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First published online 12 October 2005
doi: 10.1242/dev.02072

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Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest

¹ Department of Biochemistry and Molecular Biology, Norris Cancer Hospital, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089, USA
² Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA

View larger version (97K): [in a new window]   Fig. 1. Craniofacial dysmorphogenesis and skeletal abnormalities in Msx1/2 compound mutant mice. (A-E) Craniofacial morphology of control (A,C) and Msx1/2 double-mutant embryos (B,D,E) at E15.5. A and B show frontal views; C,D and E lateral views. Msx1–/–; Msx2–/– embryos exhibit exencephaly, a hypoplastic maxilla, cleft mandible, and defects in the fusion of the median lateral, nasal and maxillary prominences. Arrows in B, D and E indicate the whisker pad. (F-I) Alkaline phosphatase stain of coronal sections of control (F,H) and mutant (G,I) embryos. Boxed areas in F and G are enlarged in H and I. Msx1/2 mutants exhibit cleft palate (asterisk in G). Arrows in H and I indicate vibrissa follicles. Note, the mutant has divided whisker pads (brackets in I). (J-S) Skeletal analysis of Msx1/2 compound mutants. (J-Q) E18.5 Alizarin Red/Alcian Blue-stained bone and cartilage. (J,L,N,P) Normal controls; (K,M,O,Q) Msx1/2 mutants. (J,K,P,Q) Lateral views; (L,M) dorsal views; (N,O) ventral views. Note that a significant portion of the cranial skeletal components, including neural crest derivatives, is either missing or severely affected in mutant embryos. (P,Q) Fusion of hypoplastic mandible and maxilla is seen in the Msx1/2 mutant (Q). Note that tympanic ring was also hypoplastic in the mutant. (R,S) Lateral views of Alcian Blue-stained control (R) and Msx1/2 mutant (S) heads at E15.5. Note the parietal, supraoccipital and caudal processes of chondrocranium were absent in mutant. bo, basioccipital; bs, basisphenoid; c1, first cervical vertebra; e, eye; eo, exoccipital; fb, frontal bone; ip, interparietal bone; jg, jugal bone; ln, lateral nasal prominence; m, Meckel's cartilage; md, mandible; mn, median nasal prominence; mx, maxilla; na/nb, nasal bone; ns, nasal septum; oc, otic capsule; oe, outer ear; pb, parietal bone; pl, palatine; pmx, premaxilla; ps, palatal shelf; so, supraoccipital bone; sq, squamosal bone; t, tongue; tr, tympanic ring; ty, thyroid cartilage. Scale bars: 1 mm.

View larger version (121K): [in a new window]   Fig. 2. Defects in neural crest-derived cranial ganglia in Msx1/2 mutant embryos. (A-F) Whole-mount immunohistochemistry of neurofilament at E10.5. Control (A), Msx1/2 double homozygous (B,C,D), Msx1–/–; Msx2+/– (E), and Msx1+/–; Msx2–/– (F) embryos. (B-D) Double homozygous mutant embryos show a hypoplastic trigeminal nerve (V; arrows), and fused glossopharyngeal (IX) and vagus (X) nerves in the distal portion (asterisk). An abnormal connection between the trigeminal and facial (VII) nerves can be observed in Msx1/2 mutant embryos (arrowheads, B,C). Note the double-mutant embryo shown in D has impaired peripheral nerve growth and an interruption of the projection of the vagus nerve. The glossopharyngeal and vagus nerves were also fused in Msx1–/–; Msx2+/– and Msx1+/–; Msx2–/– embryos (asterisk, E,F). III, oculomotor nerve. Scale bar: 0.5 mm.

View larger version (129K): [in a new window]   Fig. 3. Cardiac outflow tract defects in Msx1/2 mutant embryos. Histological analysis of E12.5 (A,B) and E15.5 (C-H) control (A,C-E) and Msx1/2 mutant (B,F-H) embryo hearts. (A,B) At E12.5, the septation of the pulmonary trunk (PT) and the ascending aorta (Ao) is evident in the control (A), but not in the mutant (arrow in B). (C-H) Serial transverse sections from caudal to rostral show the aorta and pulmonary trunk arising from the right ventricle in the mutant (double outlet right ventricle). Msx1/2 mutants also exhibit dysmorphogenesis of the ventricular wall and myocardium. The location of the thymus (TH) was abnormal. RV, right ventricle; LV, left ventricle. Scale bars: in B, 0.5 mm for A,B; in E, 0.5 mm for C-H.

