Supplementary Material for: Altered expression of pulmonary epithelial cell markers in fetal and adult mice generated by in vitro embryo culture and embryo transfer

Lung development is impaired in mice generated through transfer of in vitro-derived blastocysts. The main objective of the current study was to determine if the composition of epithelial cells in the fetal and adult lung tissue is altered in mice generated through transfer of in vitro-derived blastocysts. The study comprised of two experimental (EGs) and two control (CGs) groups. Fetuses (18.5 d.p.c.) and adult mice (8-week-old) of the EGs (EGfetus , n=18, EGadult, n=15) were produced by the transfer of day-5 F2 blastocysts to pseudo-pregnant females. F2 fetuses and adult mice derived from naturally-ovulating females served as the CGs (CGfetus, n=18, CGadult n=15). The expression of Tuba-1a (a marker of ciliated cells), Foxj-1 (a marker of motile ciliated cells), Uch-L1 (a marker of neuroendocrine cells), Cldn-10 (a marker of Club cells), Aqp-5 (a marker of Type I alveolar cells), and Sp-C (a marker of Type II alveolar cells) was determined using western blot, immunohistochemistry/immunofluorescence and qRT-PCR analyses. Weight of fetuses as well as adult mice is decreased in mice comprising the EGs. Impaired lung development observed in EGfetus was associated with altered expression of Tuba-1a, Foxj-1, Cldn10, Uch-L1, Sp-C and Aqp-5. Morphology of the adult lung tissue was similar between the groups except for a significant increase in the thickness of the epithelia in EGadult. The expression of Cldn-10 and Sp-C was also altered in EGadult. It remains to be determined whether altered expression of these genes has any long-term impact on epithelial cell functions in the adult lung tissue.