Retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells is potentiated by leukemia inhibitory factor.

Leukemia inhibitory factor (LIF) is a cytokine that exhibits proliferation, survival and differentiation in a wide range of cell types. Here we show that LIF potentiates retinoic acid-mediated neural induction in pluripotent P19 embryonal carcinoma cells. This activity of LIF was demonstrated by a profounded neural morphology followed by increased expression of neural-specific proteins (N-CAM, III beta-tubulin, and GAP-43), up-regulation of early neural lineage-specific gene Mash-1, and down-regulation of early endoderm-specific genes -fetoprotein and GATA-4. Moreover, LIF also slows growth and increases the level of apoptosis in differentiating cells.

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum.

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Regulation of skeletal muscle stem cell behavior by Pax3 and Pax7.

Pax genes have important roles in the regulation of stem cell behavior, leading to tissue differentiation. In the case of skeletal muscle, Pax3 and Pax7 perform this function both during development and on regeneration in the adult. The myogenic determination gene Myf5 is directly activated by Pax3, leading to the formation of skeletal muscle. Fgfr4 is also a direct Pax3 target and Sprouty1, which encodes an intracellular inhibitor of fibroblast growth factor (FGF) signaling, is under Pax3 control. Orchestration of FGF signaling, through Fgfr4/Sprouty1, modulates the entry of cells into the myogenic program, thus controling the balance between stem cell self-renewal and tissue differentiation. This and other aspects of Pax3/7 function in regulating the behavior of skeletal muscle stem cells are discussed.