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Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest

Stanford J. Kwang1,*, Sean M. Brugger2,*, Arthur Lazik2,*, Amy E. Merrill2, Lan-Ying Wu2, Yi-Hsin Liu1, Mamoru Ishii2, Frank O. Sangiorgi1, Michael Rauchman3, Henry M. Sucov2, Richard L. Maas4 and Robert E. Maxson, Jr.2,{dagger}

1 Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA
2 Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center and Hospital, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA
3 Department of Medicine, Renal Division, School of Medicine, Washington University, St Louis, MO 63110, USA
4 Genetics Division, Department of Medicine, Brigham and Women’s Hospital and Harvard, Medical School, Harvard University, Boston, MA 02115, USA
* These authors contributed equally to the experimental work



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  		Fig. 1. Targeted inactivation of Msx2 but not of Msx1 suppresses the embryonic lethality of the homozygous Splotch genotype. Mice with targeted mutations in Msx1 or Msx2 were intercrossed with Splotch mutant mice as indicated in A. (B) A gross view of a newborn Msx2–/–; Pax3Sp/Sp pup. These mice typically had exencephaly (C), spina bifida (D) and craniofacial abnormalities including a foreshortened snout.

 


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  		Fig. 2. Targeted inactivation of Msx2 selectively rescues derivatives of the cardiac neural crest in Splotch mutant mice. We show a histological analysis of the influence of a targeted mutation in Msx2 on neural crest derivatives affected by the Splotch mutation. These included the cardiac outflow septum (A-I), the glossopharyngeal (IXth) ganglion (N-Q), dorsal root ganglia and sympathetic ganglia (R-U). The myocardium (J-L), though not derived from neural crest, is known to be affected by the Splotch mutation (Li et al., 1999). Note the lack of septation of the aortic and pulmonary channels in the Pax3Sp/Sp embryo (C) but normal septation in the Msx2–/–; Pax3Sp/Sp embryo (E). Note normal morphology of a Msx2–/–; Pax3Sp/Sp at the newborn stage, both at level of outflow vessels (I) and myocardium (L) compared with wild-type (G,J) and Msx2–/– (H,K) animals. Both the glossopharyngeal ganglion and the thoracic sympathetic ganglion are absent in Pax3Sp/Sp embryos (O,S). In contrast to the cardiac outflow septum, neither is rescued in Msx2–/–; Pax3Sp/Sp embryos (Q,U). The dorsal root ganglia, also reduced or absent in Pax3Sp/Sp (S), are partially rescued in Msx2–/–; Pax3Sp/Sp (U) embryos. IX, glossopharyngeal ganglion; a, aorta; aa, arch of aorta at site of entrance of ductus arteriosus; ap, aorticopulmonary trunk; drg, dorsal root ganglion; hm, hypaxial muscle; lv, left ventricle; p, pulmonary trunk; rv, right ventricle; sg, sympathetic ganglion. Scale bar: 500 µm.

 


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  		Fig. 3. Altered expression of Msx2 in Splotch mutant embryos. Embryos at E9.5 were allowed to hybridize with digoxygenin-labeled antisense probes against Pax3 (A,D,G,J) and Msx2 (B,C,E,F,H,I,K,L). (A,B,D,E,G,H,J,K) Wild-type embryos; (C,F,I,L) Pax3Sp/Sp embryos. Brackets indicate the region of the postotic hindbrain from which the cardiac neural crest originates. (D-F) are higher magnification views of hindbrain region of images shown in A-C. (G-L) Cross sections of embryos shown in A-C. Arrows to the right of D-F indicate the levels of the transverse sections. Sense probes did not hybridize detectably (not shown). Note the expansion of Msx2 expression in dorsal (arrowhead) and lateral (arrow) regions of postotic neural tube of Pax3Sp/Sp embryos (L). o, otic vesicle; r5, rhombomere 5. Scale bars: 100 µm.

