COMMD10 Is Essential for Neural Plate Development during Embryogenesis

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 Commd10Tg(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 (Commd10Null) offspring, suggesting that COMMD10 is required for embryogenesis. Analysis of Commd10Null 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, Commd10Null 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, Commd10Null embryos demonstrated higher expression of genes involved in tissue remodeling and regression processes. Taken together, our findings show that Commd10Null embryos die by day E8.5 due to COMMD10-dependent neural crest failure, revealing a new and critical role for COMMD10 in neural development.


Introduction
Endosomes are intracellular lipid bilayer organelles that regulate the trafficking of biological cargo between the plasma membrane and other subcellular compartments, including the trans-Golgi network and lysosomes. Following endocytosis, transmembrane proteins undergo sorting to be recycled back to the cell surface or sent for degradation in lysosomes. Cell surface recycling is essential for membrane receptor maintenance and is executed by two distinct protein complexes: Retromer and Retriever (reviewed in [1]). Each of these recycling complexes associates with other multi-protein structures, such as the Wiscott-Aldrich and Scar Homolog (WASH) complex and the COMMD/CCDC93/CCDC22 (CCC) complex [2,3]. Mutations in these multi-protein complexes are increasingly associated with human pathologies, including neurodegenerative and developmental disorders [4][5][6][7].
The COMMD (copper metabolism MURR1 domain)-containing subunit of the CCC complex includes several COMMD family proteins [1]. All members of this family share a unique C-terminal motif termed a COMM domain, which fosters homo-and heterodimerization of COMMD proteins and facilitates interactions with CCDC22 and CCDC93. On the other hand, the N-terminal region is unique in each COMMD protein, suggesting their diverse functions [8]. The first identified member of this family, COMMD1, was discovered to be mutated in Bedlington terriers with copper toxicosis [9]. Subsequently, COMMD1 was demonstrated to regulate the endosomal sorting of the copper transporter ATP7A [2]. COMMD1 also participates in the downregulation of nuclear factor kappa B (NF-κB)-dependent transcription [10,11].
The analysis of Commd10 conditional knockout mice with targeted deficiency to myeloid cells and macrophages demonstrated its direct role in propagating phagolysosomal maturation and clearing of monocyte-driven inflammation [12] and infection [13]. However, Commd10 is ubiquitously expressed, suggesting its role in other tissues [14,15]. Here, we examine the role of COMMD10 in the embryonic development of mice with a disrupted Commd10 gene.

Mice
Commd10 Het mice were bought from the Jackson Laboratory (B6.Cg-Commd10 Tg(Vav1-icre) A2Kio /J, Stock # 008610) [16]. Wild-type (WT) and Commd10 Null embryos were generated by interbreeding of Commd10 Het littermates. Animals were housed and bred in a specific pathogen-free animal facility and fed a standard diet. All mouse breeding and procedures were carried out according to the laboratory animal protocol approved by the IACUC. Animal genotyping was based on the detection of the intact Commd10 allele and iCre by real-time PCR using a DuPlex PCR approach with the following TaqMan assays: Commd10-Fwd: CGGGTCTTCCCATCTCATTT Commd10-Rev: TCAACTGGTTAGTCGGGATTG Commd10 Probe: CAGACACACCCAGAGGCTCATTCATT iCre-Fwd: TGGGCATTGCCTACAACA iCre-Rev: ATCAGCATTCTCCCACCATC iCre Probe: CGCATTGCCGAAATTGCCAGAATCA

Embryological Analysis
In order to harvest embryos at specified embryologic stages, timed pregnancies were set up by breeding Commd10 Het mice. The embryos were considered 0.5 days post coitus (dpc) at noon on the day of detection of the vaginal plug. At embryonic days 8.5 (E8.5), E9.5, and E10.5, females were euthanized and embryos extracted. Embryonic genotyping was performed on genomic DNA purified from yolk sacs. Whole embryo images were obtained at total magnifications of 15× and 45× (combination of magnifications of 1.5× and 4.5× objective lens with 10× ocular lens) using an AmScope microscope with a MU1003 digital camera and AmScope software (AmScope).

RNA Extraction
WT and Commd10 Null embryos at days E8.5, E9.5, and E10.5 were extracted from yolk sacs and immediately placed in Invitrogen™ RNAlater™ Stabilization Solution (Fisher Scientific, AM7023). They were kept at 4 • C for 24 h and transferred to −80 • C for long-term storage before RNA extraction. Total RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN, Hilden, Germany, 74034) and QIAshredder (QIAGEN, 79656) according to the manufacturer's instructions.

