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The pedigree family tree includes two unaffected parents, four unaffected male siblings, five unaffected female siblings, and two each female and male affected sibling. Subjects 1–6, indicated in red, were enrolled in the study. Subjects 4–6 show complex FND with ocular involvement. The eldest affected sibling (subject 4) presented with right coloboma, left microphthalmia, and bilateral Tessier 4 oblique facial clefts. Subject 5 presented with bilateral anophthalmia with fused eyelids and shallow orbits, with bilateral oblique facial clefts. Subject 6 presented with bilateral anophthalmia with open shallow orbits, absent upper and lower eyelids, exposed orbital mucosa, bilateral oblique facial clefts, and malformed nasal ala with nodular skin tags. iPSCs were generated using blood samples collected from subjects 1, 5, and 6.

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Schematic of the differentiation protocol timeline. Maintenance Medium (MM) = iPSC medium (StemFlex with 1× penicillin/streptomycin), NCC differentiation medium = DMEM‐F12, 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM penicillin/streptomycin, 1 mM nonessential amino acids, 110 μM 2‐mercaptoethanol, 10 ng/ml epidermal growth factor.

Gene expression analysis across NCC differentiation of unaffected control ALX1^165L/165L (green), heterozygous ALX1^165L/165F (magenta), and homozygous ALX1^165F/165FiPSC:ALX1, neural plate border specifier genes ZIC1, PAX7, PAX3, MSX1, MSX2, DLX5; neural crest specifier genes FOXD3, P75, TFAP2A, SNAI2, TWIST1; and lineage specifier gene HAND2. The RT–qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype. Exact P‐values are provided in Table EV1. Data from each clone were pooled, and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A P‐value < 0.05 was considered to be statistically significant.

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Homozygous ALX1^165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1^165L/165L NCC (black). The data on the left represent the mean percentage of Annexin V‐positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for 10 min. Representative experiment for each condition is shown. Data are represented as pooled mean ± SEM of three independent experiments. Data obtained of each clone from three independent experiments were pooled, and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A P‐value < 0.05 was considered to be statistically significant. *: Significantly different from the basal apoptosis rate: P = 3 × 10⁻¹² between control NCC basal apoptosis and induced apoptosis, and between ALX1^165F/165F NCC basal apoptosis and induced apoptosis. **Significantly different from control NCC (P = 0.0004).

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Mutant ALX1^165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1^165L/165L NCC (black). Data are presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24‐h period. For fluorescent pictures, cells were stained in serum‐free media containing 3.6 μM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 h using a Keyence BZ‐X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 h. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200 μm. Data obtained of each clone from three independent experiments were pooled, and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A P‐value < 0.05 was considered to be statistically significant. *Significantly different from control NCC (P < 0.0001).

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Dissected flatmount wild‐type and alx1^−/− zebrafish larvae craniofacial cartilages after Alcian blue staining, the anterior points to the left of the page in all images. The ventral cartilages appear normal, but the alx1^−/− anterior neurocranium (ANC) appears narrow, with the midline element that is derived from the frontonasal NCC being absent. Meckel's cartilage (arrow, MC) is also diminutive. Scale bar: 200 μm.

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Schematic representation of the strategy used to generate iPSC. Blood samples from an unrelated normal individual, unaffected father (subject 1), and two of the affected children (subjects 5 and 6) were processed. Isolated PBMC were infected with Sendai virus, and individual clones were picked 21 days after the infection. Following expansion until passage 10, iPSC were characterized and embryoid bodies were formed by suspension culture for 14 days.

Multilineage differentiation experiments revealed that both control and subject‐derived NCC are able to differentiate into Schwann cells, shown by the GFAP and S100B‐positive immunofluorescence staining; adipocytes, demonstrated by the Oil Red O. positive lipidic droplets; osteoblasts, shown by Alizarin Red S. positive mineralized nodules and chondrocytes, assessed by Alcian Blue‐positive cartilaginous matrix. Scale bar is 200 μm for images of Schwann cells and adipocytes, and 400 μm for images of chondrocytes and osteoblasts.

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10, 50, or 100 ng/ml of soluble BMP2 was added to the culture medium. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The data of NCC migration following the treatment with 10, 50, and 100 ng/ml soluble BMP2 are represented as the average of the percentage of closure ± SEM from three independent experiments performed with each clone. To test statistical significance, an ANOVA test was performed. A P‐value < 0.05 was considered to be statistically significant *: Significantly different from untreated ALX1^165F/165F NCC: at 12 h, P = 0.0038 when comparing BMP2 50 ng/ml and untreated ALX1^165F/165F NCC, and P = 0.0045 when comparing BMP2 100 ng/ml and untreated ALX1^165F/165F NCC. At 18 h, P = 0.0337 when comparing BMP2 10 ng/ml and untreated ALX1^165F/165F NCC; P = 0.0009 when comparing BMP2 50 ng/ml and untreated ALX1^165F/165F NCC; and P = 0.0006 when comparing BMP2 100 ng/ml and untreated ALX1^165F/165F NCC. At 24 h, P = 0.005 when comparing BMP2 10 ng/ml and untreated ALX1^165F/165F NCC; P < 0.0001. when comparing BMP2 50 ng/ml and untreated ALX1^165F/165F NCC; and P < 0.0001 when comparing BMP2 100 ng/ml and untreated ALX1^165F/165F NCC.

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Human ALX1 and zebrafish alx1 protein sequences were obtained from Ensembl and aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) under the default settings. The homeobox DNA‐binding domain is shown in bold, with the amino acid residue mutated in the subjects indicated by an outline. The transactivation domain is shaded in gray. Zebrafish alx1 CRISPR sites #1 and #2 are highlighted in yellow. The red and blue letters visually demarcate the sites.

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The number of embryos displaying craniofacial phenotypes increased with increasing concentration of Alx1DN mRNA injected into the single cell stage embryo. Overview of the relationship of the results of injections of 25, 50, and 100 pg of control mRNA and Alx1DN mRNA with the outcomes of wild‐type zebrafish (green), a craniofacial phenotype (gray), and dead zebrafish (magenta).