Identification and characterization of genes involved in mouse embryonic stem cell differentiation into neurons

Mouse Embryonic Stem (ES) cells are pluripotent, as they have the ability to differentiate into all cell types of the embryo. Many protocols have been established to differentiate ES cells into the desired cell type in vitro. However, the identification of molecular machinery involved in ES cell biology will increase the knowledge of embryo development and will improve the use of ES cell for regenerative medicine. To identify genes involved in pluripotency and differentiation we developed a systematic approach based on the screening of a shRNA library. The results of the screening shed light on an uncharacterized gene, we named Dies1 (Differentiation of Embryonic Stem cells) given its effect on ES cell differentiation. Dies1 knock-down affected proper ES cell differentiation into neurons, astrocytes and cardiomyocytes. Dies1 knock-down cells, in fact, maintained the expression of undifferentiated markers such as Oct3/4, Nanog and Alkaline Phosphatase in conditions promoting differentiation. In addition, the growth of Dies1 knock-down cells was independent from LIF. Dies1 suppression exerted its effect on ES cell differentiation in vivo and in vitro, giving rise to teratoma smaller than controls when Dies1 knock-down cells are injected into nude mice. The in silico analysis of Dies1 sequence indicated that it possesses a trans-membrane domain and an Ig-like domain in the putative extra-cellular domain of the protein, like many membrane receptors. This analysis was confirmed by the staining of Dies1 on cell surface of mouse ES cell and blastocyst. The possible role for Dies1 as receptor was also supported by the dominant negative function of Dies1 extra-cellular domain, whose effect resembled Dies1 suppression. Thus, we investigated the involvement of Dies1 in the signaling pathways controlling ES cell pluripotency: LIF and BMP4. Dies1 knock-down did not affect the expression of undifferentiated markers such as Oct3/4, Nanog, Sox2 and Rex1 and of LIF down-stream targets such as Kruppel like factors in undifferentiated conditions. On the other hand, Inhibitory of Differentiation (Id) genes, which are BMP4 targets, were down-regulated in Dies1 knock-down cells. This down-regulation was dependent on a decreased response to BMP4, as shown by luciferase assays and occurred in a Smad dependent manner, as the amount of phosphorylated Smad 1/5/8 complex was decreased in Dies1 knock-down cells. Moreover, the involvement of Dies1 in BMP4 signaling was supported by the direct interaction between Dies1 and BMP4 in vitro. The down-regulation of BMP4 targets induced also the up-regulation of Nodal/Activin targets, such as Cripto and Lefty1/2. Such a regulation between Nodal/Activin and BMP4 was observed also in consequence of the suppression by RNA interference of the BMP4-type I receptor Alk3. This phenomenon was not surprising because Nodal/Activin and BMP4 belong to the TGF- superfamily and share the common mediator Smad4 which balances the transcriptional activity of Smad 2/3 (dependent on Nodal/Activin) and Smad 1/5/8 (dependent on BMPs). Thus, we hypothesized that the up-regulation of Nodal/Activin signaling was responsible for the effect of Dies1 suppression. To verify this hypothesis we treated Dies1 knock-down cells with the specific Nodal/Activin inhibitor SB-431542. The treatment with SB-431542 restored proper differentiation ability in the absence of LIF, leading to the rescue of Dies1 knock-down effect. Taken together our results suggest that the balance between BMP4 and Nodal/Activin exerts a crucial role in regulating ES cell pluripotency and differentiation. In summary, we have identified Dies1 as novel component of BMP4 signaling pathway required for proper ES cell differentiation.