphase II eggs in subsequent reprogramming studies by adult somatic NT in zebrafish. Unfortunately, in contrast to mammals, a limited egg aging (2 h) did not improve the activation process in zebrafish NT.

Effect of gametes aging on their activation and fertilizability in zebrafish (Danio rerio).

The zebrafish represents an important model organism for biological research. In this context, in vitro collection and fertilization of zebrafish gametes are basic and widely used techniques for many topical research works. In this work, the fertilization ability and normal embryo development of gold-type zebrafish sperm and eggs were re-evaluated after being stored for different times at 8 degrees C in a modified medium (Hanks' saline supplemented with 1.5 g BSA and 0.1 g ClNa; 320 mOsm, pH 7.4). Results obtained indicated that the temporal limits usually recommended for zebrafish sperm to fertilize fresh eggs (2 h) could be extended for up to 24 h without significant differences compared with fresh sperm. In contrast, the rapid egg aging observed (even less than 1 h) recommends minimizing as far as possible the egg storage time before fertilization. These results suggest a possible strain difference in the fertilization response.