The Mafb cleft-associated variant H131Q is not required for palatogenesis in the mouse

Abstract

Background: Orofacial clefts (OFCs) are common birth defects with complex etiology. Genome wide association studies for OFC have identified SNPs in and near MAFB. MAFB is a transcription factor critical for structural development of digits, kidneys, skin, and brain. MAFB is also expressed in the craniofacial region. Previous sequencing of MAFB in a Filipino population revealed a novel missense variant significantly associated with an increased risk for OFC. This MAFB variant, leading to the amino acid change H131Q, was knocked into the mouse Mafb, resulting in the Mafb^H131Q allele. The Mafb^H131Q construct was engineered to allow for deletion of Mafb ("Mafb^del ").

Results: Mafb^del/del animals died shortly after birth. Conversely, Mafb^H131Q/H131Q mice survived into adulthood at Mendelian ratios. Mafb^del/del and Mafb^H131Q/H131Q heads exhibited normal macroscopic and histological appearance at all embryonic time points evaluated. The periderm was intact based on expression of keratin 6, p63, and E-cadherin. Despite no effect on craniofacial morphogenesis, H131Q inhibited the Mafb-dependent promoter activation of Arhgap29 in palatal mesenchymal, but not ectodermal-derived epithelial cells in a luciferase assay.

Conclusions: Mafb is dispensable for murine palatogenesis in vivo, and the cleft-associated variant H131Q, despite its lack of morphogenic effect, altered the expression of Arhgap29 in a cell-dependent context.

Keywords: Mafb; craniofacial; development; mouse; mutation.

FIGURE 1

Characterization of the Mafb H131Q and del alleles. A, Structure of the MAFB protein, including the histidine-rich domain where the H131Q patient variant is located. B, A single C to G point mutation at position 782 in the single Mafb exon identified in human nonsyndromic cleft lip and/or palate patients was engineered into the murine Mafb, resulting in protein translation and a substitution of the histidine at position 131 with a glutamine (H131Q). C, The macroscopic phenotypes of e18.5 wild-type and Mafb^H131Q/H131Q or wild-type and Mafb^del/del embryos have no noticeable difference. D, Visualization of RNA-seq mapping reads for Mafb in five wild-type (WT1–5) and five Mafb^H131Q/H131Q (H131Q1–5; left panel) and four wild-type (WT1–4), and four Mafb^del/del (del1–4; right panel) e18.5 skin. Data were visualized in the UCSC Genome Browser. Vertical axis shows read counts. Map of the single exon organization of the Mafb gene is shown on top of the panel

FIGURE 2

Palatogenesis is not altered in Mafb^H131Q/H131Q or Mafb^del/del mice. A-F, Coronal section of wild-type, A, D; Mafb^H131Q/H131Q, B, E; and Mafb^del/del, C, F; murine embryonic heads at e14.5, A-C; and e18.5, D-F. In e14.5, palatal shelves were elevated and made contact at the midline, A-C. In e18.5, palatogenesis is completed with a confluent mesenchymal bridge lined by the oral epithelium, D-F. G, H, Quantification of oral adhesions at both e13.5 and e14.5 (Mafb H131Q line) and e14.5 (Mafb del line) as described in Table 3. Percentage of oral epithelium in contact, G. Percentage of oral epithelium in contact excluding mild contact only in the commissures (H). P, palate; PS, palatal shelf; TB, tooth bud; To, tongue

FIGURE 3

Periderm morphogenesis is not altered in Mafb^H131Q/H131Q or Mafb^del/del mice. Coronal section of wild-type, A, D, G, G′; Mafb^H131Q/H131Q, B, E, H, H′; and Mafb^del/del C, F, I, I′, murine embryonic palates at e14.5 immunostained for keratin 6 (K6) and p63, A-C; and E-cadherin, D-I′. Oral epithelial (K6-negative and p63-positive) and periderm cells (K6-positive and p63-negative) are indicated in (A) by white arrows. Scale bar = 16 μm. G′, H′ and I′ are single channel images of G, H and I (Scale bar = 5 μm). MEE, medial edge epithelium; OE, oral epithelium; per = periderm; PS, palatal shelves

FIGURE 4

MAFB H131Q alters ARHGAP29 promoter- and enhancer-driven activity in a cell-dependent context. The ARHGAP29 promoter, A, C, E, or ARHGAP29 enhancer, B, D, F was cloned into the luciferase reporter. Deletion of the MAFB binding site in the ARHGAP29 promoter (A) and risk SNPs in the ARHGAP29 enhancer (B) were considered mutant (Mut). Human embryonic palatal mesenchyme (HEPM) cells (C, D) or LS-8 cells (E, F) transfected with the ARHGAP29 promoter or the ARHGAP29 enhancer were cotransfected with vector-only plasmid (WT), or plasmids containing wild-type (MAFB) or H131Q variant MAFB (H131Q). Bar chart shows luciferase activity reported as relative to the respective vector-only control. Values are presented as mean SD of three biological replicates. *P < .05, **P < .01, ***P < .005, ****P < .0001