RNA-seq and Differential Expression (DE) Analysis
Total RNA purified from WT and Commd10 Null embryos at E8.5, E9.5, and E10.5 was subjected to full transcriptome sequencing. At least three biological repeats were carried out for each condition. 3 -end RNA libraries were made using the Lexogen QuantSeq 3 mRNA-seq Library Prep Kit FWD for Illumina. Sequencing was performed from single-end 75bp on an Illumina NextSeq High Output.
Post-sequence reads were quality-filtered for length and contaminants and were trimmed for Illumina adapters using BBDuk [17]. The resulting reads were pseudo-aligned to coding regions of the mouse reference genome (mm10) using STAR [18]. Gene annotation was performed via the R package biomaRt [19]. Differential expression was calculated using the Wald test implemented in the R package DESeq2 [20]. Significantly differentially expressed genes were defined as those that had both an absolute log2Fold change ≥ 1 and a false discovery rate (FDR) adjusted p-value ≤ 0.05 for each comparison independently.

Quantitative PCR (RT-qPCR)
Whole embryo total RNA was used to measure gene mRNA levels by real-time qPCR. Reverse transcription and cDNA amplification were performed in one tube using qScript™ XLT One-Step RT-qPCR ToughMix ® , Low ROX™ (VWR Quanta Biosciences™, Beverly, MA, USA, 95134) on an Applied Biosystems 7500 Fast Real-Time PCR System (Fisher Scientific). Sample reactions were run in 3-6 replicates. Each mRNA analysis was run in a DuPlex PCR reaction with Gapdh as an internal control. Standard curves for each gene were run to verify the linear range of amplification. Input RNA was kept under 200 ng per reaction to stay within the linear range for Gapdh levels.

