Dominant mutations in GRHL3 cause Van der Woude Syndrome and disrupt oral periderm development

Abstract

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Figure 1

Mutations in GRHL3 Cause Van der Woude Syndrome (A and B) Clinical images of the probands from families VWS-II (A) and VWS-VII (B) display the cardinal feature of VWS, i.e., lip pits (arrowhead). Sequence tracks from each individual are shown to the right with an arrow pointing to the base affected by the mutation. Note that the sequence for c.1559_1562delGGAG is to be read from the reverse strand. (C) GRHL3 has four alternative transcripts variants, v1 to v4 (UCSC Genome Browser), with three alternative first exons (1, 1’, and 1”) and two alternative last exons (16 and 16’). Translation starts in the first exon of each variant (except for v4 where translation starts in exon 2) and stops in the last exon of each variant. The genomic location and cDNA change of each of the nine mutations observed are indicated (according to v3, RefSeq NM_198174.2). The mutation found in the original Finnish family (VWS-I) is indicated by a filled circle. Colors for the exons are corresponding to their coding for the GRHL3 protein domains. (D) Schematic representation of the GRHL3 protein product v2 (RefSeq NP_937816) with (at scale) the three known protein domains: the transactivation (orange), the DNA binding (green), and the dimerization (pink) domains. The position of each change in the protein sequence is also indicated. Please note that because no mutation was found in exon 16, the denomination for each amino acid change is valid both in v2 and v3. More details of the v2 full protein sequence can also be found in Figure S2.

Figure 2

VWS-Associated Alleles of GRHL3 Disrupt the Development of the Periderm when Expressed in Zebrafish Embryos (A–C) Lateral views of live sibling embryos injected with control (A), GRHL3 (B), or GRHL3 (c.1171C>T) (C) mRNA. Embryo shown in (C), injected with the GRHL3 mRNA carrying the c.1171C>T mutation, ruptured through the animal hemisphere shortly after the image was taken (67% [n = 48] of wild-type GRHL3-injected embryos reached at least 50% epiboly stage, whereas 76% [n = 115] of mutant-injected embryos burst without initiating epiboly). (D) Histogram showing fraction of embryos that ruptured when injected with indicated mRNA. Percentage is the average from 3–4 separate experiments of 20–40 embryos each. Error bars represent standard error. (E and F) Animal pole views of embryos injected with indicated mRNA and processed to detect krt4 expression. Insets, cross sections of the same embryos showing (E) krt4 expression confined to the periderm and (F) ectopically in deep cells. (G and H) Animal pole views of mosaic embryos injected with mRNA and biotinylated-dextran at 16-cell stage, fixed at shield stage, and processed for krt4 expression (blue) and biotin distribution (brown). Periderm cells possessed (black arrowhead) or lacked (white arrowhead) biotin stain, demonstrating that they were, or were not, derived from an RNA-injected cell, respectively. Daughter cells derived from the cell injected with the c.893G>A mutant variant of GRHL3 lack krt4 expression. Scale bars represent 500 μm (A–C, E, F), 100 μm (inset in E and F), and 20 μm (G, H).

Figure 3

Grhl3 Is Required for Murine Periderm and Palatal Development (A–C) Haematoxylin and eosin staining of coronal sections of posterior palate at E15.5 (A′). Wild-type embryos showed complete fusion of palatal shelves (asterisk) (A). In contrast, Irf6^−/− embryos have bilateral oral adhesions (arrows) and a fully penetrant cleft palate (asterisk) (B). Similarly, Grhl3^−/− embryos have bilateral oral adhesions (arrows) (C). However, in Grhl3^−/− embryos, adhesions were restricted to areas superficial to the tooth germ and palatal surfaces, and a cleft palate was observed in one of six embryos (asterisk) (C). (D–F) Immunostaining for Krt6 (red) and p63 (green). Krt6 was expressed uniformly in the periderm superficial to the tooth germ (arrow) of wild-type embryos (D) (from boxed structure in A) but was very weakly expressed in Irf6^−/− (E) and Grhl3^−/− (F) embryos. p63 was expressed uniformly in the basal epithelium of wild-type (D) and Grhl3^−/− (F) embryos but was expressed ectopically in suprabasal cells in Irf6^−/− embryos (E). (G–I) Loss of p63 expression marks normal dissolution of the medial edge epithelium (MEE) (arrowhead) in wild-type (G) and Grhl3^−/− (I) embryos. In contrast, p63 expression persisted around the palatal epithelium in Irf6^−/− embryos (H). (D–I) Nuclei are counterstained with DAPI (blue). Scale bars represent 2 mm for (A)–(C), 20 μm for (D)–(F), and 50 μm for (G)–(I). Labeled oral structures are mandible (mn), maxilla (mx), palatal shelf (p), tongue (t), and tooth germ (tg).

Figure 4

No Evidence for Genetic Interaction between Irf6 and Grhl3 in Murine Palatal Development (A–F) Haematoxylin and eosin staining of coronal sections of E13.5 palate anterior (A’) and posterior (D’) to the tooth germ. Compared to wild-type embryos (A, D), Irf6^+/− embryos had bilateral oral adhesions (arrowheads) at the tooth germ site (B). In contrast, Grhl3^+/− littermates had oral adhesions (arrowheads) and fusions (arrow) located predominantly posterior to the tooth germ (E). Irf6^+/−;Grhl3^+/− embryos (C, F) have oral adhesions (arrowheads) at the tooth germ (C) as well as adhesions (arrowheads) and fusions (arrow) posterior to the tooth germ (F). (G–N) Krt6 immunostaining (red) of the oral periderm. Compared to wild-type embryos (G, enlarged in K), Krt6 expression in Irf6^+/− (H, enlarged in L), Grhl3^+/− (I, enlarged in M), and Irf6^+/−;Grhl3^+/− (J, enlarged in N) embryos was markedly reduced along the oral surface of the palatal shelves and the mandible. Loss of Krt6 expression coincides with oral adhesions (arrowheads) and fusions (arrow). (O–R) p63 immunostaining (green) of the basal epithelium was continuous in wild-type (O) and Irf6^+/− (P) embryos. In contrast, p63 staining of Grhl3^+/− (Q) and Irf6^+/−;Grhl3^+/− (R) embryos was discontinuous. Oral fusions are seen between surfaces of the palate and mandible with mesenchymal communication (arrows) punctuating islands of p63-positive epithelial cells (arrowheads). Scale bars represent 2 mm (A–F, G–J, and O–R) and 20 μm (K–N). Labeled oral structures are mandible (mn), maxilla (mx), palatal shelf (p), tongue (t), and tooth germ (tg).