Targeted Deletion in the Basal Body Protein Talpid3 Leads to Loss of Primary Cilia in Embryonic Stem Cells and Defective Lineage-Specific Differentiation

Abstract

Talpid3 is a basal body protein required for the formation of primary cilia, an organelle involved in signal transduction. Here, we asked if Talpid3 has a role in the regulation of differentiation and/or self-renewal of ES cells and whether cells lacking cilia due to a deletion in Talpid3 can be reprogrammed to induced pluripotent stem (iPS) cells. We show that mouse embryonic limb fibroblasts which lack primary cilia with a targeted deletion in the Talpid3 (Ta3) gene can be efficiently reprogrammed to iPS cells. Furthermore, vector-free Ta3^-/- iPS cells retain ES cell features and are able to self-renew. However, both Ta3^-/- iPS and ES cells are unable to form visceral endoderm and differentiate poorly into neurons. The observed defects are not a consequence of reprogramming since Ta3^-/- ES cells also exhibit this phenotype. Thus, Talpid3 and primary cilia are required for some differentiation events but appear to be dispensable for stem cell self-renewal and reprogramming.

Keywords: differentiation; extraembryonic membranes; mouse ES cells; primary cilia; talpid 3.

Figure 1

Ta3^fl/fl and Ta3^−/− mouse embryo fibroblasts and pre-excision iPS cells. (A) Limb fibroblasts from E12.5 Ta3^−/− and Ta3^fl/fl mouse embryos. Scale bar: 50 µm. (B) Upper Panel: feeder-dependent Ta3^fl/fl iPS cell lines; Lower Panel: feeder-independent Ta3^fl/fl iPS cells. Scale bar: 100 µm. (C) Upper panel: feeder dependent Ta3^−/− iPS cell lines; Lower Panel: feeder independent Ta3^−/− iPS cells Scale bar: 100 µm. (D) Genotype of Ta3^fl/fl iPS cells (Fl, floxed allele; Wt, wildtype allele). positive control ‘Ctrl’ shared with panel E. (E) Genotype of Ta3^−/− iPS cells; (Del, deleted allele; Int.C, internal control PCR for an unrelated region; Ctrl, positive PCR control using mouse gDNA—Ta3^+/fl for Fl/WT PCR and Ta3^+/− mouse for Del PCR). (F) Primary cilia in R1 ES and Ta3 iPS immunostained with antibody to pericentrin and acetylated tubulin to identify the centrosome (black arrows) and axoneme of primary cilia (white arrows). Scale bar: 10 µm. (G) Quantification of primary cilia. Ta3^fl/fl cells (), R1 (), Ta3^−/− cells (). Error bars ± SEM, n ≥ 350 cells from three independent experiments. (H) Expression of pluripotency markers Nanog, Oct4, SSEA1 and Lin28 in Ta3^fl/fl and Ta3^−/− iPS cells. Scale bar: 25 µm.

Figure 2

Expression of pluripotency markers in Ta3 ES and transgene-free Ta3 iPS cells. (A) Upper panel: Ta3 ES cells and transgene-free Ta3 iPS cell lines maintained on MITC MEFs; Lower Panel: feeder-independent Ta3 ES cells and transgene-free Ta3 iPS cell lines cultured in 2i medium. Scale bar: 200 µm. (B) Immunostaining for the pluripotency markers Nanog, Oct4, SSEA1 and Lin28 in the Ta3^−/− or Ta3^fl/fl ES and iPS cell lines. Scale bar: 25 µm. (C) RT-PCR for a panel of pluripotency markers by Ta3^−/− and Ta3^fl/fl ES and transgene-free iPS cells. (D) qRT-PCR for selected pluripotency associated transcripts in R1 (), Ta3^fl/fl iPS X12 and X22 (), Ta3^fl/fl ES 9 (), Ta3^−/− iPS X10 and X17 (), and Ta3^−/− ES 6 (). (E) Expression of the limb markers HoxD13 and Meis1 in ES and iPS cell lines and in E12.5 mouse limb (Lb, ).

Figure 3

Characterisation of primary cilia in Ta3 ES and transgene-free Ta3 iPS cells. (A) Primary cilia in R1, transgene-free Ta3^fl/fl X12 or X22 and Ta3^−/− X10 and X17. Cells immunostained for pericentrin (PCNT, hollow arrows) to identify the centrosome) and acetylated tubulin to identify the axoneme of primary cilia (AcTubulin, white arrows). Scale bar: 10 µm. (B) Quantification of primary cilia. Presented as the proportion of cells determined by DAPI-positive nuclei possessing pericentrin and acetylated tubulin-positive primary cilia. (Error bars ±SEM, three independent experiments, ≥350 cells per experiment, p < 0.05). (C) The mean area of pericentrin-positive pericentriolar material in immunostained images. Area in Ta3^−/− iPS (), Ta3^−/− ES cells (), R1 (), Ta3^fl/fl iPS (), Ta3^fl/fl ES cells (). (Error bars ±SEM, three independent experiments, ≥350 cells per experiment, * p < 0.05, one-way ANOVA with Tukey’s post-hoc). (D) Primary cilia in R1, transgene-free Ta3^fl/fl X12 or X22 and Ta3^−/− X10 and X17. ES cells immunostained for pericentriolar material 1 (Pcm1, hollow arrows) and acetylated tubulin (white arrows). Scale bar: 10 µm. (E) The mean area of pericentriolar material protein Pcm1 in immunostained images. Pcm1 areas in Ta3^−/− iPS (), Ta3^−/− ES cells (), R1 (), Ta3^fl/fl iPS (), Ta3^fl/fl ES cells (). (Error bars ±SEM, three independent experiments, ≥350 cells per experiment, * p < 0.05, data were compared by one-way ANOVA with Tukey’s post-hoc). Scale bar: 10 µm.