 


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  		Fig. 4. Transgenic analysis identifies a 560 bp Pax3-responsive region within the Msx2 promoter. (A) Schematic maps of Msx2-lacZ transgene constructs. {Delta}1 and {Delta}2 contain a lacZ gene inserted in the first exon. {Delta}3 and {Delta}4 comprise the indicated promoter fragments fused to an hsp68 minimal promoter and lacZ reporter. (B-N) Effect of the Splotch mutation on transgene expression in E9-9.5 embryos. (B,C,L) Dorsal views of whole-mount preparations. Brackets indicate the approximate location of the cardiac neural crest. Note increased staining in the postotic hindbrain of {Delta}1Msx2-lacZ, {Delta}2Msx2-lacZ and {Delta}4Msx2-lacZ transgenes in Pax3Sp/Sp embryos. The embryos in L are at a slightly earlier stage than those in B,C, which accounts for the lower overall level of staining in the hindbrain and neural tube. Embryos in B and L were sectioned in the transverse plane. Shown below the whole mounts are sections (D,E,M,N) at the levels indicated by arrows to the right of whole mounts. ß-gal expression is imaged in dark field. Pink indicates a low to moderate signal, blue a more intense signal. Note the expansion of ß-gal expression into the dorsal (arrowhead) and lateral (arrow) regions of the neural tube of Splotch mutant embryos, mirroring the change in endogenous Msx2 expression (Fig. 3). (F,G) Longitudinal sections through the hindbrains of E9.5 {Delta}1Msx2-lacZ embryos. (H-K) Higher magnification views of the boxed regions in F,G. Note increased staining in both preotic crest and cardiac crest in Splotch embryos compared with wild type. cc, cardiac crest; n, neural fold; o, otic vesicle; pc, preotic crest; r5, rhombomere 5. Scale bars: 100 µm.

 


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  		Fig. 5. Mutation of a conserved Pax3-binding site causes upregulation of an Msx2 transgene in the postotic hindbrain. (A) Alignment of homologous regions of human and murine Msx2 5' flanking sequence. A stretch of 520 bp within the Pax3 responsive region matched closely (87% identity) with a region in the 5' flanking sequence of the human MSX2 gene. This stretch included a single, highly conserved Pax3 binding site (Pax site 1). We show the nucleotide sequence of Pax site 1 and surrounding DNA aligned with the human MSX2 locus. Also shown is a Pax3 site in the Tyrp1 promoter (Galibert et al., 1999), a high-affinity Pax3-binding site, Nf3' (Epstein et al., 1995) and the Pax3 consensus (Chalepakis and Gruss, 1995; Epstein et al., 1996). Pax site 1M is a base substitution mutant of Pax site 1. Bold letters show identity with the consensus sequence. (B) Protein titration EMSA analysis of Pax3 binding to Nf3', Pax site 1 and Pax site 1M. In vitro synthesized Pax3 protein (0.5, 1, 2 and 3 µl) was incubated with radiolabeled Nf3' (lanes 1-4), Pax site 1 (lanes 6-9) or Pax site 1M (lanes 12-14). Mock transcription-translation lysate (no DNA added) (1 µl) was used as a background control (lanes 5,10,11). Protein-DNA complexes were visualized by autoradiography. (C-F) Effect of Pax site 1 mutation on {Delta}4Msx2-hsplacZ transgene expression. Embryos carrying either the {Delta}4 or {Delta}4 Pax site 1M transgene were stained at E9.5 for ß-gal expression. (C,D) Dorsal views of whole mounts. The region of the neural tube from which the cardiac neural crest originates is indicated by brackets. The position of rhombomere 5 (r5) is shown to the left, the otic vesicle (o) to the right. Note elevated X-gal staining in the postotic neural tube of the Pax site 1 mutant embryo compared with the wild type. (E,F) Sections through the postotic neural tubes (arrows) of the embryos in (C,D). Note ventral and lateral expansion of X-gal stain in mutant (F) relative to wild-type (E) (arrows). Also note ectopic stain in ventromedial neural tube of mutant (F) (arrowhead). Scale bar: 100 µm.

 

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