Kir2.1 is important for efficient BMP signaling in mammalian face development

Abstract

Mutations that disrupt the inwardly rectifying potassium channel Kir2.1 lead to Andersen-Tawil syndrome that includes periodic paralysis, cardiac arrhythmia, cognitive deficits, craniofacial dysmorphologies and limb defects. The molecular mechanism that underlies the developmental consequences of inhibition of these channels has remained a mystery. We show that while loss of Kir2.1 function does not affect expression of several early facial patterning genes, the domain in which Pou3f3 is expressed in the maxillary arch is reduced. Pou3f3 is important for development of the jugal and squamosal bones. The reduced expression domain of Pou3f3 is consistent with the reduction in the size of the squamosal and jugal bones in Kcnj2^KO/KO animals, however it does not account for the diverse craniofacial defects observed in Kcnj2^KO/KO animals. We show that Kir2.1 function is required in the cranial neural crest for morphogenesis of several craniofacial structures including palate closure. We find that while the palatal shelves of Kir2.1-null embryos elevate properly, they are reduced in size due to decreased proliferation of the palatal mesenchyme. While we find no reduction in expression of BMP ligands, receptors, and associated Smads in this setting, loss of Kir2.1 reduces the efficacy of BMP signaling as shown by the reduction of phosphorylated Smad 1/5/8 and reduced expression of BMP targets Smad6 and Satb2.

Figure 1

Alizarin red and Alcian blue skeletal stains of E18.5 WT (A, C, E, G, I, K, M, O, Q, S, U) and Kcnj2^KO/KO (B, D, F, H, J, L, N, P, R, T, V) mice shows that deletion of Kcnj2 causes hypoplastic mandible (MD, yellow arrow), maxilla (MX, purple arrow), premaxilla and prenasal bones (PM, PN red arrow), components of the zygomatic arch (ZA, green arrow), and tympanic ring (TR, black arrow). Skeletons are represented in lateral views (A–F), dorsal views (G–J), ventral views with mandible attached (K, L), and without mandible attached (M, N). Mandibles are compared in lingual (O, P), and lateral (Q, R), and lingual (S, T) views. Seven Kcnj2^KO/KO skeletons were analyzed and compared to six wild type skeletons. The sagittal sutures and fontanelles are significantly larger in Kcnj2^KO/KO compared to WT siblings, p= 0.006 and p= 0.03 by T-test (U).

Figure 2

RNA in situ hybridization shows early craniofacial patterning genes in kcnj2^KO/KO mice compared to WT controls. Lateral views of WT (A) and Kcnj2^KO/KO (B) show similar expression of FoxL2 (black arrows). Ventral views of WT (C) and Kcnj2^KO/KO (D) show similar expression of Dlx5. Ventral views of WT (E) and Kcnj2^KO/KO (F) and lateral views of E10.5 WT (G) and Kcnj2^KO/KO (H) show that loss of Kir2.1 function does not affect Dlx2 expression. Ventral views of WT (I) and Kcnj2^KO/KO (J) E10.5 embryos show similar expression of Hand2. Lateral views of WT (K) and Kcnj2^KO/KO (L) E10.5 embryos show similar expression of Hand2. Lateral views of WT (M) and Kcnj2^KO/KO (N) E10.5 embryos show a slightly reduced domain of Pou3f3 expression (yellow arrow, compared to red arrow marking the eye) so that it does not extend to under the eye. Lateral views of WT (O) and Kcnj2^KO/KO (P) show posterior shift in the domain of Pou3f3 expression (yellow arrow compared to red arrow marking the eye) in E11.5 Kcnj2^KO/KO embryos. N=3 embryos of each genotype for each probe.

Figure 3

RNA in situ hybridization reveals Kcnj2 mRNA is expressed in mesenchyme of the palate shelves and in the Meckel’s cartilage at E12.5 (A and B) and in the olfactory epithelium (C). At E13.5, Kcnj2 is still apparent in the mesenchyme of the palate shelves and also in the Meckel’s cartilage (D and E). By E14.5, Kcnj2 mRNA is not apparent in the palate, but can be detected surrounding the tooth bud in wild type mouse embryos (F).

Figure 4

Alizarin red and Alcian blue skeletal stains of Kcnj2^fl/+ control (A, C, E, G, I, K, M, O, Q, S, U) compared to Wnt1-Cre; Kir2.1^fl/KO (B, D, F, H, J, L, N, P, R, T, V) E18.5 mice show that deletion of Kcnj2 from the cranial neural crest cells causes hypoplastic mandible (MD, yellow arrow), maxilla (MX, purple arrow), premaxilla and prenasal bones (PM, PN red arrow), components of the zygomatic arch (ZA, green arrow), and tympanic ring (TR, black arrow). Skeletons are represented in lateral views (A–F), dorsal views (G–J), ventral views with mandible attached (K, L), and without mandible attached (M, N). Mandibles are compared in lingual (O, P), and lateral (Q, R), and lingual (S, T) views. Six Wnt1-Cre; Kcnj2^fl/KO skeletons were stained and compared to four control siblings.

