Pramel7 mediates LIF/STAT3-dependent self-renewal in embryonic stem cells.

A unique and complex signaling network allows ESCs to undergo extended proliferation in vitro, while maintaining their capacity for multilineage differentiation. Genuine ESC identity can only be maintained when both self-renewal and suppression of differentiation are active and balanced. Here, we identify Pramel7 (preferentially expressed antigen in melanoma-like 7) as a novel factor crucial for maintenance of pluripotency and leukemia inhibitory factor (LIF)-mediated self-renewal in ESCs. In vivo, Pramel7 expression was exclusively found in the pluripotent pools of cells, namely, the central part of the morula and the inner cell mass of the blastocyst. Ablation of Pramel7 induced ESC differentiation, whereas its overexpression was sufficient to support long-term self-renewal in the absence of exogenous LIF. Furthermore, Pramel7 overexpression suppressed differentiation in ESCs in vitro and in vivo. This process was reversible, as on transgene excision cells reverted to a LIF-dependent state and regained their capacity to participate in the formation of chimeric mice. Molecularly, LIF directly controls Pramel7 expression, involving both STAT3-dependent transcriptional regulation and PI3K-dependent phosphorylation of glycogen synthase kinase 3ß. Pramel7 expression in turn confers constitutive self-renewal and prevents differentiation through inactivation of extracellular signal-regulated kinase phosphorylation. Accordingly, knockdown of Pramel7 promotes ESC differentiation in presence of LIF and even on forced STAT3-activation. Thus, Pramel7 represents a central and essential factor in the signaling network regulating pluripotency and self-renewal in ESCs.

Analysis of physiological and behavioural parameters in mice after toe clipping as newborns.

In this study, we have investigated the short- and long-term impact of toe clipping, a commonly used method for marking and simultaneously taking biopsies of pups, which is controversially discussed because of its potentially negative impact on animals. Furthermore, we have analysed animal welfare aspects such as health, behaviour, development, stress and detrimental effects in young animals and in adults after toe clipping at postnatal days 3 (P3) and 7 (P7). Our findings indicate that for both P3 and P7 pups amputations at the second phalange of one toe of each paw do not have any negative effects on growth and physical development and that the clipped pups do not suffer from rejection by their mother. Our data indicate that even though at both ages no abnormalities have been detected in histology, clipping at P7 is the preferable age for an adequate marking mostly because of the small size of the toes at P3. This was also confirmed by grip tests at the age of 12 weeks where P3 animals had lower grip strength than control animals, whereas P7 pups did not show any impairment. Hotplate tests indicated that toe clipping performed at P3 and P7 did not cause hyperalgesia at the amputation stump. Serum corticosterone analysis directly performed on P7 pups after clipping indicated that major stress was provoked mainly through the handling and not because of the clipping itself. Taken together, these data lead to the conclusion that toe clipping is from a morphological, physiological and welfare point of view an acceptable method for marking and genotyping newborn mice.