Fig. S1. Upregulation of K18 and K14 mrna levels during ectoderm specification of hescs. Quantitative real-time PCR analysis of mrna levels of OCT4

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Fig. S1. Upregulation of K18 and K14 mrna levels during ectoderm specification of hescs. Quantitative real-time PCR analysis of mrna levels of OCT4 (n=3 independent differentiation experiments for each bar), K18 and K14 (n=9 independent differentiation experiments for each bar) during hesc ectoderm specification shows a downregulation of the pluripotency marker and an upregulation of keratins.

Fig. S2. Keratinocyte specification during embryonic development in K14-H2BGFP mice. (A) Immunofluorescence analysis of K14 expression during murine epidermal specification in a K14H2BGFP reporter mouse. Embryo sections immunostained with an antibody against keratin 14 (red) reveals that the keratin 14 promoter becomes activated (green) in the surface epithelium. (B) Immunofluorescence analysis of K18 expression in K14-H2BGFP mice during epidermal specification. Sections of K14H2BGFP embryos were immunostained with antibodies against keratin 18 (red). K18 expression within the surface epithelium is downregulated from E10 to E12 as keratinocytes are specified. (C) Quantification of the total number of surface epithelial cells that are GFP + K18 +, GFP + K18 or GFP-K18 + and percentage of K14H2BGFP + cells that are K14 + or K14 within the surface epithelium at E11 in several areas of the embryos. n=3-4 embryos for each bar. The dotted line indicates surface epithelium boundary. Scale bar: 50 μm.

Fig. S3. Characterization of the embryonic murine skin populations isolated by cell sorting. At embryonic day 11 a6 integrin (red) and epidermal cadherin (E-cad, green) colocalize within the surface epithelium but do not colocalize in other areas of the embryo. Scale bar: 100 μm or 50 μm (insets).

Fig. S4. Characterization of FACS-sorted E11 murine cell populations. (A) Dot plots and quantification of dissociated cells from E11 K14-H2BGFP embryos stained with antibodies against E-cadherin and a6 integrin (n=3 independent FACS-purified cell populations for each bar). (B) E11 E-cadherin +, a6 integrin +, GFP and E11 E-cadherin +, a6 integrin +, GFP + cell populations isolated by cell sorting were characterized by immunofluorescence using antibodies for K14 and K18. Scale bar: 10 μm.

Fig. S5. Cellular localization of the Notch receptors Notch1 and Notch4 in E10 murine embryos. Immunofluorescence analysis of Notch1 (A) (green) and Notch4 (B) (green) and p63 (red) expression during murine skin development at E10. The dotted line indicates surface epithelium boundary. Scale bars: 50 μm.

Fig. S6. Notch signaling inhibition does not affect mrna levels of K18 or K14 in differentiated hescs. Quantitative real-time PCR analysis does not reveal a significant change in mrna levels for K14 (A) and K18 (B) when hescs are treated with a Notch signaling inhibitor (DAPT) compared to untreated or vehicle-treated cells throughout the ectoderm differentiation protocol (n=9 independent sorting experiments for each graph bar for K14 and n=6 independent sorting experiments for each graph bar for K18).

Fig. S7. Notch signaling inhibition does not affect the percentage of K14 + and K18 + cells after differentiation. (A) FACS analysis of hescs reveals that upon differentiation there is a significant increase in the percentage of K18 + cells, but this percentage is not affected when Notch signaling is inhibited by DAPT. (B) Similarly, there is a small increase in the levels of K14 at the end of the differentiation protocol, but this change is not affected by DAPT treatment throughout the differentiation protocol. Percentages of K18 or K14 are indicated on each plot. (C) Quantification of K14 and K18 FACS results shows that there are no significant changes in DAPT-treated cells when compared with untreated or vehicle treated cells throughout the entire differentiation protocol. All data are ± s.e.m. (n=3 independent differentiation experiments for each bar).

Fig. S8. Notch inhibition does not affect the numbers of Keratin 14 and P63 double positive cells after differentiation. Immunofluorescence analysis of differentiated hescs using antibodies for K14 (red) and P63 (green) reveals that Notch signaling inhibition by DAPT does not affect K14/P63 double positive cells when compared to untreated or vehicle-treated cells. Scale bar: 100 μm.

Fig. S9. P63 is expressed in K18 + ectoderm cells in the presence and absence of Notch signaling. Immunofluorescence analysis of differentiated hescs using antibodies for keratin 18 (red) and P63 (green) reveals that P63 + keratinocyte progenitors express keratin 18 and that this expression pattern is not affected when cells are treated with the Notch signaling inhibitor DAPT throughout the differentiation protocol. Scale bar: 100 μm.

Fig. S10. Notch signaling inhibition does not alter trophoblast formation during hesc differentiation. (A) Treatment of hescs throughout the entire differentiation protocol with the g-secretase inhibitor DAPT leads to an increase in the numbers of P63-positive cells (green) that are CDX2 (red) negative. (B) Quantitative real-time PCR reveals that the global levels of Cdx2 mrna do not significantly change when Notch signaling is inhibited by DAPT compared to untreated or vehicle-treated control experiments. All data are ± s.e.m. (n=9 independent differentiation experiments for each graph bar). Scale bar: 100 μm.

