Knockdown of IKK1/2 promotes differentiation of mouse embryonic stem cells into neuroectoderm at the expense of mesoderm

Abstract

Activation of nuclear factor kappa B (NF-κB) is accomplished by a specific kinase complex (IKK-complex), phosphorylating inhibitors of NF-κB (IκB). In embryonic stem cells (ESCs), NF-κB signaling causes loss of pluripotency and promotes differentiation towards a mesodermal phenotype. Here we show that NF-κB signaling is involved in cell fate determination during retinoic acid (RA) mediated differentiation of ESCs. Knockdown of IKK1 and IKK2 promotes differentiation of ESCs into neuroectoderm at the expense of neural crest derived myofibroblasts. Our data indicate that RA is not only able to induce neuronal differentiation in vitro but also drives ESCs into a neural crest cell lineage represented by differentiation towards peripheral neurons and myofibroblasts. The NC is a transiently existing, highly multipotent embryonic cell population generating a wide range of different cell types. During embryonic development the NC gives rise to distinct precursor lineages along the anterior-posterior axis determining differentiation towards specific derivates. Retinoic acid (RA) signaling provides essential instructive cues for patterning the neuroectoderm along the anterior-posterior axis. The demonstration of RA as a sufficient instructive signal for the differentiation of pluripotent cells towards NC and the involvement of NF-κB during this process provides useful information for the generation of specific NC-lineages, which are valuable for studying NC development or disease modeling.

Fig. 1

Retinoic acid promotes expression of markers associated with an EMT during ESC differentiation. (a) Peripherin and Tuj1 stainings of EBs 5 days after plating in media containing either 10 % FCS or N2 and B27. Asterisks indicate migrating cell populations. Scale bar: 200 μm. Scale bar, All insets: 50 μm (b) EBs were plated in medium containing 10 % FCS and immunohistochemically analyzed 2 days and 5 days after plating. Negative data for Otx2 are not shown. Scale bars: 50 μm. (c) qPCR analysis of different EMT and NC markers during RA induced differentiation of ESCs. Data indicate mean ± SD of one experiment performed as triplicate representing at least two independent experiments. In all experiments RA was applied after 4 and 6 days of EB formation. For immunohistochemical analysis in (a) and (b) EBs were plated 8 days after formation and further cultivated as indicated

Fig. 2

Neural crest related markers are expressed upon retinoic acid mediated differentiation of ESCs. (a) RA treated or untreated EBs were immunohistochemically analysed for p75, AP2α, Pax3, Pax6 and Vimentin 1 day after dissociation. Though in untreated cells Vimentin expression was detectable in form of non-filamentous cluster (inset), Vimentin filaments were only detectable in the minority of the cells (indicated by arrowheads). Scale bar: 100 μm. (b) 5 days after dissociation of RA treated or untreated EBs, cells were labeled for SMA and Tuj1. Scale bar: 200 μm. RA was applied after 4 and 6 days of EB formation as indicated. Dissociation of EBs was performed after 8 days of cultivation in suspension. For quantification of immunohistochemical analysis >300 cells were evaluated. Data indicate Mean ± SD

Fig. 3

Knockdown of IKK1 and IKK2 in ESCs. (a, b) Western blots were carried out after co-expression of flag-tagged IKK1 (a) or IKK2 (b) with the indicated sh-sequences in HEK 293FT cells. (c) Lentiviral vector for inhibition of NF-κB signaling by knockdown of IKK1 and IKK2. LTR, long terminal repeat. WRE, woodchuck post-transcriptional regulatory element. hUbiQ, human Ubiquitin C promoter. U6, murine U6 promoter. (d) Validation of the lentiviral vector. Western blots were performed after ectopic co-expression of Flag-tagged IKK1 and IKK2 together with the lentiviral vector in HEK 293FT cells. (e) J1 ESCs, transduced with the lentiviral vectors indicated, were differentiated as EBs. Scale bar: 200 μm. (f) To validate the knockdown of IKK1 and IKK2, qPCR were carried out after differentiation of ESCs as EBs. Data represent mean ± SEM of three independent experiments, each performed as triplicate. (g) Knockdown of IKK1 and IKK2 results in reduced levels of phosphorylated IκBα. ESCs were differentiated for 8 days as EBs and treated with RA after 4 and 6 days. Proteins were extracted after 8 days and Western blots were performed. (h) Oct4 stainings of transduced ESCs to confirm maintenance of pluripotency after knockdown of IKK1 and IKK2. Feeder cells were also stained by DAPI, but negative for Oct4 and GFP. Scale bar: 50 μm. (i) Expression levels of oct4 and nanog during differentiation of ESCs as EBs. After 4 days cells were treated with RA. Data indicate mean ± SD of one experiment performed as triplicate representing two independent experiments. (j) Effect of NF-κB inhibition assayed by qPCR. After transduction of J1 ESCs, cells were differentiated as EBs and treated for 4 days with RA. At the day of EB dissociation samples were collected and different markers were assayed. Data represent mean ± SEM of three independent experiments, each performed as triplicate

Fig. 4

NF-κB promotes RA mediated differentiation of ESCs into neuroectoderm at the expense of mesoderm. (a–c) Four days after formation of EBs, cells were treated for 4 days with RA and dissociated after 8 days of differentiation. Five days after dissociation of EBs and replating, cells were stained for SMA (a), Tuj1 (b) and Islet1 (c). Scale bar: 200 μm. (d) Quantification of immunostainings. Only GFP⁺ cells were evaluated. Data show mean ± SD of three independent experiments; >750 cells were quantified. (e) Scheme of a cross-sectioned Drosophila embryo. Nuclei within the syncytium are either with high level of nuclear Dorsal (black, ventral side), intermediate levels of nuclear Dorsal (grey) or with no nuclear dorsal (white). High levels of dorsal activate target genes Twist and Snail, necessary for mesoderm formation, whereas neuroectoderm is formed with intermediate levels of dorsal. (f) Hypothetical pathway of cell fate induction by NF-κB in mESCs. ESCs are devoid of translated NF-κB [4]. Forced expression of NF-κB resulted in the formation of mesoderm [4], whereas this study indicates that knockdown of IKK1 and IKK2 enhanced the formation of neuroectoderm. ES: Embryonic stem cells; NC: Neural crest cells; NE: Neuroepithelial cells