COMMD10 Is Essential for Neural Plate Development during Embryogenesis

Abstract

The COMMD (copper metabolism MURR1 domain containing) family includes ten structurally conserved proteins (COMMD1 to COMMD10) in eukaryotic multicellular organisms that are involved in a diverse array of cellular and physiological processes, including endosomal trafficking, copper homeostasis, and cholesterol metabolism, among others. To understand the role of COMMD10 in embryonic development, we used Commd10^{Tg(Vav1-icre)A2Kio}/J mice, where the Vav1-cre transgene is integrated into an intron of the Commd10 gene, creating a functional knockout of Commd10 in homozygous mice. Breeding heterozygous mice produced no COMMD10-deficient (Commd10^Null) offspring, suggesting that COMMD10 is required for embryogenesis. Analysis of Commd10^Null embryos demonstrated that they displayed stalled development by embryonic day 8.5 (E8.5). Transcriptome analysis revealed that numerous neural crest-specific gene markers had lower expression in mutant versus wild-type (WT) embryos. Specifically, Commd10^Null embryos displayed significantly lower expression levels of a number of transcription factors, including a major regulator of the neural crest, Sox10. Moreover, several cytokines/growth factors involved in early embryonic neurogenesis were also lower in mutant embryos. On the other hand, Commd10^Null embryos demonstrated higher expression of genes involved in tissue remodeling and regression processes. Taken together, our findings show that Commd10^Null embryos die by day E8.5 due to COMMD10-dependent neural crest failure, revealing a new and critical role for COMMD10 in neural development.

Keywords: COMMD10; Sox10; embryonic development; neural crest.

Figure 1

COMMD10 deficiency results in embryonic lethality. (a) Schematic drawing (up-to-scale) of the Commd10 gene on mouse chromosome 18 shown as a thick grey line. Its direction of transcription is indicated by the black arrow above. Coding exons are represented as thin black boxes. Noncoding 5′- and 3′-untranslated regions are shown as open boxes. The Vav-iCre cassette sketch is shown above the track. The sequence around the Vav-iCre cassette insertion site is shown below the gene scheme in an inset window. Flanking the cassette, GC nucleotides are marked by red bold underlined font and indicated by blue arrows. Their exact positions in the genome are designated by numbers from Reference GRCm39 C57BL/6J below the sequence window. (b) Genotyping analysis of offspring of heterozygous Commd10^Het mice (Het) mating. Commd10^Null (Null) mice had never been born but embryo genotypes show the expected Mendelian distribution. (dpc): days post-coitus. (c) Morphological analysis of WT and Commd10^Null (Null) embryos at E8.5 in dorsal (top panels) and lateral (bottom panels) views. (d) Western blot analysis of whole embryo lysates and anti-COMMD10 or anti-COMMD1 antibodies, as indicated. Anti-SP1 antibody was used as the loading control. NS: non-specific bands.

Figure 2

Commd10^Null embryos fail to develop beyond E8.5 due to impaired neural plate and neural crest development. (a) PCA plot of RNA-seq analysis in WT and Commd10^Null (C10_Null) embryos at E8.5, E9.5, and E10.5. Sample clusters are shown in different colors. Colored arrows show direction of cluster shifts through E8.5 to E10.5 developmental timeframe for both genotypes. Changing arrow colors correlate with the corresponding sample cluster in a timeframe. (b) Volcano plot of RNA-seq analysis visualizing significant DEGs in WT vs. Commd10^Null (C10_Null) E8.5 embryos: magnitude of change (x-axis) vs. statistically significant p-values (y-axis). Points that have a fold change less than 2 (log₂ = 1) are shown in grey. Genes that are transcription factors are marked in italic font. Genes that are expressed in neural crest more highly than in any other cell type are shown in Bold font. (c) Heatmap of mRNA expression levels for top 100 significant DEGs in WT vs. Commd10^Null E8.5 embryos by RNA-seq. (d) Distribution of Commd10 and Sox10 mRNA expression in WT embryos during early embryogenesis in a single-cell molecular map [22]. Presented plots were generated on a single-cell molecular map of mouse gastrulation and early organogenesis at https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/ (accessed on 2 September 2022). The full legend annotating cell clusters by different colors and the schematic map are shown in Figure S1c.

Figure 3

Six of the top ten neural crest-specific markers are differentially expressed in WT versus Commd10^Null embryos. (a) Table listing the top ten neural crest-specific markers, genes that are expressed in the neural crest more highly than in any other cell type. Six genes with differential expression in WT and Commd10^Null embryos are shown in bold font. (b) Tissue distribution of mRNA expression of different transcription factors in WT embryos during early embryogenesis in the molecular map of whole dataset, as described in (c). (c) Legend for (b) annotating cell clusters by different colors, and a single-cell molecular map of mouse gastrulation and early organogenesis [22] up to day E8.5 of embryogenesis. All presented plots were generated on a single-cell molecular map of mouse gastrulation and early organogenesis at https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/ (accessed on 2 September 2022).

Figure 4

Tissue distribution of 14 genes significantly upregulated in Commd10^Null embryos on E8.5. (a) Single-cell molecular maps of mRNA expression in WT embryos for the top 14 genes significantly upregulated in Commd10^Null embryos on E8.5. (b) Legend for (a) annotating cell clusters by different colors, and a single-cell molecular map of mouse gastrulation and early organogenesis [22] up to day E8.5 of embryogenesis. All presented plots were generated on a single-cell molecular map of mouse gastrulation and early organogenesis. at https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/ (accessed on 2 September 2022) website.

Figure 5

Tissue distribution of differentially expressed genes in Commd10^Null embryos on E8.5. The top row of table lists 29 cell lineages/tissues present in normal mouse embryos at the E8.5 stage of embryogenesis. The columns list the top 25 lineage-specific gene markers for each tissue. All lists were found on a single-cell molecular map of mouse gastrulation and early organogenesis. at https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/ (accessed on 22 October 2022) website. Genes that are significantly expressed at lower levels in Commd10^Null embryos when compared with WT are shaded in red. Genes with higher expression in Commd10^Null are shaded in yellow. Blue and green brackets below the table mark cell lineages/tissues involved in neurogenesis and heart development, respectively.