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Experiment details for dll1

Nicetto D et al. (2013) Assay

Suv4-20h histone methyltransferases promote neuroectodermal differentiation by silencing the pluripotency-associated Oct-25 gene.

Gene Clone Species Stages Anatomy
dll1.L laevis NF stage 13 neuroectoderm , neural plate

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  Figure 3. xSuv4-20h enzymes are required for differentiation of the neuroectoderm. (A) Schematic illustration of analysed markers of the different germ layers (germ layer colour code extended to in situ panels). Downregulated genes upon xSuv4-20h depletion are labelled in red. (B) Expression pattern of the neuroectodermal markers Ngnr 1a (NF12.5), Delta-like 1 (NF13), and N-tubulin (NF15). The pictures show dorsal views of the open neural plate with anterior to the left. (C) Expression patterns of XK81 (ectoderm), Sox2, Xiro1, Zic1 (neuroectoderm), Xbra, MyoD (mesoderm), Sox17 α, Endodermin (endoderm) in ctrl-MO injected or double morphant embryos. XK81 - anterior views with dorsal side to the top. Sox2 and MyoD - dorsal views, anterior to the left. Xiro1 and Zic1 - dorsal views; injected halves are lineage-traced by coinjection of LacZ mRNA and subsequent βal staining (light blue). Xbra - vegetal view. Sox-17 α - internal stain from the injected side in bisected embryos, animal pole up. Endodermin internal stain from the injected side in bisected embryos; anterior to the left. doi:10.1371/journal.pgen.1003188.g003

Gene Clone Species Stages Anatomy
dll1.L laevis NF stage 13 neuroectoderm , circumblastoporal collar , neural plate , neural plate border , neural progenitor cell

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  Figure 4. xSuv4-20h1/h2 enzymatic activity is required in the ectodermal germ layer. (A, D) Schematic illustrations of targeting microinjections into mesendodermal or ectodermal territories at 8-cell stage. (B) Injecting xSuv4-20h MOs into the mesendoderm causes no apparent morphological phenotype in the embryo. (C) Neural, mesodermal and endodermal marker genes are expressed normally. (E) xSuv4-20h MOs reduce eyes, cranial and trunk melanophores, when injected into the ectoderm. (F) Expression of all tested markers in mesoderm and endoderm is normal, except for Delta-like 1, whose expression specifically in the open neural plate is strongly reduced on the injected side. Global morphology was assessed at hatching stage (NF36), molecular markers at indicated stages during neurulation. Top row images in (B) and (E) depict whole embryos for overview. doi:10.1371/journal.pgen.1003188.g004

Gene Clone Species Stages Anatomy
dll1.L laevis NF stage 13 neuroectoderm , circumblastoporal collar , neural plate

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  Figure S8. Xenopus laevis Suv4-20h1 or h2 mRNA overexpression. (A) Overexpression of frog Suv4-20h1 and h2 enzymes causes an upregulation of H4K20me2 and H4K20me3 marks. Bulk histones from uninjected embryos or embryos bilaterally injected with increasing amounts of Suv4-20h1 or h2 mRNAs were isolated at NF11.5 and analysed by Western blot. Pan H3 antibody was used as loading control. (B, C) Morphological phenotypes of NF30-33 embryos injected with xSuv4-20h1 (B) or h2 (C) mRNA. (D) RNA In situ hybridization of NF30-33 uninjected embryos (top row) and embryos injected with Suv4-20h1 (middle row) or h2 (bottom row) mRNA using probes against Rx-1 and Pax-6. Pictures show the head of stained embryos. (E) RNA In situ hybridization analysis of uninjected embryos (top row) and embryos injected with xSuv4-20h1 (middle row) or h2 (bottom row) mRNA using probes against Ngnr-1a, Delta-like 1, N-tubulin, Xbra, MyoD, Sox17 α and Endodermin. Pictures show dorsal views of stained embryos, anterior is on the left; Xbra pictures show vegetal views of NF11 embryos; MyoD pictures show dorsal views of NF15 embryos, with the head on the left. For Sox-17 α and Endodermin sagittal sections of NF15 embryos were created; pictures show internal view of the injected halves, with anterior on the left.

Gene Clone Species Stages Anatomy
dll1.L laevis NF stage 13 circumblastoporal collar , neural plate , neural plate border , neural progenitor cell

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  Figure S9. Mus musculus Suv4-20h1 or h2 mRNA overexpression. (A) Western Blot analysis of uninjected embryos or embryos injected with Mus musculus Suv4-20h1 or h2 mRNAs at different concentrations. Bulk histones from NF11.5 embryos were isolated and analyzed as described in Materials and Methods section. Pan H3 antibody was used as loading control. (B, C) Morphological phenotypes of NF30-33 embryos injected with mouse Suv4-20h1 (B) or h2 (C) mRNA. (D) RNA In situ hybridization analysis of uninjected embryos (top row) and embryos injected with mSuv4-20h1 (middle row) or h2 (bottom row) mRNA using probes against Ngnr 1a, Delta-like 1 and Rx-1. Rx-1 pictures show the head of NF30-33 stained embryos. Ngnr 1a (NF12.5) and Delta-like 1 (NF13) pictures show dorsal view of stained embryos, anterior is on the left.

Gene Clone Species Stages Anatomy
dll1.L laevis NF stage 13 neuroectoderm , circumblastoporal collar , neural plate , neural plate border , neural progenitor cell

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  Figure S10. Cell proliferation and apoptosis in xSuv4-20h double morphants. (A) Double morphants show increased number of apoptotic cells during neurulation. Top row immunocytochemistry for active Caspase3 in unilaterally injected embryos (NF15). Middle and bottom rows - RNA in situ hybridisation for Delta-like 1 (NF13) and N-tubulin mRNAs (NF15). Pictures show dorsal views, with anterior to the left. (B) Proliferation assay immunocytochemistry for the mitotic histone modification H3S10P in Crtl-MO versus double morphant embryos. The chart shows a two-fold difference in the number of H3S10P positive cells on the injected side of double morphants. Data represent mean values of four embryos per condition from two independent experiments; error bars indicate SEM.