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doi: 10.1242/10.1242/dev.00241

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Cell autonomous requirement for PDGFR in populations of cranial and cardiac neural crest cells

Michelle D. Tallquist^*, and Philippe Soriano

Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
^* Present address: Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9148, USA

View larger version (90K): [in a new window]   Fig. 1. Pdgfra-/- cells contribute minimally to limb bud and cranial ectomesenchyme. (A-G,I) Pdgfra-/- ES cells injected into ROSA26+/- blastocysts; chimeras after staining for ß-galactosidase activity. (A-E) E10.5 chimeras that contain a high percentage of Pdgfra-/- cells phenocopy Pdgfra-/- embryos (A, CH1; B,C, CH2; D,E, CH3; see Table 1). (C,E) Transverse sections through chimeras shown in B and D at the cranial and cardiac crest level, respectively. (F) Transverse section through hindlimb bud of 50% mutant cell derived chimera (CH8). Inset shows a section through forelimb bud with predominantly Pdgfra-/- contribution to surface ectoderm (arrowhead) and wild-type contribution to the mesenchyme. (G,I) Transverse sections of branchial arches and head mesenchyme from chimeras generated by injection of Pdgfra-/- ES cells into ROSA26+/- blastocysts (CH11). (H,J) Transverse sections through similar regions to those in G and I for chimeras made by injection of Pdgfra-/-; ROSA26+/- ES cells into wild-type blastocysts (CH10). nt, neural tube; se, surface ectoderm; lb, limb bud; ov, otic vesicle; ba1, branchial arch 1; ba2, branchial arch 2; fb, forebrain. Arrows in G-J indicate regions of mesoderm-derived muscle progenitors. Arrowheads in C indicate location of occasional wild-type cells.

View larger version (53K): [in a new window]   Fig. 2. PDGFR conditional allele and analysis of Cre efficiency. (A) The wild-type PDGFR locus, the targeted floxed allele and the resulting allele after Cre-mediated inactivation of the PDGFR. The deleted region of genomic DNA in the fl allele is identical to that deleted in the PDGFR null allele R4 (Soriano, 1997). Black boxes represent exons. The floxed allele contains the neo cassette (gray box). Red arrowheads represent loxP sites, and the small black rectangle represents the probe used in Southern blot analysis. (B) Southern analysis of tissues from E13.5 PDGFRfl/fl; Wnt1Cre+ embryos. Genomic DNA was digested with NheI and probed with fragment indicated in A. Lane 1, DNA from yolk sac; lane 2, DNA from cranial tissues. (C,D) Transverse sections through first branchial arch region of E9 embryos stained for ß-galactosidase activity. (C) ROSA26R+/-; Wnt1Cre+; section was counterstained with nuclear Fast Red. (D) PDGFRfl/fl;Wnt1Cre+;ROSA26R+/-; section was not counterstained. Note that D contains a similar distribution of ß-galactosidase-expressing cells in the branchial arches when compared to the distribution of ß-galactosidase-expressing cells in C. (E) PDGFR cell-surface expression histogram. Cells from E10.5 embryos were isolated from the branchial arches, stained for PDGFR expression, and analyzed by flow cytometry. Red profile, cells stained with only secondary antibody; gray profile, cells from PDGFRfl/fl embryo; black profile, cells from PDGFRfl/fl;Wnt1Cre embryo.

View larger version (69K): [in a new window]   Fig. 3. PDGFRfl/fl; Wnt1Cre+ and Pdgfra-/- neonates and embryos. (A,B) External appearance of NCC conditional mutants after birth. Frontal and ventral view, respectively. The embryo in A is albino; therefore, it lacks pigment in the retinal epithelium. (C) E14.5, PDGFR embryo has extensive blebbing (arrowheads) and edema. (D) E13.5, PDGFRfl/fl;Wnt1Cre+ possesses the midline facial hemorrhage.

