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doi: 10.1242/10.1242/dev.00581

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The role of chordin/Bmp signals in mammalian pharyngeal development and DiGeorge syndrome

Daniel Bachiller1,2,*,{dagger}, John Klingensmith3,4,*, Natalya Shneyder2, Uyen Tran1, Ryan Anderson3, Janet Rossant4 and E. M. De Robertis1

1 Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA
2 Victor Goodhill Ear Center, Head and Neck Surgery Division, University of California, Los Angeles, CA 90095-1794, USA
3 Department of Cell Biology, Duke University Medical Center, Durham, NC 272710, USA
4 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, M5G 1X5, Canada



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  		Fig. 1. Expression pattern and targeted homologous recombination of the Chrd gene. (A-F) Whole mount in situ hybridization with Chrd probe. (A) At E7, the expression domain of Chrd extends from the rostral limit of the notochord to the node. (B,C) As gastrulation progresses and the node moves posteriorly, Chrd expression in the newly formed axial mesendoderm is maintained. (D,E) After the embryo turns, Chrd transcripts are present in the dorsal endoderm adjacent to the notochord; the arrowhead indicates the level of the section in E. (F) By late E 8.5, the axial expression of Chrd has disappeared from most of the trunk and tail, but it is still strong in the pharynx, chordoneural hinge of the tailbud and postanal gut. (G) Schematic representation of the chordin locus (top), targeting construct (middle) and mutant allele (bottom). The approximate location of the four cysteine-rich repeats is indicated by boxes. The position of the primers used in the genotyping reactions (A,A',B,B') is indicated by arrows. The thick black bars indicate the location of the probe used to distinguish between recombination events occurring 5' or 3' of the stop codons placed at the SfiI site. (H) Southern blot analysis of genomic DNA from two targeted cell lines (E2 and D9) and wild-type, heterozygous and homozygous mutant embryos digested with EcoRV. The 17 kb band corresponds to the wild type allele. The 3 kb band results from an homologous recombination event 5' of the stop codons inserted at SfiI. (I) PCR analysis of embryos obtained from matings between heterozygous mice. (A,A') PCR amplifications used in the original ES cell screening. (B,B') Amplifications used to genotype the animals during the study. (J) In situ hybridization analysis of Chrd and Bmp4 expression in wild-type and Chrd-/- embryos. Both antisense probes were transcribed from full-length cDNA clones. The arrowhead indicates the lack of Chrd transcripts in the node of the mutants. The maintenance of normal Bmp4 expression in the extra-embryonic region of the embryos serves as an internal control for the in situ procedure.

 


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  		Fig. 8. Chrd regulates Tbx1 and Fgf8 expression in mutant mice and Xenopus ventral marginal zones. (A-E) Wild-type embryos. (A'-E') Mutant littermates. In mutants, the levels of Tbx1 expression are diminished at E7.5 (A,A'), E8.0 (B,B'), E8.5 (C,C') and E9 (D,D'). Arrowheads indicate the level of sections depicted in F-H'. At E9 Tbx1 transcripts are absent of otic vesicle (arrowhead in D'). At E9, Fgf8 expression in the mutant endoderm has disappeared and mesodermal expression is restricted to a small area in the most posterior part of the neck (arrowhead in E'). (F-H') Nomarski optic photographs of section through wild-type (F-H) and Chrd mutant littermates (F'-H'). Note the reduction of Tbx1 expression in the splanchnic (arrowheads) and somatic mesoderm (arrows), as well as in the mesenchyme of the head and peripharyngeal region. I, first pharyngeal arch; II, second pharyngeal arch; fp, frontonasal prominence; is, isthmus; pe, pharyngeal endoderm; sv, sinus venosus; bc, bulbus cordis; v, ventricular chamber; a, atrial chamber; fe, foregut endoderm; hd, hepatic diverticulum. (I) Xenopus VMZ assays. RT-PCR analyses of RNAs isolated from whole embryos (WE), dorsal marginal zones (DMZ), uninjected ventral marginal zones (VMZ co) and Chrd injected ventral marginal zones (VMZ chrd). Tbx1 and Fgf8 transcripts are high in the whole embryo and DMZ, but not in uninjected VMZ (lanes 1, 2 and 3, respectively). Lane 4 shows that Tbx1 and Fgf8 are induced in VMZs by Chrd injection. Note that the levels of endodermin (Edd), a pan-endodermal marker, are not affected. Ornithine decarboxylase (ODC) is expressed uniformly during embryonic development and serves as a loading control.

 


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  		Fig. 2. Gastrulation phenotype of Chrd-/- embryos. (A) Wild-type and (A') mutant embryos at early somite stage. In the mutant, the body is reduced and the allantois (al) proportionally enlarged. (B-D') Sections through wild-type (B-D) and mutant embryos (B'-D') at the levels indicated in A and A'. Note the poorly differentiated neural plate (np) of the mutant (B') and its lack of trunk mesoderm (C'). (D') The increase in extra-embryonic mesodermal cells in the allantois of the mutant. so, somite.

 


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  		Fig. 3. Morphological and histological analysis of Chrd-/- newborn mice. (A,A') External appearance of wild-type (A) and homozygous mutant (A') mice. The mutants appear cyanotic; their external ear is reduced and set closer to the eye than in wild type. (B,B') Sagittal sections of wild-type (B) and mutant (B') mice. In the mutant, the secondary palate (p) and thymus (t) are absent and the laryngeal cartilages (l) are severely reduced in size. The overall morphology and size of the central nervous system was not affected. (C,C') Coronal sections of wild-type (C) and mutant (C') mice at the level of the neck. Note the absence of the inner ear (ie) and oesophagus (oe), and the reduction in size of the trachea (tr) and thyroid (th). pi, pituitary gland.

