Rao A, LaBonne C.

R01 GM116538 NIGMS NIH HHS

             stem cell population maintenance

	Fig. 1. HDAC activity is required for neural crest formation. (A-D) In situ hybridization examining the expression of neural crest factors snail2, foxd3,ets1 and myc following treatment with vehicle or inhibitor [200 nM TSA (A,C) or 20 mM VPA (B,D)]. Embryos were treated at mid-gastrula stages (stage 11) and collected at mid-neurula stages (stage 15). (E,F) In situ hybridization examining expression of epk, sox2 and six1 following treatment with vehicle or inhibitor [200 nM TSA (E) or 20 mM VPA (F)]. (G,H) Explant assay examining snail2 and foxd3 expression in Pax3/Zic1-induced explants treated with vehicle or inhibitor [200 nM TSA (G) or 10 mM VPA (H)]. Explants were cultured alongside sibling embryos grown until late neurula stages (stage 18). (I) In situ hybridization examining the expression of neural crest factor snail2 in embryos after morpholino-mediated knockdown of HDAC1, and rescued with co-injection of HDAC1, HDAC2 or HDAC1+HDAC2 mRNA. Embryos were injected at the eight-cell stage and collected at mid-neurula stages (stage 15). Scale bars: 250 μm.
	Fig. 2. HDAC activity is essential for proper gene expression in pluripotent blastula cells. (A,B) In situ hybridization examining tfap2, id3, oct25, vent2 and sox3 expression in pluripotent blastula cells following treatment with vehicle or inhibitor [500 nM TSA (A) or 20 mM VPA (B)]. Embryos were treated at the two-cell stage and collected at the late blastula stage (stage 9). (C) qRT-PCR of animal pole explants examining the expression of pluripotency genes after treatment with vehicle or inhibitor (500 nM TSA) (***P<0.005). Explants were cultured alongside sibling embryos grown until late blastula stages (stage 9). (D) In situ hybridization examining epk and sox3 expression in animal pole explants treated with vehicle or inhibitor (500 nM TSA). Explants were cultured alongside sibling embryos until late gastrula stages (stage 13). Scale bars: 250 μm.
	Fig. 3. HDAC activity is required for the pluripotency of blastula stem cells. (A,B) In situ hybridization examining nrp1 and sox11 expression in animal pole explants induced with chordin mRNA and treated with vehicle or inhibitor [500 nM TSA (A) or 10 mM VPA (B)]. Explants were cultured alongside sibling embryos grown until late neurula stages (stage 18). (C-F) In situ hybridization examining expression of mesodermal markers xbra and myod and endodermal markers endodermin and Sox17 in animal pole explants induced with Activin and treated with vehicle (DMSO or water) or inhibitor [500 nM TSA (C,E) or 10 mM VPA (D,F)]. Explants were cultured alongside sibling embryos grown until mid-gastrula stages (stage 11.5) for xbra, endodermin and sox17 expression and late neurula stages (stage 18) for myod expression. Scale bars: 250 μm.
	Fig. 4. HDAC inhibition leads to aberrant expression of multiple lineage markers. (A-C) In situ hybridization examining the expression of lineage markers: endoderm (sox17; A), mesoderm (myod; B) and neural (sox11; C) in aging animal pole explants treated with vehicle or inhibitor (500 nM TSA). Explants were cultured alongside sibling embryos and grown until late blastula (stage 9), mid-gastrula (stage 11) and neural plate (stage 13) stages. (D,E) qRT-PCR examining markers of specific lineages [endoderm (sox17, vegt), mesoderm (myod, xbra) and neural (olig2, sox11)] in pluripotent and aged animal pole explants following treatment with vehicle or inhibitor (500 nM TSA) (*P<0.05, **P<0.01, ***P<0.005). Explants were cultured alongside sibling embryos until late blastula (stage 9) and neural plate (stage 13) stages. Scale bars: 250 μm.
	Fig.5. Histone acetylation increases as cells become lineage restricted. (A,B) Western blot analysis of lysates of aging animal caps examining H3K9Ac and H3K27Ac alongside total H3 levels via chemiluminescence (A) and quantified using Odyssey (B) (*P<0.05, **P<0.01). Explants were cultured alongside sibling embryos until late blastula (stage 9), mid-gastrula (stage 11) and neural plate (stage 13) stages. (C,D) Western blot analysis of lysates of aging animal caps treated with vehicle (DMSO) or inhibitor (500 nM TSA) examining H3K9Ac and H3K27Ac alongside total H3 and actin levels via chemiluminescence (C) and quantified using Odyssey (D) (**P<0.01, ***P<0.005).
	Fig. 6. HDAC1 activity promotes pluripotency gene expression at the expense of lineage restriction. (A) Western blot analysis of lysates of aging animal pole explants from control embryos or embryos injected with HDAC1 mRNA examining H3K9Ac and H3K27Ac alongside total H3 and actin levels. Explants were grown alongside sibling embryos until late blastula (stage 9) and neural plate (stage 13) stages. (B,C) In situ hybridization examining epk and sox3 expression in aging animal pole explants from control embryos or embryos injected with HDAC1 mRNA. Explants were cultured alongside sibling embryos until late blastula (stage 9), mid-gastrula (stage 11) and neural plate (stage 13) stages. (D) qRT-PCR of explants from control embryos or embryos injected with HDAC1 mRNA examining expression of pluripotency genes (*P<0.05, ***P<0.005). Explants were cultured alongside sibling embryos until the neural plate stage (stage 13). Scale bars: 250 μm.
