Wyatt BH et al. (2021), Using an aquatic model, Xenopus laevis, to unco...

XB-ART-57345

Genesis 2021 Feb 01;591-2:e23394. doi: 10.1002/dvg.23394.

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Using an aquatic model, Xenopus laevis, to uncover the role of chromodomain 1 in craniofacial disorders.

Wyatt BH , Raymond TO , Lansdon LA , Darbro BW , Murray JC , Manak JR , Dickinson AJG .

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The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders.

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Species referenced: Xenopus laevis
Genes referenced: aldh1a2 chd1 grap2 rab40b rarg rgmb slc45a2 tfap2a uqcc6
GO keywords: neural crest cell development [+]
???displayArticle.antibodies??? Casp3 Ab1
???displayArticle.morpholinos??? aldh1a2 MO1 chd1 MO1 chd1 MO2
gRNAs referenced: chd1 gRNA1 slc45a2 gRNA2

???displayArticle.disOnts??? orofacial cleft [+]
???displayArticle.omims??? CLEFT SOFT PALATE [+]
Phenotypes: Xla Wt + BMS453 (Fig. 7 a b) [+]

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	FIGURE 1. (a) Identification of a heterozygous deletion that removes CHD1 and RGMB in a patient with bilateral cleft lip and cleft palate (CLP) using aCGH analysis. Each dot in the segmentation plot represents an oligonucleotide probe co‐hybridized with case (Cy3) and control (Cy5) DNA. Probes shifted downward (highlighted in pink) represent a heterozygous deletion of the region. (b) Pedigree of patient with CNV in a.
	FIGURE 2. In situ hybridization of chd1 in X. laevis. (a,b) Stage 24–25, showing enrichment of chd1 throughout the head and eye. The black arrow indicates labeling in the head. (c,d) Stage 29–30 showing enrichment of chd1 in the eye, brain, otic vesicle, and branchial arches. (e,f) Stage 34–35 showing enrichment of chd1 in the eye, head, and presumptive heart. (g,h) Lateral and frontal view at stage 40–41 showing chd1 expressed in throughout the head with enrichment around the eye and brain. (i–l). Embryo hybridized with a sense probe. M) The head of an embryo at stage 29–30 showing the locations of sections in n and o. (n,o) Sections through the anterior head. (p) The head of an embryo at stage 40–41 showing the locations of sections in Q and R. ba, branchial arch; br, brain; cg, cement gland; no, nostril; ov, otic vesicleoc = oral cavity; st, stomodeum
	FIGURE 3 Knockdown of Chd1 in the embryo. (a) Schematic that shows the predicted binding target of Chd1.SMO1 morpholinos (red bar) and the expected spliced products of control (wild type) and morphant chd1.S mRNA. (b) RT‐PCR indicates that the chd1 mRNA sequence between primers targeting exon 1 and 3 is smaller in Chd1.SMO1 morphants compared to control (CMO) embryos. (c) Schematic that shows the predicted binding target of Chd1.MO2 (red bar) to chd1.L and chd1.S mRNA. (d) Schematic that shows a 16‐cell stage embryo injected into a dorsally fated blastomere creating mosaic knockdown. Representative image where fluorescently labeled Chd1.MO2 (green) cells have reduced Chd1 protein (pink). (e) Schematic that shows the target of the chd1Crispr sgRNA (red bar) in chd1.L and chd1.S genes. (f) High resolution melt curves that show different melt profiles in Chd1Crispr embryos with a phenotype compared to a control (purple) indicating possible mutations (g) Sequences of DNA from control and mutant embryos using primers that bind to sequences flanking the predicted sgRNA binding site. Crispr target sequence is highlighted in red and changes in gene sequence is highlighted in yellow. (h–k). Frontal and lateral views of control embryos. For the lateral views, anterior is to the left. (l–o) Frontal and lateral views of Chd1.SMO1 morphants. (p–s) Frontal and lateral views of Chd1.MO2 morphants. (t–w) Frontal and lateral views of Chd1Crispr mutants. Pink dots outline the mouth. White arrows indicate tail defect. cg, cement gland
