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- Fig. 1.
Ptx1 expression in the branchial area of wild-type, Hoxa2-/- and Msx2::Hoxa2 transgenics. (A-F) Ptx1 transcripts were detected in E9.5 (A,D), E10.5 (B,E) and E11.5 (C,F) wild-type (A-C) and Hoxa2-/- (D-F) embryos. In all cases, expression was detected in the first branchial arch (I). In the second arch (II) Ptx1 was detected only in the Hoxa2 mutant embryos after E10.5. (E, inset) Section through the second arch of a E10.5 Hoxa2-/- embryo showing that Ptx1 expression was localized to the mesenchyme. The asterisks in C and F indicate the location of the external acoustic meatus. (G-J) E10.5 wild type (G,H) and Msx2::Hoxa2 transgenics (I,J) were cut in halves. The right sides (G,I) were hybridized with a probe for Hoxa2 and the left sides (H,J) with a probe for Ptx1. In the first arches (arrowheads) Hoxa2 was expressed in the Msx2::Hoxa2 transgenic (I) but not in wild-type (G) embryos. The Ptx1 expression domain is larger in wild-type (H) than in Msx2::Hoxa2 transgenic (J) embryos.
- Fig. 4.
Lhx6, Fgf8, Hoxa2 and Bmp4 expression in the branchial area. (A,B) Expression of Lhx6 in E10.5 wild-type (A) and Hoxa2-/- (B) embryos. The arrow indicates the extra expression domain in the second arch mesenchyme beneath the rostral epithelium. The inset in B shows a section through the Lhx6-positive domain of the Hoxa2 mutant second arch, to demonstrate that expression is in the mesenchyme. (C,D) Expression of Fgf8 in E 10.5 wild-type (C) and Hoxa2-/- (D) embryos. The arrow indicates expression in the epithelium of the first cleft/pouch. (E) Expression of Hoxa2 in E10.5 embryos. The arrow indicates the strong expression in the mesenchyme beneath the cleft/pouch epithelium. (F) Expression of Bmp4 in E9.5 wild-type embryos. The arrow indicates the expression in the cleft/pouch.
- Fig. 2.
Regulation of Ptx1 expression by Fgf. Early E9.5 branchial arches were dissected out and incubated for 24 hours on isopore filters, and Ptx1 expression was determined by in situ hybridization (A-F,I,J), or apoptosis was determined by TUNEL (G,H). (A) Wild-type first arch mesenchyme incubated without epithelium. (B) Wild-type first (I) and second (II) arches incubated with epithelium. (C) Wild-type first and second arches incubated without epithelium in the presence of a bead soaked in PBS. (D) Wild-type first and second arches incubated without epithelium in the presence of a bead soaked in 1 mg/ml Fgf8. (E) Wild-type first and second arches incubated with epithelium in the presence of a bead soaked in DMSO. (F) Wild-type first and second arches incubated with epithelium in the presence of a bead soaked in 13.5 mM SU5402. There is only residual Ptx1 expression in the first arch. (G) TUNEL assay on wild-type first and second arches incubated with epithelium in the presence of a bead soaked in DMSO. (H) TUNEL assay on wild-type first and second arches incubated with epithelium in the presence of a bead soaked in 13.5 mM SU5402. (I) Hoxa2-/- first and second arches incubated with epithelium. (J) Hoxa2-/- first and second arches incubated with epithelium in the presence of a bead soaked in 13.5 mM SU5402.
- Fig. 3.
Fgf8 is required for mesenchymal Ptx1 expression. (A-C) Ptx1 expression was analyzed by in situ hybridization at E10.5 in wild-type (A) and Fgf8;Foxg1-cre mutant (B,C) embryos. (A,B) A whole-mount staining; (C) a section through the first branchial arch of a stained embryo. Although the ectodermal expression in the rostral first arch epithelium (arrows A-C) was conserved in the mutant, no mesenchymal expression (asterisks, A-C) was observed. (D) Dlx2 is expressed in the branchial arches and frontonasal mass of E10.5 wild-type embryos. Asterisk indicates the area where Ptx1 is also expressed. (E) In E10.5 Fgf8;Foxg1-cre mutant embryos, Dlx2 expression is still detected (asterisk), regardless of the smaller size of the first branchial arch (I). FN, frontonasal mass.
- Fig. 6.