Results and Discussion
To examine the role of COMMD10 in embryonic development, we used B6.Cg-Commd10 Tg(Vav1-icre)A2Kio /J mice (Jackson Laboratory; stock #008610). In these mice, the Vav1-iCre transgene is integrated into the intron between exons 5 and 6 of the Commd10 gene on chromosome 18 (Figure 1a) [16]. The insertion resulted in a functional knockout of Commd10 in homozygous (Commd10 Null ) mice [21]. Crossbreeding of Commd10 heterozygous (Commd10 Het ) littermates produced no Commd10 Null newborn mice, while WT and heterozygous genotypes were born at the expected Mendelian ratio (Figure 1b). These results are consistent with those of a viability primary screen phenotypic assay performed on another Commd10 mutant mouse strain (Commd10 tm1a(EUCOMM)Wtsi ) from the EUCOMM consortium (strain #EPD065) at https://www.mousephenotype.org/data/genes/MGI:1916706 (accessed on 17 July 2022). However, the phenotype of these mice has not been reported in the literature. Thus, the essential role of COMMD10 in embryonic development was confirmed by using two different mouse strains with deficient COMMD10 expression.
another Commd10 mutant mouse strain (Commd10 tm1a(EUCOMM)Wtsi ) from the EUCOMM consortium (strain #EPD065) at https://www.mousephenotype.org/data/genes/MGI:1916706 (accessed on 17 July 2022). However, the phenotype of these mice has not been reported in the literature. Thus, the essential role of COMMD10 in embryonic development was confirmed by using two different mouse strains with deficient COMMD10 expression. E8.5 Commd10 Null embryos were visually abnormal and displayed abnormal neural plate morphology and growth retardation, but still remained comparable in size and yielded a comparable amount of RNA for analysis ( Figure 1c). E9.5 and E10.5 mutant embryos showed progressive degradation and signs of tissue resorption ( Figure S1a). Western blot analysis of E8.5 embryo lysates demonstrated lower levels of COMMD10 protein in Commd10 Het embryos and its complete absence in Commd10 Null embryos compared with WT embryos (Figure 1d).
To examine the root cause of the developmental failure of Commd10 Null embryos, we carried out comparative transcriptome analyses of mutant and WT embryos ( Figure S1b). E8.5 Commd10 Null embryos were visually abnormal and displayed abnormal neural plate morphology and growth retardation, but still remained comparable in size and yielded a comparable amount of RNA for analysis (Figure 1c). E9.5 and E10.5 mutant embryos showed progressive degradation and signs of tissue resorption ( Figure S1a). Western blot analysis of E8.5 embryo lysates demonstrated lower levels of COMMD10 protein in Commd10 Het embryos and its complete absence in Commd10 Null embryos compared with WT embryos (Figure 1d).
To examine the root cause of the developmental failure of Commd10 Null embryos, we carried out comparative transcriptome analyses of mutant and WT embryos ( Figure S1b). Figure 2a shows the gene expression principal component analysis (PCA) plot. The cluster of WT samples on E8.5 appears stretched compared with other clusters, indicating some variability among WT samples on that day. The rest of the clusters are tight without any overlap. Importantly, the direction of embryonic development from E8.5 through E10.5 is reflected in the WT cluster distribution on the PCA plot (WT arrow). Interestingly, Commd10 Null E8.5 and E9.5 clusters are located on opposite sides of the WT E8.5 samples. Importantly, both of these clusters are far from each other and from E10.5 samples (Figure 2a, C10_Null arrow). This segregation pattern suggests that the divergence point between WT and Commd10 Null embryos took place not long before day E8.5. Thus, the Commd10 Null E8.5 transcriptome represents an inflection point in embryogenesis from development to tissue resorption. Figure 2b shows a volcano plot visualizing differentially expressed genes (DEGs) in WT vs. Commd10 Null embryos at E8.5 and displaying wide areas of scattered genes on both sides of the y-axis. We sorted all significant DEGs by the absolute value of log2FoldChange and chose the top 100 DEGs to plot on a heatmap (Figure 2c). Among these top 100 DEGs, only 15 were upregulated in Commd10 Null embryos, and the 85 remaining genes were downregulated in contrast to those in WT embryos. Interestingly, the 85 DEGs that are downregulated in mutant embryos include 20 transcription factors, at least 11 cytokines/growth factors/cell surface receptors, and 30 genes with unknown function. The rest of these DEGs encode structural proteins, modifying enzymes, and proteins involved in ion channel function, cell adhesion, and other metabolic cellular processes.
To find the specific embryonic lineage where each of these DEGs is expressed, we searched a single-cell molecular map of mouse gastrulation and early organogenesis at https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/ (accessed on 2 September 2022) [22]. This interactive atlas demonstrates specific mRNA expression profiles during mouse embryonic development between E6.5 and E8.5. As shown in Figure 2d, Commd10 is broadly expressed in all lineages during embryogenesis. The top most significantly (461-fold) downregulated gene in Commd10 Null embryos at E8.5 is Sox10, a transcription factor with a central role in neural crest development and maturation of glia [23]. We have also validated Sox10 mRNA expression in WT and Commd10 Null embryos at E8.5, E9.5, and E10.5 by RT-qPCR and found the highest Sox10 expression and the most drastic difference between the two genotypes at E8.5 ( Figure S1d). In normal developing mouse embryos, Sox10 expression emerges after E8.0 almost exclusively in the neural crest (Figure 2d). The table in Figure 3a lists the top ten neural crest-specific markers according to the interactive atlas. Interestingly, six of those markers were differentially expressed in WT versus Commd10 Null embryos, suggesting that there is a defect in neural crest development in Commd10 Null embryos (Figures 2b-d and 3b). Moreover, a list of significant DEGs, which define the trajectory of neurogenesis, includes numerous transcription factors critical for neural plate development, starting from rostral neuroectoderm at E6.5 and subsequent development of caudal neuroectoderm, spinal cord, forebrain/midbrain/hindbrain, and neural crest by E8.5.
Besides Sox10, Commd10 Null embryos exhibit significantly lower expression of transcription factors Tfap2b [24,25], Nr2f1 [26], Msx3 [27], Dbx2 with Pax6 [28,29], Sox1 [30], Gbx2 [31], Zic1 [32], Pou3f2 [33][34][35], Prdm13 [36], Fezf1 [37], Six6 [38,39], Foxg1 [40], and Foxi2 [41] (Figure 3). They all participate in the early stages of central nervous system development. Also significantly downregulated in Commd10 Null embryos are genes encoding cytokines/growth factors involved in early embryonic neurogenesis, such as Ptn [42,43], Mdk [43,44], and Grem1 [45] (Figure 2b). In addition, transcription factors such as Meox2 [46], expressed in paraxial and somatic mesoderm, and Bhlha9 [47,48], expressed in surface ectoderm, are important for the expression of genes involved in signaling pathways essential for the formation and morphogenesis of somites and limbs in developing embryos (Figure 3). Taken together, these data are in agreement with the observation that WT embryos at E8.5 undergo continuous embryogenesis by means of cell proliferation, migration, and differentiation, particularly in the process of primary neurulation. This highly orchestrated process is defined by the expression of a number of transcription and growth factors that are coordinated in place and time. Significantly lower levels of these molecules in Commd10 Null embryos may result in the termination of embryonic development. reflected in the WT cluster distribution on the PCA plot (WT arrow). Interestingly, Commd10 Null E8.5 and E9.5 clusters are located on opposite sides of the WT E8.5 samples. Importantly, both of these clusters are far from each other and from E10.5 samples ( Figure  2a, C10_Null arrow). This segregation pattern suggests that the divergence point between WT and Commd10 Null embryos took place not long before day E8.5. Thus, the Commd10 Null E8.5 transcriptome represents an inflection point in embryogenesis from development to tissue resorption.   