Fig 2: ALX4 partly complements ALX1 function in regulating frontonasal development. Lateral (A–C) and frontal (D–F) views of whole mount embryo heads showing patterns of Pax7 mRNA expression in the control (A, D), Alx1 del/del Alx4 +/- (B, E), and Alx1 del/del Alx4 -/- (C, F) embryos at E10.5. Arrow points to the domain of reduced Pax7 expression in the lateral nasal process of the Alx1 del/del Alx4 +/- (B, E) and Alx1 del/del Alx4 -/- (C, F) embryos, respectively. (G–L) Lateral (G–I) and frontal (J–L) views of whole mount embryo heads showing patterns of Lhx6 mRNA expression in the control (G, J), Alx1 del/del Alx4 +/- (H, K), and Alx1 del/del Alx4 -/- (I, L) embryos at E10.5. Arrows and arrowheads point to the domain of ectopic Lhx6 expression in the lateral and medial nasal processes, respectively, in the in the Alx1 del/del Alx4 +/- (H, K) and Alx1 del/del Alx4 -/- (I, L) embryos. (M–R) Lateral (M–O) and frontal (P–R) views of whole mount embryo heads showing patterns of Lhx8 mRNA expression in the control (M, P), Alx1 del/del Alx4 +/- (N, Q), and Alx1 del/del Alx4 -/- (O, R) embryos at E10.5. Arrows and arrowheads point to the domain of ectopic Lhx8 expression in the lateral and medial nasal processes, respectively, in the Alx1 del/del Alx4 +/- (N, Q) and Alx1 del/del Alx4 -/- (O, R) embryos. e, eye. Scale bars, 500 µm.

Fig 3: Alx1 expression during early craniofacial development. (A) Lateral view of a Wnt1-Cre;Rosa26 mTmG embryo at somite stage (SS)10 showing GFP (green) labeled dorsal midbrain neuroepithelium and cranial neural crest cells. (B, C) Lateral views of wildtype SS10 (B) and SS12 (C) embryos showing the patterns of Sox10 mRNA expression detected by whole mount in situ hybridization (blue/purple color). White arrowheads in (C) point to the Sox10 expression in the trunk neural crest cells. (D–F) Lateral views of wildtype SS9 (D), SS11 (E), and SS12 (F) embryos showing the patterns of Alx1 mRNA expression detected by whole mount in situ hybridization (blue/purple color). The asterisk in A-F mark the location of the optic placode. Arrowheads in (D–F) point to the Alx1 expression in the lateral plate mesoderm. Scale bar, 500 µm. ba1, branchial arch 1; mb, midbrain; r2, rhombomere 2; r4, rhombomere 4.

Fig 4: Cranial neural crest migration to the facial primordia and expression of frontonasal neural crest marker genes appeared normal in Alx1 del/del embryos. (A–D) Representative lateral views of whole mount embryo heads showing GFP (green) labeled neural crest cells in the developing facial primordia from Wnt1-Cre;Rosa26 mTmG/+ (A, C) and Wnt1-Cre;Rosa26 mTmG/+ ;Alx1 del/del (B, D) embryos at E9.5 (A, B), and E10.5 (C, D). White arrow in A and B points to the frontonasal prominence, whereas the white arrowhead in (A–D) points to the mandibular arch. (E–H) Lateral views of whole mount embryo heads showing expression (blue/purple color) of Alx3 (E, F) and Alx4 (G, H) mRNAs in control (E, G) and Alx1 del/del embryos (F, H) at E9.5. Arrow in E-H points to the frontonasal prominence, whereas the arrowhead points to the mandibular arch. e, eye. Scale bars, 500 µm.

Fig 5: Generation of Alx1-deletion mice using CRISPR/Cas9-mediated genome editing. (A) Schematics of the strategy for generating Alx1 del/+ mice using the CRISPR/Cas9 technology. The top row shows the genomic organization of the mouse Alx1 locus. Exons 1–4 are boxed with coding regions filled in grey color and the domains of ALX1 protein represented in the second row. Lines point to the exons of the Alx1 gene with the corresponding region in the ALX1 protein. The DNA-binding Homeodomain and the OAR domain, a conserved domain in the C-terminal region of several paired-like homedomain proteins including Drosophila Orthopedia and Aristaless and vertebrate Rax, are marked. The locations of sgRNA target sequences are indicated in intron-1 and intron-2 regions, respectively, in the schematic in the third row, whereas the fourth row depicts the Alx1 del allele that lacks exon-2 and flanking sequences in between the two sgRNA target sites. The exon-2 deletion results in a frameshift and premature translational STOP codon in the exon-3 sequence. (B) PCR genotyping of the embryos from the intercross of heterozygous Alx1 del/+ mice. The amplicons from wildtype and Alx1 del alleles are 497 bp and 446 bp, respectively. (C) RT-PCR analysis using primers from exon1 to exon4, respectively, reveals a wildtype amplicon of 673 bp and an Alx1 del allele-specific product of 370 bp. (D) Western blot analysis using a polyclonal anti-ALX1 antibody raised against the full-length ALX1 protein reveals a lack of the full-length ALX1 protein in the Alx1 del/del embryos. Alx1 del/del embryos produced a truncated N-terminal product of around 10.5 kDa +/-, Alx1 del/+ ; +/+, wildtype; -/-, Alx1 del/del .