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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01197

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Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp^2H) mutant effect

¹ Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
² Neural Development Unit, Institute of Child Health, University College London, London WC1N 1EH, UK

View larger version (57K): [in a new window]   Fig. 1. (A) Drawing of the dorsal view of an E8.5 Institute of Cancer Research (ICR) mouse embryo with 5 somites. Labelling/graft sites (*) were located on the left side of the embryo at the lateral margin of the neural plate, where the neural tube had not yet completely closed [prorhombomere b (ProRhB), prorhombomere c (ProRhC) and at levels of somites 1-3], or in the dorsal part of the closed neural tube [at levels of somites 4 (S4) and 5 (S5)]. The neural crest of ProRhB was injected or grafted just rostral to the position of the otic placode. The definition of prorhombomeres follows Osumi-Yamashita et al. (Osumi-Yamashita et al., 1996). PreOs, preotic sulcus; PostOs, postotic sulcus. (B) Lateral view of an E8.5 ICR embryo immediately following grafting with a DiI-labelled neural crest fragment (arrow). (C) Dorsal view of an embryo 48 hours following grafting. The graft site is indicated by the arrow. Scale bars: 250 µm in B; 160 µm in C. ot, otic vesicle.

View larger version (69K): [in a new window]   Fig. 2. Lateral views (A-C,E) and transverse sections (D,F) of ICR embryos after 48 hours in vitro. (A) An embryo which was injected with DiI at prorhombomere b (ProRhB) (arrow marks injection site) appears normal at 48 hours. (B) In this embryo, a stream of DiI-labelled cells (white arrows) is observed extending from the labelling site (black arrow) to the second pharyngeal arch (2). (C) Following injection of another embryo in prorhombomere c (ProRhC) (black arrow), DiI-labelled cells (white arrows) are seen extending into the third pharyngeal arch (3). (D) A section through the level of the labelling site in C shows DiI-labelled cells (white arrows) in a dorsolateral pathway that extends from the dorsal side of the neural tube to the level of the developing pharynx (ph). (E) Grafting of a DiI-labelled neural crest fragment into ProRhC (black arrow), produces a narrow stream of labelled cells (white arrows) passing from the graft site to the third pharyngeal arch (3). (F) A section through the graft site of the embryo in E shows a similar distribution of labelled cells (white arrows) as in the DiI-injected embryo (C,D). Scale bars: 160 µm in A-C,E; 80 µm in D,F. da, dorsal aorta; ot, otic vesicle; ph, pharynx.

View larger version (18K): [in a new window]   Fig. 3. Percentage of ICR embryos with DiI-labelled cells in the cardiac outflow tract 48 hours after injection with DiI or grafting with a DiI-labelled neural crest fragment at different axial levels of the neural crest. n, number of embryos analysed.

View larger version (83K): [in a new window]   Fig. 4. Lateral views (A,B,D) and transverse section (C) of ICR embryos after 48 hours in vitro. (A,B) DiI-labelled cells (white arrows) are seen spreading from the labelling site (black arrow) to the third (3), fourth (4) and sixth (6) pharyngeal arches after injection at the level of the first somite (A), or after grafting with a DiI-labelled neural crest fragment at the level of the fourth somite (B). Some labelled cells are found in the aortic sac and cardiac outflow tract (oft) (arrowheads in B). (C) Section through the level of the graft site of the embryo in B confirms that some labelled cells (arrows) have reached the proximal end of the oft ventral to the pharynx (ph). (D) DiI injection of neural crest at the level of the fifth somite (black arrow) produces labelled cells in the foregut (arrowhead) but not in the pharyngeal arches nor any cardiac structures. Scale bars: 160 µm in A,B,D; 80 µm in C. nt, neural tube; ot: otic vesicle.

View larger version (151K): [in a new window]   Fig. 5. Transverse sections of ICR embryos through mid-prorhombomere c (ProRhC) (A,C) or somite 2 (B,D-I). Embryos were fixed at different time points after labelling at the 5-6 somite stage by WGA-Au injection (A-G,I) or grafting of a WGA-Au-labelled neural crest fragment (H). At the 7-somite stage, WGA-Au-labelled cells, which carry dark silver granules (arrowheads in A,B), are seen leaving the dorsal part of the neural tube and some labelled cells are found in the mesenchyme beneath the surface epithelium (arrows in A) or at the dorsal tip of the somite (arrows in B; so, somite). At 11 somites after labelling at ProRhC (C), labelled cells (arrows in C) are located in the dorsolateral pathway beneath the surface epithelium and lateral to the anterior cardinal vein (acv in C). At the same stage after labelling at the somite-2 level (D,E), labelled cells (arrows in D) are observed in the dorsolateral pathway, between somite and surface epithelium, and also in the medial pathway (arrowheads in D) between neural tube (nt) and the somite (so). Another embryo shows WGA-Au-labelled neural crest cells in the intersomitic pathway (arrows in E). By the 13-somite stage, labelled cells (arrows in F) are more ventrally located, in the region adjacent to the dorsal aorta (da). At 17 somites (G), some labelled cells (arrows in G) have migrated beyond the somite to the region lateral to the developing pharynx (ph). By 21 somites, labelled cells (arrows in H) have reached the ventral side of the pharynx. Note that the spatio-temporal distribution of the labelled cells following grafting of a piece of neural crest tissue (H) is very similar to that observed after WGA-Au injection (G), although the number of labelled cells is smaller after grafting than labelling. At 23 somites (I), no more labelled cells are seen emigrating from the dorsal part of the nt, which exhibits a smooth contour on its basal surface. Scale bars: 50 µm in A-H; 20 µm in I.