Figure 4

Differentiation of Ta3 ES and transgene-free iPS cells to EBs and distribution of cell polarity, VE and PaE endodermal markers. Representative immuno- and histochemically stained cryosections of Ta3^fl/fl and Ta3^−/− ES-derived embryoid bodies at the indicated time points of three independent experiments. (A) Phase-contrast images of R1, Ta3^fl/fl 9, Ta3^−/− 6 ESC and transgene-free Ta3^fl/fl (X12 and X22) or Ta3^−/− (X10 and X17) iPS cell derived EBs. Scale bars: 200 µm. (B) Toluidine blue, dotted lines demarcate densely stained PaE-like cells. Scale bars: 50 µm. (C) Immunostaining for apical membrane marker PKCζ and tight junction marker ZO-1. Scale bars: 25 µM. (D) Immunostaining for apical membrane marker PKCζ and LamB1. Scale bars: 25 µM. (E) Immunostaining for CK8 counterstained with DAPI. Cropped insets and asterisks indicate PrE/VE outer single cell squamous epithelia and a dotted line demarks PaE from PrE/VE and EB interior. Scale bar: 50 µm. (F) Immunostaining for CK19 counterstained with DAPI. Cropped insets and asterisks indicate PrE/VE outer single cell squamous epithelia and a dotted line demarks PaE from PrE/VE and EB interior. Scale bar: 50 µm.

Figure 5

Expression of differentiation markers in Ta3 ES and transgene-free iPS cells EBs. (A) Immunostaining for CK8 and Arl13b was counterstained with DAPI. Dotted line demarks PaE from PrE/VE and EB interior. Single cell highlighted by white boxed area and shown enlarged on right of each panel. White arrows indicate cilia present in the CK8 positive epithelia, and black arrows indicate cilia in the EB interior. Scale bar: 50 µm. (B) Quantification of primary cilia frequency on CK8+ cells in Ta3^fl/fl EBs across the differentiation time course. Data from five EBs, each from three independent experiments. Error bars St. Dev. No cilia were observed in Ta3^−/− EBs at any stage. (C) qRT-PCR for expression of visceral endoderm marker AFP, primitive endoderm marker Ihh and primitive hematopoiesis marker Bh1. R1 (), Ta3^fl/fl ES 9(), Ta3^fl/fl iPS X12 (), Ta3^fl/fl iPS X22 (), Ta3^−/− ES 6 (), Ta3^−/− iPS X10 () and Ta3^−/− iPS 17 (). n = 3. Error bars ± SEM. qPCRs were performed in triplicate for each time point. Data from 3 independent experiments.

Figure 6

Expression of Shh pathway components and processing of Gli transcription factors. (A). qRT-PCR for pluripotency marker Nanog and Hedgehog pathway components Shh, Ptch1/2, Smo, Gli1, Gli2 and Gli3 in Ta3^fl/fl ES 9 and Ta3^−/− ES 6 EBs. n = 3. Error bars: ±SEM. qPCRs were performed in triplicate for each time point. Data from 3 independent experiments. (B) Immunoblots of EB lysates for Gli expression and processing during differentiation. GAPDH-loading control. (C) Quantification of the relative expression levels of Gli3A (190 kDa) and Gli3R (83 kDa) in Ta3^fl/fl and Ta3^−/− EBs relative to GAPDH. Quantified from three independent experiments. Error bars ± SEM. (D) Expression of Gli1 in Ta3^−/− and Ta3^fl/fl EBs co-stained with acetylated tubulin. Co-localised Gli1 and acetylated tubulin highlighted by arrow heads. Scale bar: 25 µm.

Figure 7

Neuronal Differentiation of EBs on Matrigel. Embryoid bodies were formed from Ta3^−/− and Ta3^fl/fl ES and iPS cells and differentiated on Matrigel with RA. (A) Immunostaining shows nestin-positive precursors on day six and nestin-positive immature neurites extending from differentiating colonies. (B) Higher magnification of colonies shows that in comparison to Ta3^fl/fl, nestin-positive cells in Ta3^−/− cultures remain in a more immature state. Ta3^−/− nestin-positive neuroepithelial cells maintain a more rosette-like morphology, while Ta3^fl/fl cells have acquired a neuroblast-like morphology and are migrating outwards. Staining for neurofilament (NF) identifies mature neurons (C), while Islet (Isl1/2l) and Peripherin (Per) identify post-mitotic motor neurons (D). The inclusion of the hedgehog agonist purmorphamine increases the frequency of motor neuron specification in Ta3^fl/fl differentiations but fails to rescue Ta3^−/− cultures (E). Scale Bar: 100 µm. Representative images from three independent experiments.