Fig 1.
Loss of Gsα in NCCs results in severe craniofacial malformations.
(A, B) P0 Wnt1-cre;Gsαf/f mutant and control. Wnt1-cre;Gsαf/f mutant mice become cyanotic and die within hours after birth, exhibit domed skull, shortened maxilla and mandible and exposed tongue. (C-F) The gross appearance of Wnt1-cre;Gsαf/f mutants (C and E) and controls (D and F) are identical at E10.5 and E12.5 respectively. (G, H) E14.5 Wnt1-cre;Gsαf/f mutant and control. Wnt1-cre;Gsαf/f embryos show short snout, round face and hypertelorism. (I-L) E16.5 and E18.5 Wnt1-cre;Gsαf/f mutants (I and K) display exposed tongues and shortened maxilla and mandible compare to controls (J and L).
Fig 2.
Wnt1-cre;Gsαf/f mutants exhibit craniofacial skeleton defects.
(A-F) Neonates skeleton preparations of newborn Wnt1-cre;Gsαf/f mutant and control stained with alcian blue and alizarin red; dorsal (A, B), lateral (C, D) and ventral (E, F) views. A suture between frontal bones in control (asterisk in B), but craniosynostosis (A, arrowhead) inWnt1-cre;Gsαf/f mutant. (C-F) In Wnt1-cre;Gsαf/f mutant, the premaxilla(pmx), maxilla (mx) and mandible (ma) are hypoplastic and deformed, the nasal capsule cartilage (nc) is missing, the body of hyoid bone (b-hy) is over-ossified, and the tympanic rings are thickened and deformed. (G, H) The mandible is removed to enhance the view of palatal bone. Palatal bones are fused to form the secondary palate in control (H, dashed line), but severely hypoplastic and cleft in Wnt1-cre;Gsαf/f mutant (G, dashed lines and asterisk). (I-K) Dissected hyoid and laryngeal skeletons from E14.5 (I), E15.5 (J), and P0 (K) skeletal staining samples. Wnt1-cre;Gsαf/f mutants exhibit premature ossification in the body of hyoid bone at E15.5, and abnormal ossification in hyoid bone and thyroid cartilage at P0. (L-N) Dissected mandibles from the same embryos shown in Fig 2 I-K. Arrowheads in (L) and (M) indicate the abnormal ossification of incisor tip in Wnt1-cre;Gsαf/f mutants. The NCCs-derived mandible and tympanic rings are severely malformed, but the mesoderm-derived otic capsule is normal in P0 Wnt1-cre;Gsαf/f mutant (N). agp, angular process; b-hy, body of the hyoid bone; bo, basioccipital; cdp, condylar process; crp, coronoid process; eo, exoccipital; fr, frontal; gh-hy, greater horn of the hyoid bone; ip, interparietal; lh-hy, lesser horn of the hyoid bone; ma, mandible; mx, maxilla; na, nasal bone; nc, nasal capsule; oc, otic capsule; pmx, premaxilla; pr, parietal; rpMC, rostral process of Meckel’s cartilage; so, supraoccipital; th, thyroid cartilage; tr, tympanic ring.
Fig 3.
Wnt1-cre;Gsαf/f mutants exhibit cleft palate.
(A, B) Ventral view of palates after removal of mandible in P0 Wnt1-cre;Gsαf/f mutant and control. Fused palate in control (B), but cleft palate in Gsα knockout mice (A, arrow). (C-H) H&E staining of head coronal sections in Wnt1-cre;Gsαf/f mutants and controls from different embryonic stages. The palatal shelves grow vertically at two sides of tongue at E12.5 and E13.5 (C-F); and then the palatal shelves have been elevated horizontally above the tongue and fused with remnant medial edge epithelium in control at E14.5 (H, arrow), however they fail to fuse and a cleft was observed in Wnt1-cre;Gsαf/f mutant (G, asterisk), the initiation of palatal bone formation are indicated by arrowheads in (G and H). (I-L) The palatal shelves have completely fused with flat tongues and disappearance of midline epithelium in E16.5 and E18.5 controls (J and L), in contrast to the cleft palates with arched tongues in Wnt1-cre;Gsαf/f mutants (I and K). (M, N) In vitro organ culture shows Wnt1-cre;Gsαf/f mutant palatal shelves are able to fuse, asterisk indicates the disappearance of midline epithelium. P, palate; T, tongue.
Fig 4.
Conditional deletion of Gsα in NCCs does not affect neural crest migration or cell proliferation.
(A, B) Whole-mount X-gal staining of E9.5 and E14.5 embryos. Normal distribution of migratory neural crest cells inWnt1-cre;Gsαf/f mutants. FNP (asterisk), BA1 (arrow) and BA2 (arrowhead). (C, D) Immunofluorescent labeling (C) and quantification (D) of BrdU positive cells in coronal maxilla sections. At least thirty-five sections were randomly selected from six pairs of E13.5 Wnt1-cre;Gsαf/f mutants and controls. n.s, no significant difference. BA1, first branchial arch; BA2, second branchial arch; FNP, frontonasal prominence.
Fig 5.
Loss of Gsα in NCCs results in abnormal ossification.
(A-H) Von Kossa and nuclear red staining of heads coronal sections in Wnt1-cre;Gsαf/f mutants and controls from different embryonic stages. The aggregated mesenchymal cell in maxilla are similar between E12.5 Wnt1-cre;Gsαf/f mutant and control (arrows in A and B); however, the ossification region in E14.5 Wnt1-cre;Gsαf/f mutant is larger than that in control (arrows in C and D), and this phenotype are much more severe at later embryonic stages (arrows in E-H). (I-L) Von Kossa staining (I, J) and alcian blue staining (K, L) show the abnormal ossification and malformation of nasal septum cartilage in E17.5 Wnt1-cre;Gsαf/f mutants (arrows in I and K).
Fig 6.
Loss of Gsα in NCCs leads to accelerated osteochondrogenic differentiation.
(A) RT-qPCR analysis of relative mRNA levels in E16.5 isolated mandible and maxilla tissues. Data shown are normalized ratio of Wnt1-cre;Gsαf/f/Control (mean±SEM); student’s t-test; *p < 0.05; **p <0.01; ***p <0.001; n≥6. (B) Western blot analysis of protein expression of Gsα, Runx2 and Sox9 in E16.5 isolated mandible and maxilla tissues. Data shown are from 3 pairs of mutants and controls. (C) Alcian Blue and Alizarin Red staining show accelerated in vitro chondrogenic and osteogenic differentiation in Wnt1-cre;Gsαf/f mutant cells. Craniofacial mesenchymal cells from Wnt1-cre;Gsαf/f mutants and controls were subjected to osteochondrogenic differentiation in differentiation medium (complete medium supplemented with 10mM β-glycerophosphate, 50 μg/ml ascorbic acid and 2.5 μM retinoic acid) for indicated days. (D) Western blot analysis of protein expression of Gsα, P-CREB, CREB, P-Smad2 and P-Smad5 in E16.5 isolated mandible and maxilla tissues. Data shown are from 3 pairs of mutants and controls.
Table 1.
Table 2.