Figure 5

Alizarin red and Alcian blue skeletal stains of Kcnj2^fl/+ control (A) compared to Crect; Kir2.1^fl/KO (B) E18.5 mice show that deletion of Kcnj2 from the cranial neural crest cells does not significantly affect mandible (MD, yellow arrow), maxilla (MX, purple arrow), premaxilla and prenasal bones (PM, PN red arrow), components of the zygomatic arch (ZA, green arrow), and tympanic ring (TR, black arrow). Skeletons are represented in lateral views (A–F), dorsal views (G–J), ventral views with mandible attached (K, L), and without mandible attached (M, N). Mandibles are compared in lingual (O, P), and lateral (Q, R), lingual (S, T) views. Five Crect; Kcnj2^fl/KO skeletons were stained and compared to five Kcnj2^fl/+ control sibling skeletons.

Figure 6

H&E staining of E14.5 WT (A) and Kcnj2^KO/KO (B) shows that the palate has fused at this stage for WT, but for Kcnj2^KO/KO mice, palate shelves (PS) have elevated, but remain small and have not fused. H&E staining of E14.5 Wnt1-Cre; Kcnj2^fl/+ (C) and Wnt1-Cre; Kcnj2^fl/KO (D) shows that the palate shelves are small and elevated and fail to fuse in Wnt1-Cre; Kcnj2^fl/KO when it has fused in Wnt1-Cre; Kcnj2^fl/+ siblings at the same stage. H&E staining of E14.5 Crect; Kcnj2^fl/+ (E) and Crect; Kcnj2^fl/KO (F) shows that the palate fuses for both genotypes by E14.5. EdU staining of proliferating cells in E13.5 WT (G), Kcnj2^KO/+ (H), Kcnj2^KO/KO show that there is less proliferation in palate mesenchyme in Kcnj2^KO/KO animals compared to WT and heterozygous siblings. A graph quantifies the percentage of cells marked with EdU in WT, Kcnj2^KO/+, and Kcnj2^KO/KO (J), p< 0.05 by two-tailed T-test comparing each genotype. EdU was quantified in 3 sections for 3 animals of each genotype. TUNEL staining marks apoptotic cells in E13.5 WT (K), Kcnj2^KO/+ (L), Kcnj2^KO/KO (M) palate shelves. The number of TUNEL positive cells out of 75 mesenchymal and 75 ectodermal DAPI stained nuclei were quantified in 3 sections for 3 animals of each genotype. There was no significant difference in TUNEL positive cells between Kcnj2^KO/KO compared to WT siblings and heterozygous siblings, p>0.05, two tailed T-test.

Figure 7

Quantification of expression of BMP and TGF-β ligands shows that expression of BMP5 is significantly increased in Kcnj2^KO/KO E13.5 palatal shelves compared to WT controls (p=0.007, T-Test). (A). Quantification of expression of BMP and TGF-β receptors shows that expression of receptors in E13.5 palatal shelves is not significantly different between Kcnj2^KO/KO compared to WT controls (B). Expression of SMADs in E13.5 palatal shelves is not significantly different between Kcnj2^KO/KO compared to WT controls (C). Quantification of mRNA levels encoding BMP and TGF-β inhibitors shows that Chordin, Tgfbi and Tgfbrap1 are significantly increased in Kcnj2^KO/KO compared to WT controls (D). Expression of BMP target genes Smad6 and Satb2 are significantly reduced in E13.5 Kcnj2^KO/KO palatal shelves compared to WT, n= 6 WT and 4 Kcnj2^KO/KO, T-test p= 0.007 and 0.01 respectively (E). A western blot shows that phosphorylated Smad 1/5/8 is reduced in Kcnj2^KO/KO E13.5 craniofacial structures compared to WT sibling controls (F). Phosphorylated Smad 1/5/8 is quantified and normalized to Histone H3 (G) Phosphorylated Smad 1/5/8 is quantified and normalized to total Smad 1/5 (H). Protein was quantified from lysates from 3 animals of each genotype. A western blot shows that phosphorylated Smad 2 is not significantly different in Kcnj2^KO/KO E13.5 palate shelves compared to WT sibling controls (I). Phosphorylated Smad 2 is quantified and normalized to Histone H3 and is not significantly different between Kcnj2^KO/KO and WT controls. (J). Phosphorylated Smad 2 is quantified and normalized to total Smad 2 (K).