Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase ␣ and ␤ isoforms during mouse preimplantation development, only the ␣1 and ␤1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/KATPase ␤1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealth TM Na/K-ATPase ␤1 subunit oligos or nonspecific Stealth TM siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase ␤1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase ␤1 subunit downregulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase ␤1 subunit mRNAs and protein were not detectable in ␤1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between ␤1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase ␣1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase ␤1 subunit siRNA-microinjected embryos. Our results demonstrate that the ␤1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.

SummaryAdult forebrain definitive neural stem cells (NSCs) comprise a subpopulation of GFAP-expressing subependymal cells that arise from embryonic fibroblast growth factor (FGF)-dependent NSCs that are first isolated from the developing brain at E8.5. Embryonic FGF-dependent NSCs are derived from leukemia inhibitory factor (LIF)-responsive, Oct4-expressing primitive NSCs (pNSCs) that are first isolated at E5.5. We report the presence of a rare population of pNCSs in the periventricular region of the adult forebrain. Adult-derived pNSCs (AdpNSCs) are GFAP−, LIF-responsive stem cells that display pNSC properties, including Oct4 expression and the ability to integrate into the inner cell mass of blastocysts. AdpNSCs generate self-renewing, multipotent colonies that give rise to definitive GFAP+ NSCs in vitro and repopulate the subependyma after the ablation of GFAP+ NSCs in vivo. These data support the hypothesis that a rare population of pNSCs is present in the adult brain and is upstream of the GFAP+ NSCs.

GFAP was markedly elevated in cerebrospinal fluid and serum in children after severe TBI and serum GFAP measured on pediatric intensive care unit day 1 correlated with functional outcome at 6 months. Hypothermia therapy did not alter serum GFAP levels compared with normothermia after severe TBI in children. Serum GFAP concentration, together with other biomarkers, may have prognostic value after TBI in children.