	FIGURE 4 Quantifying face malformations in Chd1MO1 morphants. (a) Schematic indicating intercanthal distance and mouth width. (b,c) Box and whisker plots to visualize the difference in intercanthal distance and mouth width in Chd1.SMO1 morphants and controls (CMO). (d) Schematic that shows the positions of the landmarks utilized for morphometric analysis. (e) Transformation grid that shows the changes in landmark positions between Chd1.SMO1 morphants compared to control embryos (Procrustes distance 0.1378, p‐value <.0001). The blue arrows indicate prominent narrowing in the midface between the eyes. (f) Principle component analysis of landmark coordinates showing statistical differences between control and Chd1.SMO1 morphant embryos, p‐value <.0001. (g) Schematic of morphant face transplants. (h–k) Frontal views of embryos that received either control MO or Chd1.SMO1 morphant tissue. The green overlay in i and k show the location of morpholino containing tissue. The mouth is outlined in pink dots. (l) Box and whisker plot summarizing measurements of the mouth width of embryos that received face transplants
	FIGURE 5 Chd1 is required for cranial cartilages and neural crest development. (a) Schematic of the experimental design for b–e. (b–e) Alcian blue labeling of cartilage in Control and Chd1.SMO1 morphant embryos (stage 45–46). Blue arrow indicates the missing ethmoid cartilage in Chd1.SMO1 morphants. (f) Schematic of the experimental design for tfap‐2α in situ hybridization experiments, where one cell was injected at the 2‐cell stage in g–j. (g–h) Lateral views of Control MO or Chd1.SMO1 injected sides of embryos labeled with tfap‐2α probe (st. 24–25). Blue arrows indicate branchial neural crest streams. (i,j) Dorsal and frontal views of Chd1.SMO1 injected embryos labeled with tfap‐2α (st. 24–25). Black arrowhead indicates the Chd1.SMO1 injected side. Yellow arrow indicates the mandibular neural crest stream. (k) Schematic of the experimental design for qRT‐PCR experiment in l. (l) Relative expression of tfap‐2α by qRT‐PCR. Asterisk indicates statistical significance. Bh, Basihyal; cg, cement gland; Ch, ceratohyal; Eth, Ethmoid; Mk, Meckel's; Pq, palatoquadrate
	FIGURE 6 Decreased Chd1 results in increased apoptosis. (a) Schematic of the experimental design for flow cytometry and cleaved caspase 3 immunohistochemistry. (b,c) Average percentages of cells in each phase of the cell cycle. Blue arrow emphasizes the increase in the SubG1 phase. (d,e) Representative cell cycle profiles. Blue arrow emphasizes the increase in the SubG1 phase (f–i) Control or Chd1.SMO1 morphant embryos (stage 29–30) labeled with cleaved caspase‐3. Purple arrows indicate increased cleaved caspase‐3 immunohistochemistry in the eye (h) head (i) and tail (i). cg, cement gland
	FIGURE 7 Retinoic acid regulates chd1 during orofacial development. (a) Schematic of the experimental design for RT‐PCR experiments to measure relative chd1 levels. (b) Relative chd1 expression levels (normalized to actin) following exposure to a RAR antagonist or RAR agonist compared to controls (c). Asterisks designate statistical significance. (c) Schematic of the experimental design for RAR antagonist and CHD1.SMO1 synergy experiments. (d–g) Frontal views of embryos exposed to Control MO (d), sub‐phenotypic concentration of RAR inhibitor (e), sub‐phenotypic concentration of CHD1.SMO1 (f) and combination of sub‐phenotypic concentrations of RAR inhibitor and CHD1.SMO1 (g). Pink dots outline the mouth. (e) Schematic of the chd1.S and chd1.L homeologs indicating the location of putative retinoic acid response elements (RAREs)
	Suppl. Fig 1: RT-PCR of chd1 during developmental stages in X. laevis
	Suppl. Fig. 2: slc452a Crispr/Cas9 mutagenesis A. Representative embryos. B. High resolution melt showing the presence of different melt profiles in 5 mutant embryos compared to a control (purple) demonstrating possibility of induced mutations. C. Sequence results and chromatographs showing changes in the slc45a2 sequence of mutant embryos demonstrating the presence of induced mutations surrounding the Crispr target site (shaded grey).
	Suppl. Fig. 3: Bar graph showing the number of RAREs surrounding chd1 in the vertebrates examined
	Supp. Fig. 4: Representative images of embryos with deficient RA signals (A-F). In situ hybridization of chd1 and RARy showing similar patterns of labeling (G-I). Mouth is outlined in pink dots. Abbreviations; cg=cement gland. Black arrows indicate similar regions of gene expression

References [+] :

Albino, Gene expression profiling identifies eleven DNA repair genes down-regulated during mouse neural crest cell migration. 2011, Pubmed