A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells.

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17(+) endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17(+) cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP(+) cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17(+) cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.

Tetratrichomonas spp. and Pentatrichomonas hominis are not persistently detectable after intravaginal inoculation of estrous heifers.

Virgin heifers (44) were intravaginally inoculated at estrus with low (10(6)) or high (10(8)) doses of live Tetratrichomonas sp., Pentatrichomonas hominis (P. hominis), or Tritrichomonas foetus (T. foetus). Controls were inoculated with Diamond's trypticase yeast extract maltose media. Genital infection was determined by culture of cervico-vaginal mucus (CVM) in Schneider's media and InPouch TF as well as by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). The presence of trichomonads in fecal samples was determined by culture in Schneider's medium and PCR/RFLP. In CVM samples, tetratrichomonads were found by PCR/RFLP and Schneider's culture only sporadically at intermittent weeks. The presence of tetratrichomonads was not associated with the dose in the experimental vaginal inoculation since Tetratrichomonas sp. appeared more frequently in heifers inoculated with a low dose of tetratrichomonads than in heifers inoculated with a high dose of tetratrichomonads. Moreover, Tetratrichomonas spp. were isolated not only in heifers inoculated with tetratrichomonads but also in control heifers and in heifers inoculated with P. hominis. In feces, Tetratrichomonas spp. were frequently identified by culture in Schneider's and by PCR/RFLP in heifers of all groups. P. hominis was never found in CVM or feces by any method. Based on the common appearance of tetratrichomonads in feces and vaginal secretions, it appears that tetratrichomonads were detected periodically in the vagina of heifers as a consequence of repeated contamination from feces and not as a result of experimental infection. In summary, in this study, the strains of Tetratrichomonas sp. and P. hominis did not establish persistent infection in heifers.