Schwenty-Lara J , Nehl D , Borchers A .

             histone methylation

                Kabuki syndrome

           CHARGE SYNDROME
           KABUKI SYNDROME 2; KABUK2

Xla Wt + kmt2d MO (Fig 1 C row2 col1)
Xla Wt + kmt2d MO (Fig 2A row3, col2)
Xla Wt + kmt2d MO (Fig 2D row2 col2)
Xla Wt + kmt2d MO (Fig. 3. B, C)
Xla Wt + kmt2d MO (Fig. 4A col3, E)
Xla Wt + kmt2d MO (Fig. 4A col4, E)
Xla Wt + kmt2d MO (Fig. 4B col3, F)
Xla Wt + kmt2d MO (Fig. 4B col4, F)
Xla Wt + kmt2d MO (Fig. 4C col3, G)
Xla Wt + kmt2d MO (Fig. 4C col4, G)
Xla Wt + kmt2d MO (Fig. 4D col3, H)
Xla Wt + kmt2d MO (Fig. 4D col4, H)
Xla Wt + kmt2d MO (Fig. 5C, E)
Xla Wt + kmt2d MO (Fig. 5D,E)
Xla Wt + kmt2d MO (Fig. 5 I, J)
Xla Wt + kmt2d MO (Fig. 6A)
Xla Wt + kmt2d MO (Fig. 7A row col2, B)