	Fig. 7. HDAC activity promotes neural crest formation. (A,B) In situ hybridization (A) and qRT-PCR (B) examining snail2 and foxd3 expression in animal cap explants induced with levels of Pax3/Zic1 titrated for weak neural crest establishment, with/without co-expression of HDAC1 (*P<0.05, ***P<0.005). Explants were cultured with sibling embryos until late neurula stages (stage 18). (C,D) Western blot analysis of lysates of control explants and explants induced with Pax3/Zic1 examining H3K9Ac and H3K27Ac alongside total H3 levels via chemiluminescence (C) and quantified using Odyssey (D) (*P<0.05, **P<0.01). Explants were cultured alongside sibling embryos until late blastula (stage 9) and neural plate (stage 13) stages. Scale bars: 250 μm.
	Supplementary Figure 1. (A) (B) Morphological changes in embryos treated with increasing concentrations of TSA (100nM, 200nM). Embryos were treated at mid-gastrula stages (stage 11).
	Supplementary Figure 2. (A) In situ hybridization examining HDAC1 expression in early embryos from 2-cell to late neurula stages. (B) In situ hybridization examining neural plate border markers Msx2, Ap2, and Zic1 expression after treatment with vehicle or inhibitor (TSA- 200nM or VPA- 20mM). Embryos were treated at mid-gastrula stages (stage 11) and collected at mid-neurula stages (stage 15). (C) Explant assay examining Snail2 and FoxD3 expression in Wnt/Chordin induced explants that were treated with vehicle or inhibitor (TSA-200nM or VPA-10mM). Explants were cultured alongside sibling embryos grown until late neurula stages (stage 18). (D) Explant assay examining Snail2 and FoxD3 expression in Wnt/Chordin induced explants that were treated with vehicle (DMSO) or inhibitor (RMD – 15uM). Explants were cultured alongside sibling embryos cultured until late neurula stages (stage 18).
	Supplementary Figure 2. (A) In situ hybridization examining HDAC1 expression in early embryos from 2-cell to late neurula stages.
	Supplementary Figure 3. (A) In situ hybridization examining Oct60 expression in animal cap explants treated with vehicle (DMSO) or inhibitor (TSA-500nM). Explants were cultured alongside sibling embryos until neural plate stages (stage 13). (B) TUNEL assay examining cell death in the vehicle and TSA treated animal cap explants alongside positive control DNMT3B injected embryos. Explants were cultured alongside sibling embryos until neural plate stages (stage 13). (C),(D) qRT-PCR examining Xbra(C) and Sox17(D) in animal pole explants after vehicle or inhibitor (TSA-500nM) or Activin induction. Explants were cultured alongside sibling embryos until mid-gastrula stages (stage 11.5).
	Supplementary Figure 4. (A) Immunofluorescence staining on Tubb2-GFP transgenic embryos costaining GFP with N-Tubulin E7 antibody. (B) Immunofluorescence staining on Tubb2-GFP transgenic embryos co-staining GFP with muscle actin 12101 antibody after treatment with vehicle or increasing amounts of TSA (50nM-200nM). Embryos were treated at mid-gastrula stages (stage 11). (C) Tubb2-GFP transgenic embryos following treatment with vehicle or TSA (50nM – 200nM). Embryos were treated at mid-gastrula stages (stage 11). (D) Immunofluorescence examining 12101- muscle actin staining on embryos after treatment with vehicle or TSA (50nM – 200nM). Embryos were treated at mid-gastrula stages (stage 11).
	Supplementary Figure 5. (A) Western blot analysis of lysates of aging animal caps treated with vehicle or inhibitor (TSA-500nM) examining H3K9Ac and H3K27Ac alongside total H3 and actin levels via via LiCOR-Odyssey (P value: ** < 0.01). (B) In situ hybridization examining Sox3 expression in animal cap explants from control embryos or embryos injected with HDAC1/ HDAC2 or HDAC1+HDAC2 mRNA. (C) In situ hybridization examining Nrp1and Sox11 expression in aging animal cap explants from control embryos or embryos injected with HDAC1 mRNA or positive control Chordin mRNA. (D) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of explants from control embryos or embryos injected with HDAC1 mRNA examining expression of pluripotency genes (P value : * <0.05). Explants were grown alongside sibling embryos until late blastula (stage 9). (E) Western blot analysis of lysates from control embryos or embryos induced with Pax3/Zic1 at blastula stages (stage 9) and quantified using LiCOR-Odyssey.

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