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Requirement for endoderm and FGF3 in ventral head skeleton formation

Nicolas B. David¹, Laure Saint-Etienne¹, Michael Tsang², Thomas F. Schilling^3, and Frédéric M. Rosa^1,*,

¹ U 368 INSERM, Ecole Normale Supérieure, 46, rue d’ Ulm, F-75230 Paris Cedex 05, France
² Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, NIH, Bethesda MD, USA
³ Developmental and Cell Biology, University of California, Irvine CA 92697, USA
These two authors contributed equally to this work

View larger version (132K): [in a new window]   Fig. 1. Chondrogenic neural crest cells do not form pharyngeal cartilage elements in endoderm mutants. (A,C) (ventral views) Cartilages from 4-day-old embryos were stained with Alcian Blue, revealing the viscerocranium (green labels) and the neurocranium (red labels). Pharyngeal arch cartilages and trabeculae (arrowhead) are absent (B) or reduced (C) in cas and bon. (D,E) Transverse sections as indicated by dashed lines in A and B. Stars indicate the heart/hemicardia and arrowheads indicate arch cartilages. (F,G) radar expression in 48 hpf embryos (lateral view). (H-O) Expression of the marker dlx2. (H-M) Dorsal views. (H,I) Formation of the three streams I, II and III of dlx2-positive neural crest cells is initially normal in cas embryos. (J,K) At 24 hpf, stream III, which is already segmented in wild type, is no longer detectable in mutants. (L,M) At 30 hpf, expression of dlx2 in cas embryos is limited to stream I. (N,O) Lateral views. At 48 hpf, expression of dlx2 in cas embryos is restricted to a thin line posterior to the eye. (P-S) At 24 hpf, expression of fkd6 (P,Q) as well as Hu (R,S) appears unaffected in cas. b, branchial arches; e, eye; ep, ethmoid plate; f, fin; h, hyoid; m, mandibular; mo, mouth; no, notochord; ov, otic vesicle; pc, parachordal; t, trabeculae.

View larger version (88K): [in a new window]   Fig. 2. Fate of chondrogenic neural crest cells in wild-type and cas embryos. (A,B) Dorsal view of the head of live 24 hpf embryos. Note in cas an additional lobular structure (arrowhead). (C,D) Dorsal neuroectoderm and neural crests posterior to the otic vesicle were labelled by activation of DMNB-caged fluorescein (see Materials and Methods). By 28 hpf, neural crest stream III (white arrowheads) has initiated segmentation in wild-type but not in cas embryos (D). (E-H). Same as in C,D but DMNB-caged fluorescein was activated in precursors of streams II (black arrowhead) and III. Embryos were fixed at 28 hpf and processed for detection of fluorescein (E-H, orange) and dlx2 (G,H, purple). (E-H) Streams I, II and III are present but dlx2 expression is downregulated in stream III in cas embryos (H). (I-L) Dorsal views. hoxb2 (I,J) and hoxb3 (K,L) expressions at 28 hpf reveal the correct AP value of stream II and III, which adopt an ectopic lateral position in cas embryos. (M,N) Lateral view. 30 hpf embryos were labelled with Acridine Orange, revealing increased cell death in the region of the three cranial neural crest streams in cas embryos. (O-Q) These results were confirmed by TUNEL analysis and sectioning (Q). e, eye; ov, otic vesicle.

View larger version (61K): [in a new window]   Fig. 3. cas neural crest cells differentiate into cartilage in wild-type embryos. (A) Diagram of the transplant procedure. (B) At the 10-somite stage, a few FITC-labelled (green) premigratory neural crest cells were transplanted from a cas embryo into a wild-type host. (C) At 3 days, in similarly transplanted embryo, mandibular and hyoid cartilage can be seen close to the eye. (D) cas neural crest have contributed to these cartilages in a characteristic stack-of-penny arrangement. e, eye; h, hyoid; m, mandibular.

View larger version (93K): [in a new window]   Fig. 4. Restoration of endoderm rescues arch cartilage formation in cas embryos. (A) At blastula stage, wild-type cells expressing Tar* were transplanted to a cas host. (B) Side view of the head region at 30 hpf. Grafted cells (green) have differentiated into endodermal derivatives, here pharyngeal endoderm. The arrow indicates a forming pouch. (C) These pouches restored by grafted cells (black nuclei) were stained with the Zn5 antibody (brown). (D,E) At 4 days, grafted cas embryos (E) but not controls (D) develop arch cartilage elements revealed by Alcian Blue staining. Anterior to the left. (F) Horizontal section of a grafted cas embryo showing the differentiation of cartilage elements (blue) lined by endodermal pouches derived from transplanted cells (brown). Anterior to the top. (G) Close-up of the region containing grafted cells and rescued cartilage. (H,I) 30 hpf grafted cas embryos rescues dlx2 expression in posterior cranial neural crest cells (H, dorsal view, anterior left). (I) Cross section (indicated in H) showing that wild-type transplanted cells (brown) abut the cluster of dlx2-positive neural crest cells (blue). Note the absence of dlx2 expression on the control ungrafted side of the embryo. Dorsal to the top. b, branchial arch; h, hyoid; m, mandibular; no, notochord; nt, neural tube.

View larger version (128K): [in a new window]   Fig. 5. Expression of fgf3 in pharyngeal pouches. Dorsal (A-C) and lateral (D-G) views of embryos stained for fgf3 expression. Endodermal expression is indicated on one side (arrowheads and labelling). Anterior to the left. (I) The endodermal expression of fgf3 is absent in cas embryos. (J) Section of a 12-somite embryo showing expression of fgf3 in flat cell abbutting the yolk sack, thereby adopting the normal position and shape of endoderm cells. (K) Enlarged lateral view from the otic region. 24 hour-old embryos were double stained for fgf3 (blue) and dlx2 (red). fgf3 is expressed in the posterior part of posterior pharyngeal pouches (white dots) and abuts dlx2-expressing neural crest cells. ov, otic vesicle.

View larger version (109K): [in a new window]   Fig. 6. fgf3 is required in endoderm for the formation of posterior arches. (A-H) Alcian Blue staining of 4-day-old embryos. Ventral views, anterior to the left. (A-F) Wild-type embryos were either left untreated (A), immersed in Su5402 (B,C) or injected with morpholinos as indicated in lower right corner (D-F). (B) Su5402 prevents head cartilage formation, except for some very reduced anterior elements (arrowhead in C). (D,E) Cartilage in fgf4 morphants appears normal but fgf3 morphants do not develop branchial cartilage (except the fifth one, arrowhead), and develop a hyoid cartilage with inverted AP polarity. (F) Localisation of fgf3 morpholinos to anterior mesendodermal tissues is sufficient to disrupt branchial cartilage formation. The arrow indicates the injected side of the embryo. (G,H) Wild-type cells expressing Tar* and fgf3 morpholinos were grafted to cas embryos as in Fig. 4. At 4 days, compared to control cas embryos (G), grafted embryos developed mandibular and hyoid arch cartilages but not branchial cartilages. (I-N) Dorsal and lateral views, anterior to the left. Embryos were either injected with fgf3 morpholino or left untreated. In fgf3 morphants, cranial neural crest of stream III downregulates dlx2 expression (I,J). However pharyngeal endoderm differentiates normally as revealed by the expression of foxA2 (K,L) and the staining of pharyngeal pouches with Zn5 antibody (M,N).