On the other hand, there are no transcription factors or cytokine/growth factors among the top 15 DEGs upregulated in Commd10 Null embryos at E8.5 as compared with their WT littermates (Figure 2c). While some of these genes, such as Anxa8 and Anxa1 [49], are modestly expressed in notochord, caudal neuroectoderm, and neural crest of the WT embryos, most are not expressed in developing neural tissue (Figures 4 and 5). Instead, the majority of those genes are expressed in blood progenitors and erythroid tissue in particular (Gypa, Hbq1b, Epb42, Trim10 [50,51], Spta1). Interestingly, some of the upregulated DEGs in Commd10 Null embryos may be involved in tissue remodeling and regression. Granzyme C (Gzmc) is increased 48-fold in Commd10 Null embryos compared with WT, while Inhibin beta A chain (Inhba), a member of the inhibins/activins network of proteins, is increased 49fold. Thus, embryonic cell death leading to tissue regression in E8.5 Commd10 Null embryos may be caused by two main events. The first event is a failure of the neural plate and neural crest processes due to a substantial deficiency of transcription factor Sox10, together with lower expression of other transcription factors and cytokines/growth factors involved in early embryonic neurogenesis. The second event is based on the increased expression of proteins with potential embryo resorption abilities.
To verify our conclusions further, we examined the expression of statistically significant DEGs with the top 25 gene markers representing each embryonic cell type present in the mouse embryo at E8.5 ( Figure 5). A single-cell molecular map of mouse gastrulation and early organogenesis [22] lists 29 different cell/tissue types for the E8.5 mouse embryo. The gene analysis revealed that the majority of genes with low expression in Commd10 Null embryos are found in cells involved in early neural and heart development ( Figure 5). Since recent studies demonstrated that neural crest cells develop into cardiomyocytes and contribute to heart development [52,53], gene expression deficiency in cardiomyocytes may be due to failed neural crest differentiation and/or cell migration.
We also performed gene functional enrichment analysis for the top 15-20 upregulated or downregulated DEGs using ToppGene Suite (https://toppgene.cchmc.org (accessed on 16 February 2023) [54]. We analyzed the top 20 genes downregulated in Commd10 Null embryos and came up with a "GO: Biological Process" list of positive regulation of RNA biosynthetic process, epithelium development, animal organ morphogenesis, and brain and head development. We also analyzed the top 15 genes upregulated in Commd10 Null embryos and selected the two top biological processes with the highest number of genes from the list: hemopoiesis and immune system development (Supplementary Tables).
Mice deficient in other members of the COMMD family, COMMD1 or COMMD9, were shown to be embryonically lethal. Commd1 −/− embryos died between E9.5 and E10.5 due to defects in placenta vascularization [55]. Using genome-wide gene expression microarray analysis of embryonic RNA, the authors identified transcriptional upregulation of hypoxiainducible factor 1 (HIF1) target genes in Commd1 −/− embryos compared with their WT counterparts. Moreover, they demonstrated that COMMD1 may inhibit HIF1A stability and HIF1 activation by the physical association between the two proteins. Despite similarities in the timing of embryonic development failure between Commd1 −/− and Commd10 Null embryos, there were no similarities in gene expression patterns in the present study. Only Pfkp, one of eighteen hypoxia-associated DEGs upregulated in Commd1 −/− versus WT embryos, was slightly upregulated in Commd10 Null E8.5 embryos. Thus, the failure of Commd10 Null embryos to thrive appears to have different underlying reasons compared to Commd1 −/− embryos.    To verify our conclusions further, we examined the expression of statistically significant DEGs with the top 25 gene markers representing each embryonic cell type present in the mouse embryo at E8.5 ( Figure 5). A single-cell molecular map of mouse gastrulation and early organogenesis [22] lists 29 different cell/tissue types for the E8.5 mouse embryo. The gene analysis revealed that the majority of genes with low expression in Commd10 Null embryos are found in cells involved in early neural and heart development ( Figure 5). Since recent studies demonstrated that neural crest cells develop into cardiomyocytes and contribute to heart development [52,53], gene expression deficiency in cardiomyocytes may be due to failed neural crest differentiation and/or cell migration.
We also performed gene functional enrichment analysis for the top 15-20 upregulated or downregulated DEGs using ToppGene Suite (https://toppgene.cchmc.org (accessed on 16 February 2023) [54]. We analyzed the top 20 genes downregulated in Commd10 Null embryos and came up with a "GO: Biological Process" list of positive regulation of RNA biosynthetic process, epithelium development, animal organ morphogenesis, and brain and head development. We also analyzed the top 15 genes upregulated in Commd10 Null embryos and selected the two top biological processes with the highest number of genes from the list: hemopoiesis and immune system development (Supplementary Tables). In contrast to Commd10 Null embryos, Commd9 −/− embryos die by E13.5 [56]. The authors found low levels of Hey1, Hey2, and Hes1 mRNA in the hearts of Commd9 −/− embryos and concluded that the embryonic lethality of these mice was due to complex cardiovascular changes with signs of Notch deficiency. There were no differences in the mRNA expression of Notch or the genes listed above in Commd10 Null embryos compared with WT. Taken together, these data indicate that COMMD1-, COMMD9-, and COMMD10deficient mice display different underlying reasons for failed embryonic development and suggest that COMMD proteins play different critical roles during embryogenesis.
No direct connection between COMMD10 and Sox10 has been described in the scientific literature. We can only speculate as to how the absence of COMMD10 may lead to lower expression of Sox10 and, sequentially, other genes during embryogenesis. During normal embryogenesis, Sox10 mRNA appears in late gastrulating embryos (mouse E7.5) in the neural crest-forming region, and its gene expression depends on Wnt signaling [57,58]. Sox10 protein was also found to directly interact with β-catenin [59], which is activated in the canonical Wnt signaling pathway (reviewed in [60]). Wnt protein ligands bind to Frizzled family receptors (cell surface Fzd proteins and co-receptor Lrp5/6). Commd10 Null embryos show significantly lower expression of Fzd3 and Fzd9 suggesting lower Wnt signaling potency. In addition, several Wnt ligands themselves were also dysregulated. There were higher levels of Wnt3 and Wnt9b while there were significantly lower levels of Wnt1, Wnt7a, and Wnt8b, suggesting dysregulation of Wnt signaling pathways in Commd10 Null embryos. Wnt1-deficient mice exhibit a range of phenotypes, from early embryonic lethality to survival with severe ataxia [61]. Wnt7a signaling also controls multiple steps of neurogenesis [62]. It is plausible that by being part of the endosomal trafficking process inside the cell, COMMD10 may be involved in Wnt signaling regulation through as yet unknown mechanisms of Fzd receptor recycling or Wnt ligand secretion.