View larger version (96K): [in a new window]   Fig. 4. Cranial and frontonasal defects in PDGFRfl/fl;Wnt1Cre embryos. (A-D) Ventral views of skeletal preparations from E17.5 wild type and NCC conditional embryos. (C,D) The mandible was removed for visualization of the palate. as, alisphenoid bone; bo, basioccipital bone; bs, basisphenoid bone; h, hyoid bone; m, maxilla; ps, palatal shelf; th, thyroid cartilage. *Cartilage remnants potentially from the hyoid horns. Note blood vessels prominent in D because of filling with India ink. (E,F) Frontal view of E11.5 PDGFRfl/fl and PDGFRfl/fl; Wnt1Cre+ embryos, respectively. The frontonasal masses fail to meet at the midline.

View larger version (118K): [in a new window]   Fig. 5. Proliferation in craniofacial mesenchyme. (A,B) Transverse sections though frontal nasal process of E11.5 embryos. Punctate staining pattern indicates phosphohistone 3 positive cells. Scale bar: 200 µm.

View larger version (78K): [in a new window]   Fig. 6. Aortic arch defects in E18.5 embryos. (A) Corrosion cast of PDGFRfl/fl; Wnt1Cre-. (B-D) PDGFRfl/fl; Wnt1Cre+ embryos. (A) Architecture of normal aortic arch where the ascending aorta has three branches. The brachiocephalic artery bifurcates into the right subclavian (a) and right common carotid (b) arteries. The next branches are the left common carotid (c) and left subclavian (d) arteries. The ductus arteriosus (da) branches off the descending aorta (dAo) with the two pulmonary arteries (pa) branching from the pulmonary trunk. (B) Persistent truncus arteriosus (PTA) and a retro-esophageal subclavian artery. Arrow indicates to the ectopic origins of the right subclavian artery. Inset in B is a higher magnification and is taken from a slightly different angle, illustrating that the pulmonary arteries are emerging from the persisting truncus arteriosus. (C) Double outlet right ventricle and retro-esophageal right subclavian artery. The left common carotid artery was broken during manipulation. (D) Right subclavian artery originates from the proximal pulmonary trunk, where it bifurcates into the pulmonary arteries. The left pulmonary artery was broken during manipulation.

View larger version (55K): [in a new window]   Fig. 7. Ventricular septal defects. (A-C) Transverse sections through hearts of E14.5 PDGFRfl/fl; Wnt1Cre+ embryos. (A,B) Sections through region of membranous ventricular septum. (C) More caudal section through the heart of embryo in B but containing an intact muscular ventricular septum. a, atrium; s, septum; vsd, ventricular septal defect.

View larger version (141K): [in a new window]   Fig. 8. Vascular anatomy of the branchial arch arteries. (A-D) Intracardiac Indian ink injection of the left ventricle. All images are of the right side of the embryo. (A-C) E11.5 embryos. (D) Normal vessel architecture in a mutant at E9.5. I-IV, first, second, third and fourth branchial arch arteries, respectively; VI, sixth branchial arch artery. *A missing sixth arch artery. In C, the vessels are distended while in the control (A), the vessels have become narrow.

View larger version (86K): [in a new window]   Fig. 9. NCC fate mapping. E9.5 embryos carrying the ROSA26 Cre reporter were stained for ß-galactosidase activity. (A,B) Wholemounts. (A, left) PDGFRfl/+;Wnt1Cre+. (A, right) PDGFRfl/fl;Wnt1Cre+. (B) Higher magnification of the migrating NCCs in the PDGFRfl/fl;Wnt1Cre+ embryo. Arrows indicate NCCs that have migrated to form part of the peripheral nervous system. (C,D) Transverse sections through conotruncal area of (C) PDGFRfl/+;Wnt1Cre+ and (D) PDGFRfl/fl;Wnt1Cre+ embryos. Arrows indicate NCCs migrating into the truncus arteriosus. Abbreviations: s, aortic sac; ta, truncus arteriosus.

View larger version (93K): [in a new window]   Fig. 10. Mutant arch arteries contain normal numbers of endothelial and VSMC. Transverse sections through fourth arch artery region of E11.5 control (A,C) and mutant (B,D,E,F) embryos. Endothelial and VSMC of the branchial arch arteries were detected using antibodies directed against CD31 (PECAM; A,B,E) and smooth muscle actin (AMSA; C,D,F), respectively. The higher magnification images (E,F)are from different sections from those shown in B,D to illustrate that vessels appeared normal at many levels. Scale bars: in A, 200 µm for A-D; in E, 25 µm for E,F.