View larger version (83K): [in a new window]   Fig. 4. Altered expression of Ap-2 in the premigratory neural crest of Msx1/2 mutant embryos. (A-F) Whole-mount in situ hybridization analysis of pharyngeal arch development. Ap-2, Dlx5, and Twist expression was unaltered in pharyngeal arches in Msx1/2 mutant embryos (B,D,F) compared with controls (A,C,E). (G,H) Wnt1-Cre/R26R neural crest lineage tracer analysis at E9.5. There was no gross deficiency of neural crest in Msx1/2 mutants (H). Cardiac neural crest cells emigrate into the outflow tract (oft) in both control (G) and mutant (H). Reduced ß-galactosidase staining was evident in the peripharyngeal region (bracket) and in the stream of neural crest from r6 and r7 (arrowhead) in the mutant embryo. These differences may be due to the delayed timing of crest migration. (I-P) Analysis of premigratory and migratory neural crest development. Enlarged views of squares in I,J,M and N are shown in K,L,O and P. Expression of Ap-2 was significantly reduced in the neural fold caudal to the preotic sulcus in the mutant (arrowheads in L,P). (Q-T) In wild-type, the expression of Msx1 and Msx2 overlapped with that of Ap-2 at E8.5. pos, preotic sulcus; br1, first pharyngeal arch; br2, second pharyngeal arch. Scale bars: in B (for A,B), D (for C,D), F (for E,F), H (for G,H), J (for I,J), N (for M,N), S (for Q-T), 0.2 mm; in L (for K,L), P (for O,P), 0.1 mm.

View larger version (99K): [in a new window]   Fig. 5. Ectopic neural crest marker gene expression in the dorsal neural tube and mesenchyme cells adjacent to r3 of E9.5 Msx1/2 mutant embryos. (A-L) Dorsal views of embryos analyzed by whole-mount in situ hybridization. (A,B) Expression of Msx1 and Msx2 in E9.5 wild-type embryos. (C-L) Expression of the neural crest markers Ap-2 (C-F), Sox10 (G-J) and cdh6 (K,L) in control (C,E,G,I,K) and Msx1/2 mutant (D,F,H,J,L) embryos at the indicated stages. Note the aberrant expression of each marker at the level of r3 in mutant embryos (arrowheads in D,H,J,L). Intense ectopic expression of Ap-2 in the midline of the dorsal neural tube was also evident in mutant embryos (arrows in D,F). (M-P) Serial sections of Ap-2 stained whole-mount embryos. Ap-2 transcripts in the mutant are located in the mesenchyme adjacent to r3 (arrowhead in P). Arrows in N and P indicate ectopic Ap-2 expression in the dorsal neural tube of the mutant embryo. nc, neural crest; nt, neural tube; ot, otic vesicle. Scale bars: in B (for A,B), D (for C,D), H (for G,H), in L (for K,L), 0.2 mm: in F (for E,F), in J (for I,J), 0.4 mm; in O (for M-P), 50 µm.

View larger version (117K): [in a new window]   Fig. 6. Mispatterning and retarded migration of neural crest cells in E9.5-E10.5 Msx1/2 mutant embryos. (A-P) Lateral views of E9.5 (A-H) and E10.5 (I-P) embryos hybridized with digoxigenin-labeled RNA probes for Ap-2 (A-D,I-L), Sox10 (E,F,M-P) and cdh6 (G,H). Note the abnormal expression in mutants of each marker at the level of r3, as indicated by arrowheads (B,F,H,N). Also note the retarded production and migration of neural crest cells from rhombomeres 2, 4, 6 and 7 at E9.5 (B,D). Ap-2 and Sox10 expression in the trigeminal ganglion was significantly reduced (B,F,J,N arrows). In mutant embryos, merging of the migrating neural crest from r6 and r7 at E9.5 (D,F,H) results in the fusion of neural crest streams migrating into third and fourth arch arteries at E10.5 (asterisk in L,P). Boxed areas in I and J are shown at higher magnification in K and L. Scale bars: in A (for A,B), C (for C,D), F (for E,F), H (for G,H), 0.2 mm; in J (for I,J), L (for K,L), N (for M,N), P (for O,P), 0.4 mm.