Fig. S11. Inhibition of Notch signaling during BMP treatment is required to increase P63 expression in hescs. (A) hescs were treated with vehicle or DAPT either during the BMP4 treatment (first 3 days) or after BMP4 treatment (days 4-10). (B,C) Quantitative real-time PCR analysis of mrna levels of HES5 and P63 shows that inhibition of Notch signaling during BMP4 incubation was sufficient to induce an increase in the levels of P63 mrna. All data are ± s.e.m. (**0.001<P<0.01) (n=3 independent differentiation experiments for each bar). (D) Immunofluorescence analysis of psmad1/5/8 (green) expression in ectoderm specified hescs reveals that inactivation of Notch signaling with DAPT does not affect the levels of psmad1/5/8. Scale bars: 50 μm.

Table S1. Mouse (Mus musculus) primers used for quantitative real-time PCR analysis Gene Fw primer Rev primer keratin 18 GACGCTGAGACCACACT TCCATCTGTGCCTTGTAT keratin 14 AGGGAGAGGACGCCCACCTT CCTTGGTGCGGATCTGGCGG Trp63 ACGCCCCGCCTCTTTGCAAAT TGAGCTGGGGTTTCTATGAAACGCT Eya4 ACAGCTGTACCCCTCCAAGCCC TAGACGGCCGGCTGCTGCAT Gata2 CCGCCTCCAGCTTCACCCCTA TGCACAGGTAGTGGCCCGTG Irf8 GCAACGCGGTGGTGTGCAAG ACAGCTGCTCTACCTGCACCAGA Gbx2 GCAAGTTCGCTCCACAGCCAC AGCTCTCCTCCTTGCCCTTCGG Six1 GGCCAAGGAAAGGGAGAACACCG TGAGCTGGACATGAGCGGCTTG Notch1 GGCTGCACAGAAGCGAGGCAT CTGCCCGTGTAGCCTGCCTG Notch2 TTCGTGTCCCCCAGGCACCC AATCCGGTCCACGCACTGGC Notch3 GCACCCCCTTGTCTGGATGGA GTGCCCGCCACCACTGAACTC Notch4 ACCTGTGTGCCTCAGCCCAGT GGGCTGGGACTGACAAGCGTC Jag1 TGGACTGGCCCCACGTGTTC GGGCGGGCACACACACTTGAA Jag2 ACCCGGGCCTCGTCGTCAT TGCAGGCTCTTCCAGCGGTC Dll1 CGGGCCAGGGGAGCTACACA AGCTGTCCTCAAGGTCCGTGC Dll3 TGCCCTTCCGCGATGCTTGG CTCCCATGTGCCTGTGCGCT Dll4 CAGCATCCCCTGGCAGTGTGC GCTGGCACACTTGCTGAGTCCC Dlk1 CCCCCTGCGCCAACAATGGA CCGTGCTGGCAGGGAGAACCAT Dlk2 CCTGCCAGAGCGGATGACTGC CTCACAGTGCAGCCCCTCCCA Dner GCCCAGCTGGTGGACTTCTGC GGCCATGGTAACCTGGATCGC Hey1 GCGCCGACGAGACCGAATCAA CAGGGCGTGCGCGTCAAAAT Hes5 GCTCCGCTCGCTAATCGCCT CCGGCTTCCGCAGTCGGTTTTT

Table S2. Human (Homo sapiens) primers used for quantitative real-time PCR analysis Gene Fw primer Rev primer keratin 18 TGAGACGTACAGTCCAGTCCTT GCTCCATCTGTAGGGCGTAG keratin 14 TCAGCATGAAAGCATCCCTGGAGAA ATTTGGCGGCTGGAGGAGGTCA TRP63 AGCCAGAAGAAAGGACAGCAGCATT CTGTGCGGGCCTGGGTAGTC OCT4 CCCCTGGTGCCGTGAAGCTG CCCCAGGGTGAGCCCCACAT CDX2 CGGCGGAACCTGTGCGAGT TGGCGGCTAGCTCGGCTTT SOX1 TCTATGCTCCAGGCCCTCTCCTCG GGACCACACCATGAAGGCGTTCA FOXA2 GAGCAGCAGCGGGCGAGTTA CCCAGGCCGGCGTTCATGTT NOTCH1 CTACGTGTGCACCTGCCGGG CGTTTCTGCAGGGGCTGGGG NOTCH2 GCACTCGGGGCCTACTCTGTGAAGA AGGGGTTGGAGAGGCACTCGT NOTCH3 GTGGACGAGTGTGCTGGCCC CGGCGAAACCAGGGAGGCAG NOTCH4 TCCCCAGCTCTCCCTCTCCATTG CAGAAGTCCCGAAGCTGGCACT JAG1 TGCGAGCCAAGGTGTGTGGG CGTGGACCCTGAGCCGAAGC JAG2 ATCAACGTCAACGACTGTCGCGGG TATAGCAGCGAGCGCCGTTCC DLL1 GCAGCCCTGGCAGTGCAACT CGAGATCCGTGCAGCTCCCT DLL3 ATCTACGCTCGGGAGGCCTGAC AGACTGGGCACCACCGAGCAA DLL4 ACCTTGAGCTGCGCCGACTC CACTGTCCCCCGTTGGCACA DLK1 ACCTGCGTGAGCCTGGACGA GCAGGGGGAGCCGTTGATCAC DLK2 GAGGTGTCCACGCGTCCGGC CGCTCACAGTGCAGCCCCTC DNER GGGATCTCCGGCGCCAACTG AAGCTGTCGGGGTGCCATGG HES1 ATGACGGCTGCGCTGAGCAC TAACGCCCTCGCACGTGGAC HES5 CCGGTGGTGGAGAAGATG GACAGCCATCTCCAGGATGT HEY1 TGAGCTGAGAAGGCTGGTACCCA TGCGCGTCAAAGTAACCTTTCCC

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