 


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  		Fig. 4. Skeletal preparations of wild-type and mutant neonates. (A-E) Wild-type neonates. (A'-E') Mutant littermates. Bone is stained with Alizarin Red and cartilage with Alcian Blue. (A,A') Lateral view of the skull showing microcephaly and the lack of the squama temporalis (st) in the mutant (A'). (B,B') Tracheal and laryngeal cartilages in the wild type (B) and mutant (B'); th, thyroid; cr, cricoid cartilages; hy, Hyoid bone. (C,C') Lateral view of the mandibles; note the lack of the coronoid (cor), condylar (con) and angular (an) processes in the mutant (C') jaw. (D,D') Dorsal view of the base of the skull. as, alisphenoid; pl, palatine; ps, presphenoid; bs, basisphenoid; bo, basioccipital; tr, tympanic ring; oc, otic capsule. (E,E') Ventral view of the cervical vertebral column. The anterior arch of the atlas (aaa) is missing in the mutant and the ossification centres of the vertebral bodies (vb) are reduced.

 


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  		Fig. 5. Phenotype of Chrd-/- embryos at E14.5. (A,A') Skeletal preparation of wild-type (A) and (A') mutant littermates. Arrowheads in A' indicate underdeveloped vertebral neural arches. (B,B') Dorsal view of the base of the cranium. Arrows in B indicate the presence of the anterior notochord. Arrowheads in B' indicate the cartilaginous bridge that links the primordia of the basisphenoid (bs) and basioccipital bones (bo). oc, otic capsule; lc, laryngeal cartilages. (C,C') External view of wild-type (C) and mutant (C') animals. Note the severe oedema (arrowheads) and haemorrhage in the Chrd-/- embryo. (D,D') Wild-type (D) and Chrd-/- (D') mutant hearts. ao, aorta; pt, pulmonary trunk; ta, truncus arteriosus. (E-F') Coronal sections of wild-type (E,F) and mutant (E',F') embryos. In the thorax of the mutant (E') the undivided truncus arteriosus is clearly visible. In the mutant, an enlarged anterior spinal artery (asa, inset in F') is seen instead of a notochord (no). Note the striking reduction of the pharynx (ph) and the absence of the eustachian tube (eu) in the mutant. da, descending aorta.

 


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  		Fig. 6. Arterial defects in Chrd-/- newborns. Frontal (A-C) and posterior (D-F) views of the outflow tract and great vessels of wild-type (A,D) and two Chrd-/- (B,C,E,F) neonates. The auricles have been removed to facilitate observation. In the wild-type (A,D) the aorta (Ao) and the pulmonary trunk (Pt) are separate. The aorta begins at the left ventricle and turns to the left. The descending aorta (dAo) is located on the left side of the oesophagus (oe). The brachiocephalic artery (bc) branches from the right side of the aortic arch giving rise to the right common carotid (rcc) and the right subclavian arteries (rs). The left common carotid (lcc) and the left subclavian (ls) emerge directly from the aortic arch. (B,E) Mutant animal with left-turning aortic arch. The left and right common carotids originate in the truncus arteriosus (Ta). The brachiocephalic artery is absent and the right subclavian is abnormally located posterior to the oesophagus. The left (lpa) and right (rpa) pulmonary arteries arise from the most proximal part of the truncus. (C,F) Mutant animal with right-turning aortic arch. Forty percent of the mutants present abnormal right turning of the aorta. The descending aorta is placed on the right side of the oesophagus and the left subclavian runs posterior to it. Several vessels have been outlined to facilitate observation. rl, right lung; ll, left lung; lpv, left pulmonary vein; rpv, right pulmonary vein.

 


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  		Fig. 7. Pharyngeal defects in Chrd-/- embryos at mid-gestation. (A,A') External view of wild-type (A) and mutant (A') E9.0 embryos; mutants present a fully penetrant phenotype consisting in reduction of the otic vesicle (arrowheads), absence of second (hyoid) pharyngeal arch and a conspicuous indentation in the neck (arrow). (B,B') Whole-mount in situ hybridization of E9.5 embryos with a Sox10 probe that labels glial cells. The trigeminal (tr) and vestibulocochlear (vc) ganglia are deformed and displaced in the mutant (B'). (C,C') Pax3 whole-mount in situ hybridization of E10.5 embryos. Neural crest cells (arrowheads) migrating through the peripharyngeal region into the proximity of the heart (h) are absent in the mutant embryo (C'). md, mandibular component of the first pharyngeal arch; hy, hyoid or second pharyngeal arch; dm, dermomyotomes; fl, forelimb. Abnormal axonal projections from the trigeminal into the vestibulo-cochlear are indicated (arrowhead). The epibranchial placode-derived geniculate (g), petrosal (p) and nodose (n) ganglia are absent in the mutant. ov, otic vesicle; drg, dorsal root ganglia. (D-F') Whole-mount in situ hybridization with Pax9 probe. (D,D') Lateral view of E9.5 wild-type (D) and mutant (D') embryos made transparent with benzyl benzoate. Pax9 pharyngeal expression is reduced in the mutant. pe, pharyngeal endoderm; pg, postanal gut. (E,E') Dorsal view of the same embryos; in the mutant the pharynx is reduced and pharyngeal pouches II, III and IV are absent. (F,F') Lateral view of E10.5 wild-type and mutant embryos. Note the lack of Pax9 expression specifically in pharyngeal endoderm (pe) of the mutant. fm, facial mesenchyme; sc, sclerotome.

 

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