View larger version (108K): [in a new window]   Fig. 6. Transverse sections through ICR embryos with 26 somites (A) and 32 somites (B) after labelling with DiI at the level of the second somite. The most ventrally located labelled cells (arrows) are in regions lateral and ventral to the pharynx (ph) at 26 somites, and by 32 somites, labelled cells have reached the wall of the cardiac outflow tract (oft). Scale bars: 40 µm in A; 20 µm in B. nt, neural tube.

View larger version (32K): [in a new window]   Fig. 7. Number of WGA-Au-labelled cells in different regions of ICR and splotch mutant embryos at different somite stages following labelling of the neural crest in prorhombomere a (ProRhA) or prorhombomere b (ProRhB) (A-C) or at the level of the second somite (D-F). Note that the scale on the y-axis in D is different from other graphs. ICR embryos and wild type (+/+), heterozygous (Sp2H/+) and homozygous (Sp2H/Sp2H) embryos from splotch litters all show similar numbers of labelled cells in the trigeminal ganglia (A), the facio-acoustic ganglia (B) and the second pharyngeal arches (C) at the 9-, 12-13, 16-17 and 20-21-somite stage, following labelling of neural crest at ProRhA or ProRhB. In contrast, labelling of the neural crest at the level of the second somite produced significantly fewer labelled cells in splotch mutants in the following regions: medial pathway (i.e. sclerotomal mesenchyme between the somite and the neural tube) of the homozygous (Sp2H/Sp2H) embryos with 9, 12-13, 16-17 and 20-21 somites (D); peri-aortic mesenchyme of heterozygous (Sp2H/+) and homozygous (Sp2H/Sp2H) embryos with 12-13, 16-17 and 20-21 somites (E); and the mesenchymal region lateral to the developing pharynx of homozygous (Sp2H/Sp2H) embryos with 12-13 somites and heterozygous (Sp2H/+) and homozygous (Sp2H/Sp2H) embryos with 16-17 and 20-21 somites (F). The number of labelled cells at each time point was computed from at least 5 embryos (bars: s.e.m.). *Significantly different from the value for ICR embryos with the same somite number by Student's two-tailed t-test (P<0.05).

View larger version (110K): [in a new window]   Fig. 8. Transverse sections through wild type (+/+) (A,D,G,J), heterozygous (Sp2H/+) (B,E,H,K) and homozygous (Sp2H/Sp2H) (C,F,I,L) splotch embryos with 7-8 somites (A-C), 12-13 somites (D-F), 16-17 somites (G-I) and 20-21 somites (J-L) after labelling with WGA-Au at the level of the second somite. At 7-8 somites, labelled neural crest cells (arrows in A,B) are seen emigrating from the neural tube into the dorsal mesenchyme underneath the surface epithelium in both +/+ and Sp2H/+ embryos. In contrast, Sp2H/Sp2H embryos do not show any labelled cells in the same mesenchymal region (C). At 12 to 13 somites (D,E), more labelled cells can be seen migrating in the dorsolateral and medial pathways (arrows in D,E) in +/+ (D) and Sp2H/+ embryos (E). Some labelled cells have already reached the peri-aortic region. Sp2H/Sp2H embryos at the same stage (F) show fewer labelled cells in the cranial mesenchyme, which are mainly located dorsal to the dorsal aorta (arrow in F). At 16-17 somites, +/+ (G) and Sp2H/+ (H) embryos contain labelled cells (arrows in G,H) that have passed beyond the dorsal aorta and are localised lateral to the developing pharynx. In contrast, labelled cells in Sp2H/Sp2H embryos (arrows in I) are now present in regions lateral to the dorsal aorta. By 20 to 21 somites, labelled cells have traversed to the ventral side of the pharynx in both +/+ (J) and Sp2H/+ (K) embryos whereas, in Sp2H/Sp2H (L) embryos, labelled cells are located dorsolateral to the pharynx, but not at more ventral locations. Scale bar: 50 µm. da, dorsal aorta; nt, neural tube; ph, pharynx.

View larger version (117K): [in a new window]   Fig. 9. Transverse sections through recipient embryos 24 hours after receiving an orthotopic WGA-Au-labelled neural crest graft at the level of the second somite. Graft combinations (donorrecipient) are: (A,B) +/++/+; (C,D) Sp2H/Sp2H+/+; (E,F) Sp2H/Sp2H Sp2H/+; (G) Sp2H/Sp2HSp2H/Sp2H. Note that all grafts (outlined by arrowheads in A,C,E,G) successfully incorporated into the dorsal neural tube of recipient embryos. In the +/++/+ (B) and Sp2H/Sp2H+/+ (D) embryos, labelled graft-derived cells (arrows) can be seen migrating lateral to the dorsal aorta (da), reaching as far ventrally as the pharynx (ph). In the Sp2H/Sp2HSp2H/+ embryo (F), labelled cells (arrows) are migrating, but have not progressed beyond the dorsal aorta. Most strikingly, in the Sp2H/Sp2HSp2H/Sp2H embryo (G), only one labelled cell (arrow in G) has emerged from the graft, and has migrated only a short distance lateral to the neural tube (nt). Scale bars: 25 µm in A-C,E; 13 µm in D,F,G. da, dorsal aorta; nt, neural tube; ph, pharynx.