|
FIGURE 1
Loss of Adam13 affects gene expression and splicing in the cranial neural crest (CNC). (A) Volcano plot showing differential gene expressions in CNC cells dissected from stage 17 embryos from Adam13 knockdown (MO13) versus wild-type (WT) embryos. Log2(fold change) is plotted on the x axis, with negative values indicating downregulation and positive values indicating upregulation. The significantly differentially expressed genes (DEGs; adjusted p < 0.01) are highlighted in blue, and selected genes of interest are marked in red (adjusted p < 0.05). The identity and fold changes of these genes are provided in the bar graph on the right; Tfap2α-L and S are represented by two purple dots. The complete list of genes identified is provided in Supplementary Table S1. (B) Gene set enrichment analysis (GSEA) of the biological processes affected by the DEGs in Adam13 protein knockdown CNC cells. The dot size corresponds to the number of genes in the pathway, while the dot color represents the p-value. The enriched biological processes are related to protein and RNA modifications. (C) Schematic representation of the Tfap2α gene showing the three alternate transcription start sites (TSSs; S1–S3) that result in different protein isoforms with distinct N-terminal sequences. (D) Bar graph depicting the relative expressions of Tfap2α variants at the S1, S2, and S3 TSSs in response to Adam13 knockdown in the CNC cells. (E) Venn diagram showing the overlap between differentially spliced genes (DSGs) and DEGs, with Tfap2α serving as an example gene with both significant (adjusted p < 0.01) splicing and expression changes. |
|
FIGURE 2
Loss of Adam13 leads to changes in histone H3 methylation. (A) Immunofluorescence images showing H3K4me3 (red), Snai2 (green), and DAPI (blue) staining of the CNC explants from control and Adam13 knockdown (MO13) embryos. The maximum projection images from confocal microscopy are shown here. (B) Bar graph quantifying the H3K4me3 fluorescence intensities normalized to DAPI. Snai2 staining confirms the identity of the CNC. The data represent at least nine explants (three independent experiments with 100+ cells per explant). Statistical significance is indicated as ****p < 0.0001. (C) Bar graph quantifying H3K9me2_3 fluorescence intensities normalized to DAPI. The statistical analysis was the same as in (B). (D–G) Chromatin immunoprecipitation (ChIP) from Xenopus embryos at the neurula stage 20. The bar graphs represent the relative abundances of chromatin associated with different proteins by ChIP-qPCR analysis: (D) H3K4, (E) H3K9, (F) Arid3a, and (G) Adam13. The results were normalized to WT embryos and compared to homozygote knockout embryos for Adam13.L (KO). Chromatin was immunoprecipitated using antibodies against H3K4me3, H3K9me2/3, Arid3a, and Adam13, as indicated on the x axis. The associated chromatin was amplified using primers upstream of the S1 and S3 transcription start sequences of Tfap2α.L. Six replicates were used for the statistical analyses, and statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. |
|
FIGURE 3
Luciferase assay using Tfap2α reporter upstream of the S1 and S3 transcription start sites. (A) Schematic representation of the Xenopus laevis Tfap2α.L gene and S1/S3 transcription start sites used for luciferase reporter constructs. The sequences used to produce the luciferase reporter (red) are aligned to the genome. (B, C) Bar graphs showing the relative luciferase activities of S1 and S3 reporter constructs transfected into HEK293T cells together with a CMV Renilla construct with or without Xenopus Adam13 and Arid3a. (B) Three biological replicates were used for each condition, and protein expression was confirmed by Western blotting. Statistical significance was determined by one-way ANOVA (*p < 0.05, **p < 0.01, and ****p < 0.0001). (C) Bar graph showing relative luciferase activities of the S1 and S3 reporters in the presence of shRNA targeting human Arid3a. The data are normalized to the CMV Renilla reporter. Statistical significance was determined by one-way ANOVA (*p < 0.05, **p < 0.01, and ****p < 0.0001). |
|
FIGURE 4