View larger version (92K): [in a new window]   Fig. 7. Altered expression of Krox20 and Epha4 in Msx1/2 mutant hindbrain. Whole-mount in situ hybridization analysis of E8.5 and E9.5 embryos with the indicated probes. (A-F) Expression of Krox20 at E8.5 (A-D) and E9.5 (E,F). The box in A and B demarcates the region shown in C and D. Note the reduction in the level of Krox20 transcripts in the caudal portion of r5 in mutant embryo at E8.5 (B,D), but normal expression at E9.5 (F). (G-L) Expression patterns of Hoxb1 in r4 (G,H), Hoxd4 in the region caudal to r7 (I,J), and Crabp1 from r2 to r6 (K,L) at E9.5 show no apparent change in the mutant hindbrain. (M-P) Epha4 expression at E9.5. Enlarged view of rectangles in M and N is shown in O and P. Epha4 expression was significantly increased in r1, r2 and r3 of the Msx1/2 mutant (arrowheads in P). Scale bars: in B (for A,B), F (for E,F), H (for G,H), J (for I,J), L (for K,L), 0.2 mm; D (for C,D), 0.1 mm.

View larger version (93K): [in a new window]   Fig. 8. Altered expression of Bmp4 in Msx1/2 mutant embryos. (A,B) Wnt1 expression was maintained in hindbrain, dorsal neural tube, and isthmus in Msx1/2 double-mutant embryos. (C-F) Unchanged expression of Fgf8 in the median nasal prominence (mn), isthmus (is) and pharyngeal arch1 in Msx1/2 mutant (D,F) compared with control (C,E) embryos. Also note that expression of Fgf8 (F) and Tbx1 (H) in the peripharyngeal region was not significantly affected in the mutant (arrows). (I-N) Bmp4 was upregulated in the forebrain, cranial mesenchyme, body wall epithelium (arrows), maxillary prominence, distal first pharyngeal arch, and proximal second pharyngeal arch (arrowhead). (K-N) Section analysis of Bmp4 whole-mount embryos. Enlarged views of boxes in K and L are shown in M and N. The cranial mesenchyme and migratory neural crest (arrow in N) express Bmp4 highly in Msx1/2 mutants. Scale bars: in B (for A,B), D (for C,D), F (for E,F), H (for G,H), J (for I,J), 0.2 mm.

View larger version (85K): [in a new window]   Fig. 9. Msx1/2 are required for the survival of cranial neural crest cells. (A-F) Double-label immunostaining of apoptotic and proliferating cells. Immunostaining of phosphorylated histone H3 (Rhodamine, red) and TUNEL (FITC, green) were carried out on the same sections to detect cell proliferation and apoptosis in Msx1/2 mutants. Nuclei were counterstained with DAPI. Arrowheads indicate increased apoptotic neural crest-derived cells in the region of the trigeminal ganglion (B), the proximal portion of pharyngeal arch1 (maxillary prominence; D), and the distal tip of pharyngeal arch1 (mandiblular prominence; F) of Msx1/2 mutants at E9.5. Cell proliferation within these sites was not noticeably altered. (G-L) Nile Blue-stained E9.5 control (G,I,K) and Msx1/2 mutant (H,J,L) embryos; lateral (G-J) and dorsal (K,L) views. Increased cell death was detected in neural crest-derived craniofacial structures of the Msx1/2 mutant (arrows, arrowheads and open arrowheads in H and J) consistent with results of the TUNEL assay. We did not detect increased cell death in cardiac neural crest cells in mutant embryos (J). Note the lack of discernable change of cell death in the mutant hindbrain (L). Scale bar in L: 0.2 mm (for G-L).