Bone morphogenetic protein 2 signaling negatively modulates lymphatic development in vertebrate embryos.

RATIONALE: The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. OBJECTIVE: Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. METHODS AND RESULTS: BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem cell-derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox protein 1 during development. CONCLUSIONS: Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development.

Transmission of Pseudoloma neurophilia in Laboratory Zebrafish (Danio rerio) When Using Mass Spawning Chambers and Recommendations for Chamber Disinfection.

Pseudoloma neurophilia, a microsporidium that primarily infects neural tissues, is a common pathogen in laboratory zebrafish. The risk of parasite transmission with different spawning apparatuses and the effectiveness of disinfection are unknown. In this study, we spawned uninfected zebrafish with P. neurophilia-infected zebrafish in either 50 L mass spawning chambers (MSCs) or 1 L standard breeding tanks (BTs). Fish were spawned once or thrice, with and without chamber disinfection between uses, to evaluate risk of vertical and horizontal transmission. Six disinfection protocols were tested to determine which effectively eliminated residual spores. We demonstrated that three consecutive uses of an MSC significantly increased the risk of transmission to other fish when compared to the use of BTs or only one spawning event in an MSC (both p < 0.0001). Vertical transmission was not detected with any method. Disinfection with ∼100 ppm bleach soak (pH ∼7.0), 75 ppm Wescodyne® soak, and 175 ppm Wescodyne Plus spray was 100% effective in eliminating spores from the MSCs. Disinfection of MSCs before spawning did not decrease P. neurophilia transmission when infected fish remained present in the breeding population. Researchers should avoid using endemically infected fish in MSCs to minimize transmission of pathogens within their colonies.

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.