	Figure 1. Kmt2d loss-of-function causes craniofacial malformations. (A) Phenotypic analysis of craniofacial defects at stage 42. Embryos were injected with 2.5–5 ng of mmMO or Kmt2d MO or 5 ng control (Co) MO or splice MO into one blastomere at the two-cell stage. 150 pg lacZ mRNA was co-injected as a lineage tracer. The injected side of the embryo is marked (asterisk). Both the Kmt2d MO and the splice MO cause severe craniofacial malformations (arrows), compared to mmMO- and Co MO-treated controls. Scale bars = 500 μm. (B) Graph summarizing the results from at least four independent experiments; ±SEM and the number of analyzed embryos are given for each condition. A two-tailed unpaired Student’s t-test was applied. (C) Cartilage staining. Schematic representation of the cranial cartilage structures of a Xenopus laevis embryo (upper left panel). Embryos were injected with 2.5 ng mmMO or Kmt2d MO in combination with 50 pg mGFP mRNA as a lineage tracer into one blastomere at the two-cell stage, and the cranial cartilage was visualized by Alcian Blue staining at stage 44. Kmt2d knockdown leads to reduced cranial cartilage (arrowhead) on the injected side (asterisk). Scale bar = 500 μm.
	Figure 2. Kmt2d is required for cranial NC cell migration. (A) Embryos were injected with 2.5 or 5 ng mmMO or Kmt2d MO and 100 pg lacZ mRNA into one blastomere at the two-cell stage. At stage 27 NC migration was analyzed by twist whole mount in situ hybridization. Kmt2d loss-of-function leads to an inhibition of NC migration (arrows), compared to mismatch controls. The injected side of the embryo is marked (asterisk). Scale bar = 500 μm. (B) Graph summarizing the data from three independent experiments; ±SEM and the number of analyzed embryos are indicated for each condition. A two-tailed unpaired Student’s t-test was applied. (C–E) Rescue experiments. (C) Schematic view of motifs and functional domains of the human KMT2D-SET construct used for rescue experiments. (D) Embryos were injected into one blastomere at the two-cell stage with 3 ng mmMO or Kmt2d MO in combination with 400–500 pg mGFP or KMT2D-SET mRNA and 100 pg lacZ as a lineage tracer. NC migration was analyzed by twist in situ hybridization at stage 25. Overexpression of the human KMT2D-SET construct partially restores NC migration in Kmt2d morphants, compared to embryos injected with Kmt2d MO and mGFP mRNA (arrows). The injected side of the embryos is marked (asterisk). Error bar = 500 μm. (E) Graph summarizing the percentage of embryos with twist defects from six independent experiments; ±SEM and number of analyzed embryos are given for each condition. A two-tailed unpaired Student’s t-test was applied. (F) Schematic view of the Xenopus laevis Kmt2d-ATG-HA mRNA which contains the Kmt2d MO binding site. (G) To analyze the binding efficiency of the Kmt2d MO, embryos were injected at the one-cell stage with 1 ng of Xenopus Kmt2d-ATG-HA mRNA either alone or in combination with 2.5–5 ng mmMO or the translation-blocking Kmt2d MO. Protein extracts were prepared at stage 20, and the expression of Kmt2d-ATG-HA was assessed by Western Blot analysis. Injection of 2.5 and 5 ng Kmt2d MO led to a depletion of the Kmt2d-ATG-HA protein, while the expression was unaffected upon co-injection of 5 ng mmMO. GAPDH expression was used as a loading control.
	Figure 3. Kmt2d is required for cell dispersion in explanted NC cells. (A and B) Transplantation assay. Embryos were injected with 2.5 ng mmMO or Kmt2d MO in combination with 50 pg mGFP mRNA into one blastomere at the two-cell stage. Fluorescently labeled NC cells from injected embryos were transplanted into a wild-type host embryo, and their migration behavior was analyzed at stage 27. Scale bar = 500 μm. (A) While transplanted mismatch control NC cells migrate normally, (B) the migration of Kmt2d-depleted NC cells was inhibited. (C) Graph summarizing three independent experiments with ±SEM and the number of analyzed embryos given for each condition. A two-tailed unpaired Student’s t-test was applied. (D–G) Explant assay. (D and E) Embryos were injected with 2.5 ng mmMO or Kmt2d MO in combination with 50 pg mGFP and 250 pg H2B-mCherry mRNA to label the cell membrane and the nucleus, respectively. NC cells were explanted, cultured on fibronectin, and their migration behavior was imaged for 8 h. Cell dispersion was assessed using ImageJ. Scale bar = 50 μm. (D) mmMO NC explants show normal cell dispersion after 5 h of cultivation, while (E) Kmt2d MO-treated cells display inhibited dispersion. (F and G) Embryos were injected with 2.5 ng mmMO or Kmt2d MO in combination with 50 pg mGFP and 300 pg lifeact-RFP mRNA to label the cell membrane and f-actin, respectively. Higher magnification of explanted NC cells reveals normal protrusion formation (arrowheads) in both (F) mmMO- and (G) Kmt2d MO-treated NC cells. Scale bar = 10 μm. (H) Dispersion of mmMO- and Kmt2d MO-injected NC explants defined as the mean triangle size per explant (μm2) calculated by Delaunay triangulation after 5 h of cultivation in three independent experiments. Box plots show median and 25th to 75th percentiles, and Whiskers display min to max values. Mann-Whitney U test was applied (n = 33 mmMO explants and n = 28 Kmt2d MO explants analyzed). (I) Analysis of circularity showed no difference in cell shape between mmMO- and Kmt2d MO-treated NC cells (complete circle = 1). Box plots show median and 25th to 75th percentiles. Whiskers display min to max values. Mann-Whitney U test was applied (n = 53 cells analyzed from 16 mmMO NC explants; n = 105 cells analyzed from 27 Kmt2d MO NC explants; data from three independent experiments).