Biological activities of the S1 and S3 Tfap2α isoforms. (A) Schematic representation of the experimental setup. Top view of an embryo at the 8-cell stage with its dorsal side down (lighter pigment). The injection targets the dorsal animal cells, and these cells produce the red-labeled tissue at the neurula stage (dorsal view stage 18) corresponding to the dorsal anterior quadrant of the embryo that includes the CNC (Moody, 1987). Plasmids, morpholino, and mRNA including lineage tracers can be injected. (B) Lateral view with anterior to the left and dorsal side up for representative embryos at stage 24 showing typical scoring. The embryo to the left is a typical sample injected with red fluorescence protein mRNA showing clear CNC migration on the ventral side in all available segments. The embryo to the right is typical of a sample injected with the Adam13 morpholino, in which no migrating neural crest is seen on the ventral side despite correct targeting of the dorsal anterior quadrant. Only embryos identical to the example on the right side are scored as no migration. Embryos with partial migration in any of the posterior segments are scored as migration to obtain a simple binary data point. (C) Bar graph representing the luciferase activity of the calpain8 reporter injected as in (A). The reporter activity decreased by approximately 50% when injected in embryos lacking Adam13 (MO13). Embryos selected at the neurula stage based on expression of the lineage tracer in the dorsal anterior quadrant as in (A) were tested for luciferase activity and normalized to Renilla. Three to five biological replicates are plotted. Statistical analysis was performed using a t-test with **p < 0.01. (D) Bar graph showing the relative calpain8 luciferase reporter expressions in HEK293T cells transfected with the empty vector (CS2), Tfap2α-L-S1, or Tfap2α-L-S3 constructs. Statistical analysis was performed by one-way ANOVA from three biological repeats (*p < 0.05 and **p < 0.01; ns, p > 0.05). (E) Bar graph depicting the percentage of embryos with CNC migration in the three biological repeats based on images shown in (B). The number of embryos is as follows: MbC (64), MO13 (53), MO13 + Tfap2α-S1 (60), MO13 + Tfap2α-S3 (56). Statistical analysis was performed using ANOVA with **p < 0.01 and ****p < 0.0001. |
|
FIGURE 5
Adam13 binds to multiple nuclear proteins, including histone methyltransferases. Proteomic analyses of proteins (A) associated with the cytoplasmic domain of Xenopus Adam13 and (B) common to Xenopus Adam13 and human ADAM9. The protein interactions were analyzed using STRING. Proteins involved in splicing are shown in blue, translation regulation in red, and key interactors are highlighted with colored arrowheads (green, SH3-domain proteins; blue, importins; red, Arid4a and KMT2C). (C) Co-immunoprecipitation of Adam13 and endogenous human KMT2A. HEK293T cells were transfected with either the empty vector (CS2) or Adam13 (A13), and the extract was immunoprecipitated using an anti-KMT2A antibody and blotted (WB) with an anti-Adam13 monoclonal antibody (mAb 7C9). The total extracts were blotted with antibodies to Adam13, KMT2A, and GAPDH. (D) AlphaFold modeling of the interaction between the cytoplasmic domain of Adam13 (green) and Xenopus KMT2D SET domain (cyan). Potential interaction sites are highlighted in yellow (Adam13 amino acids) and purple (KMT2D amino acids). The image on the right side is a magnified version in the same orientation. (E) Capillary Western blotting showing co-immunoprecipitation of HA-tagged KMT2D SET domain and Adam13 in HEK293T cells. The upper panel shows co-immunoprecipitation with HA blotted with Adam13 (mAb 7C9), while the lower panel shows the total extract (TE) for Adam13 detection. The Adam13 mutant lacking its cytoplasmic domain (ΔCyto) does not bind KMT2D, while the mutant missing the nuclear localization signal delta KR binds KMT2D. The mAb 7C9 binds to the extracellular domain of Adam13 present in all Adam13 constructs. |
|
Supplementary Figure S1
[[Cranial neural crest (CNC) explants from control and MO13 (Adam13 morpholino (MO13)) embryos. Immunofluorescence for H3K9me2/3 (green) and DAPI (blue) staining]] |
|
Supplementary Figure S2 |