Limitations of the Study
The results described here characterize the timing of embryonic lethality of Commd10 Null mice and also begin to demonstrate that neural plate developmental delay is the most likely cause of Commd10 Null failed embryogenesis. The differential gene expression profile of Commd10 Null as compared to normally developing WT embryos after E8.5 does not necessarily imply direct associations with COMMD10 deficiency. They rather verify the timing of embryonic failure by E8.5. Broader approaches and detailed analyses of earlier embryos are needed to pinpoint the exact role of COMMD10 in mouse embryogenesis, which are subjects of continued study and outside the scope of the present study.

Conclusions
Our study demonstrated that COMMD10 deficiency leads to embryonic lethality by day E8.5, most likely due to impaired neural plate and neural crest development processes resulting from the decreased expression of transcription factor Sox10 and several other genes. The molecular mechanism by which COMMD10 upregulates Sox10 expression remains unknown and merits further investigation. Institutional Review Board Statement: All housing and experimental use of mice were carried out at AAALAC-accredited facility in accordance with United States federal, state, local, and institutional regulations and guidelines governing the use of animals and were approved by OUHSC Institutional Animal Care and Use Committee (animal study protocol No. 20-028, approval date 8 May 2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: Most data generated or analyzed during this study are included in this published article and its Supplementary Materials. Unprocessed RNA-seq raw data files and processed data files have been deposited on NCBI Gene Expression Omnibus (https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE216492 (accessed on 13 March 2023). Further information and requests for materials should be directed to and will be fulfilled by the lead contact, Ian F. Dunn (ian-dunn@ouhsc.edu).