Skeletal phenotype of Hoxa2-/-, Ptx1-/- single and Hoxa2-/-;Ptx1-/- double mutants. The base of the cranium (A-D), mandible (E-H), squamous bone (IL) and otic vesicle (M-P) of wild type (A,E,I,M), Ptx1-/- (B,F,J,N), Hoxa2-/- (C,G,K,O) and Hoxa2-/-;Ptx1-/- (D,H,L,P) mutants are shown. In the base of the cranium of Hoxa2-/- and Hoxa2-/-;Ptx1-/- embryos, an extra element associated with the basisphenoid was observed (white arrow in C,D). The mandibles of Ptx1-/- and Hoxa2-/-;Ptx1-/- embryos were smaller (F,H). The squamous bone of Hoxa2 and Hoxa2-/-;Ptx1-/- embryos showed an extra element (arrows in K,L). The styloid process (St) and the stapes (S) (which sits in the oval window) were not present in the Hoxa2-/- and Hoxa2-/-;Ptx1-/- embryos (white asterisks in O and P). At least four embryos corresponding to each genotype have been analyzed and showed similar phenotypes.
- Fig. 5.
Middle ear skeletal phenotype of Hoxa2-/-, Ptx1-/- and Hoxa2-/-;Ptx1-/- mutants. Stained middle ear skeletal elements from wild-type (A), Hoxa2-/- (B), Ptx-/- (C,E) and Hoxa2-/-;Ptx1-/- (D,F) were dissected out. (B) In Hoxa2-/- embryos the malleus (M), incus (I) and tympanic ring (TR) were duplicated in mirror image disposition (M*, I* and TR*). The gonial bone was abnormally extended (G*). (C) In Ptx1-/- embryos, the tympanic ring was slightly deformed and there was an extra ossified mass (asterisk). (D) In Hoxa2-/-;Ptx1-/- only the tympanic ring was not duplicated. The arrowhead points to a small chondrogenic mass associated to the duplicated malleus. (E,F) The same structures as in C and D but without the extra ossified mass. At least four embryos corresponding to each genotype have been analyzed and showed similar phenotypes.
- Fig. 7.
Hoxa2-/- phenotypic rescue in Hoxa2-/-;Ptx1-/- mutants. (A-C) Skeletal preparations of E16.5 wild-type (A), Hoxa2-/- (B) and Hoxa2-/-;Ptx1-/- (C) embryos to show the tympanic ring (TR), which is duplicated (TR*) in Hoxa2-/- and Hoxa2-/-;Ptx1-/- mutant embryos. (D-I) Histological analysis of the ear region of wild-type (D,G), Hoxa2-/-(E,H) and Hoxa2-/-;Ptx1-/- (F,I) newborns. Wild-type and Hoxa2-/-;Ptx1-/- double mutants have only one EAM (arrow) reaching to the tympanic ring (TR). Hoxa2-/- mutants have an additional EAM (arrowhead) associated with the duplicated ring (TR*). The insets in D and F show a small blind extension (arrowhead) close to the EAM. (G) The tympanic membrane is build up from the EAM (arrow), and epithelium of the middle ear cavity, which entrap the manubrium of the malleus (MM). (H) In Hoxa2-/- embryos, the tympanic membrane is distorted by the presence of the two EAMs (arrow and arrowhead). (I) The appearance of the tympanic membrane is quite normal in Hoxa2-/-;Ptx1-/- mutants. (J-L) Tongue phenotype of wild-type (J), Hoxa2-/- (K) and Hoxa2-/-;Ptx1-/- (L) newborns. In Hoxa2-/- mutants, there is a medial cleft (asterisk), which is lost in the double mutant. Note that the sections are at slightly different levels with respect to the hyoid bone (Hy) to show equivalent areas of the tongue. All sections are frontal. In D-H, lateral is towards the left. (D,E) More posterior areas than G,H. At least three embryos corresponding to each genotype have been analyzed and showed similar phenotypes.
- Fig. 8.
Cbfa1 expression in the branchial arches. Cbfa1 expression was analyzed by in situ hybridization on wild-type (A), Hoxa2-/- (B) and Hoxa2-/-;Ptx1-/- (C) E11.5 embryos. The asterisks indicate the location of the external acoustic meatus (in the first pharyngeal cleft). The arrow indicates the extra domain of Cbfa1 expression in the second arch (not seen in the wild-type embryo). 1st, first branchial arch.
- Fig. 9.
The relationship between Fgf signaling and Hoxa2. (A) Fgfs act on target cells to elicit a response (gray dots in the square). Active Fgf signaling blocks Hoxa2 expression; conversely, Hoxa2 blocks Fgf signaling. (B) When neural crest cells expressing high amounts of Hoxa2 are exposed to Fgfs, no response to the signal is obtained (white box); these cells remain Hoxa2 positive and Fgf unresponsive. (C) When neural crest cells expressing low (or no) amounts of Hoxa2 are exposed to Fgfs, responses to the signal are obtained (gray box). These signals will further reduce the Hoxa2 levels, thus increasing the responsiveness of the cells to Fgfs (black box). These cells will turn Hoxa2 negative and Fgf responsive.
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