	Figure 4. Kmt2d knockdown affects NPB formation and NC specification in a dose-dependent manner. (A–D) Embryos were injected with 2.5 or 5 ng mmMO or Kmt2d MO into one blastomere at the two-cell stage. 100 pg lacZ mRNA were used for lineage tracing. At neurula stages, the expression of several NC marker genes was analyzed by whole mount in situ hybridization. Injection of 5 ng Kmt2d MO leads to (A) a broader pax3 expression in the NPB region and (B) a severe reduction of foxd3, (C) slug/snai2 and (D) twist expression in the pre-migratory NC. (E–H) Graphs summarizing the results from three independent experiments for each NC marker gene analyzed in (A–D). Kmt2d loss-of-function affects the expression of (E) pax3, (F) foxd3, (G) slug/snai2 and (H) twist in a dose-dependent manner; ±SEM and the number of analyzed embryos are indicated for each condition. A two-tailed unpaired Student’s t-test was applied.
	Figure 5. Kmt2d loss-of-function leads to an expansion of the neural plate as indicated by altered epidermal keratin and sox2 expression. Two-cell stage embryos were injected with 2.5 or 5 ng of mismatch (mm) MO or Kmt2d MO in combination with 100 pg lacZ mRNA as a lineage tracer. The injected side of the embryo is marked (asterisk). Scale bars = 500 μm. (A–D) In situ hybridization for the non-neural ectoderm marker epidermal keratin (epk) was performed at stage 14. (A) Uninjected control embryos and (B) mmMO treated controls show normal epk expression in the epidermal ectoderm. (C) While 2.5 ng Kmt2d MO has no effect in the majority of embryos, (D) 5 ng Kmt2d MO leads to a dorsolaterally reduced epk expression. (E) The graph summarizes the mean percentage of embryos with epk expression defect from two independent experiments. The total number of analyzed embryos is indicated for each condition. (F–I) In situ hybridization for the neural plate marker sox2 was performed at stage 19. (F) Uninjected control embryos and (G) mmMO treated controls show normal sox2 expression in the neural plate. (H) While 2.5 ng Kmt2d MO has no effect in the majority of embryos, (I) 5 ng Kmt2d MO leads to an expansion of the sox2-positive territory. (J) Data from four independent experiments. Graph summarizes the mean percentage of embryos with sox2 expression defect. The total number of analyzed embryos and ±SEM are indicated for each condition. A two-tailed unpaired Student’s t-test was applied.
	Figure 6. Loss of Kmt2d leads to reduced H3K4me1 and H3K27ac levels in Xenopus embryos. One-cell stage Xenopus embryos were injected with 5 ng mmMO or Kmt2d MO or 7.5 ng control MO or splice MO in combination with 50 pg mGFP mRNA as a lineage tracer. Protein extracts were prepared at neurula stage 20. (A and B) Western Blot analysis of the H3K4me1 mark reveals a reduction of the H3K4me1 level in Kmt2d MO treated embryos compared to controls. Graph summarizes three independent experiments and ±SEM is indicated. A two-tailed unpaired Student’s t-test was applied. (C and D) H3K27ac Western Blot analysis shows that injection of the Kmt2d MO leads to a decrease in the H3K27ac level, compared to mismatch controls. Graph summarizes the results from four independent experiments and ±SEM is indicated; P-value = 0.0519 in a two-tailed unpaired Student’s t-test.
	Figure 7. Kmt2d loss-of-function inhibits Sema3F expression and ectopic Sema3F can partially substitute for Kmt2d. (A) Sema3F expression is reduced in Kmt2d morphants. Embryos were co-injected with 5 ng mmMO or Kmt2d MO and lacZ mRNA as a lineage tracer. In situ hybridization for sema3f confirms that Kmt2d knockdown inhibits Sema3F expression in Xenopus embryos (arrow). (B) Graph summarizing the data from three independent experiments. Total number of analyzed embryos and ±SEM are indicated for each condition. A two-tailed unpaired Student’s t-test was applied. (C) Two-cell stage embryos were co-injected with 2.5–3 ng Kmt2d MO and 250 pg sema3F mRNA or mGFP mRNA and subjected to twist in situ hybridization. Overexpression of Sema3F results in a partial rescue of NC migration in Kmt2d-depleted embryos. (D) Graph summarizing the data from six independent experiments. Total number of analyzed embryos and ±SEM are indicated for each condition. A Fisher’s exact test was applied.
	Figure 8. Working model of how KMT2D may function to activate NC-specific enhancers. KMT2D deposits H3K4me1 marks at genomic targets. p300 interaction is mediated via the KMT2D/KDM6A complex, finally leading to H3K27 acetylation and a fully active enhancer state.

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