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First published online 12 October 2005
doi: 10.1242/dev.02078

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Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis

Fabio Santagati^*, Maryline Minoux^*, Shu-Yue Ren and Filippo M. Rijli

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, UMR 7104, BP 10142, CU de Strasbourg, 67404 Illkirch Cedex, France

View larger version (107K): [in a new window]   Fig. 1. Neural crest-specific Hoxa2 knockout. (A-C) In situ hybridization on wild-type (A), Hoxa2flox/flox (B) and Hoxa2flox/flox;Wnt1-Cre (C) whole-mount 10.5 dpc embryos using an antisense Hoxa2 probe. In C, Hoxa2 expression is selectively lost in the NC-derived mesenchyme of the second (arrow) and posterior branchial arches. (D-I) Middle ear (D-F) and hyoid (G-I) skeletal preparations from wild-type (D,G), Hoxa2–/– (E,H) and Hoxa2flox/flox;Wnt1-Cre (F,I) 18.5 dpc fetuses. Normal structures are indicated: MC, Meckel's cartilage; M, malleus; I, incus; T, tympanic bone; Go, gonial bone; Hy, hyoid bone, with lesser (LH) and greater (GH) horns. In F,I, Hoxa2flox/flox;Wnt1-Cre mutants show an identical phenotype to the conventional Hoxa2–/– mutants (Rijli et al., 1993), i.e. homeotic duplication of malleus (M2), incus (I2), tympanic bone (T2), partial duplication of Meckel's cartilage (MC2), transformation of gonial bone (Go*), and loss of lesser horns of the hyoid bone (asterisk in H and I).

View larger version (64K): [in a new window]   Fig. 2. Tamoxifen-induced deletion of the Hoxa2 locus. (A-F) In-situ hybridization on whole mounts (A,B) and frontal cryosections (C-F) of mouse embryos using an antisense Hoxa2 probe. Wild-type embryos at 10.5 (A), 13.5 (C) and 14.5 (E) dpc show strong Hoxa2 expression in the second (BA2) and posterior branchial arches (A), as well as in the neural tube (NT) and middle ear regions (C,E), respectively. Significant loss of Hoxa2 transcripts is observed in 10.5 dpc Hoxa2flox/flox;Cre-ERT2 embryos (i.e. carrying two alleles to be excised) after tamoxifen (TM) treatment at 9.5 dpc (B), as well as in 13.5 (D) and 14.5 (F) dpc Hoxa2del/flox;Cre-ERT2 embryos (i.e. carrying only one allele to be excised) after TM treatment at 11.5 and 12.5 dpc, respectively. (G,H) Immunostaining on frontal cryosections of Hoxa2EGFPfloxNeo;CMV-Cre (G) and Hoxa2EGFPfloxNeo;Cre-ERT2 (H) 13.5 dpc mouse embryos using an anti-GFP antibody. Note that the distribution of TM-induced GFP expression in H is comparatively similar to the constitutive GFP expression in G. OC, otic capsule; S, stapes.

View larger version (99K): [in a new window]   Fig. 3. Middle ear and hyoid skeletal changes in tamoxifen-induced Hoxa2 mutant mice. Middle ear (A-H) and hyoid (I-L) skeletal preparations from 18.5 dpc fetuses are shown. (B-G) Hoxa2flox/flox;Cre-ERT2 homozygous mutant fetuses in the absence (B) and presence (C-G) of TM treatment at different time points from 7.0 dpc up to 11.0 dpc, as indicated. Although untreated Hoxa2flox/flox;Cre-ERT2 mice have normal middle ear bones (B; compare with Fig. 1D), homeotic duplications comparable to those of the conventional Hoxa2–/– mutant (A) are observed in C,D. In E-G, the gonial bone (Go, arrow in E) is no longer transformed in fetuses treated from 9.5 dpc onwards, while the remaining cartilage and dermal bone elements are duplicated. By contrast, Hoxa2flox/flox;Cre-ERT2 fetuses treated at 11.5 dpc (H) do not show any duplication. In H, malformed second arch structures, including stapes (S) and styloid process (St), are often fused to additional ectopic cartilages (arrows), while duplication of dermal bones is not observed. Asterisks in J-L show the loss of the lesser horns (LH) of the hyoid bone in Hoxa2flox/flox;Cre-ERT2 18.5 dpc fetuses treated at 9.5 dpc (J), 11.0 dpc (K) and 11.5 dpc (L), similar to in conventional Hoxa2–/– mutants (Fig. 1H). These structures are present in untreated Hoxa2flox/flox;Cre-ERT2 mice (arrow in I).

View larger version (137K): [in a new window]   Fig. 4. Morphological changes of external ear pinna in conditional Hoxa2 mutant mice. (A-F) Analysis of the external ear phenotype in newborn mice. Lateral view of the head of wild-type (A), Hoxa2–/– (B) and Hoxa2flox/flox;Wnt1-Cre (C), as well as Hoxa2flox/flox;Cre-ERT2 (D) and Hoxa2del/flox;Cre-ERT2 (E,F) newborn mice treated with tamoxifen (TM) at various stages, as indicated between brackets. The arrows show absent (B-E) or hypoplastic (F) ear pinna in mutant mice, according to the stage of induction. P, pinna.

View larger version (77K): [in a new window]   Fig. 5. Molecular changes in the second branchial arch of conventional and tamoxifen-induced Hoxa2 mutant embryos. (A-L) Whole-mount in situ hybridization on wild-type (A,D,G,J), Hoxa2–/– (B,E,H,K) and TM-treated Hoxa2flox/flox;Cre-ERT2 (C,F,I,L) 10.5 dpc embryos using Alx4 (A-C), Bapx1 (D-F), Msx1 (G-I) and Six2 (J-L) antisense probes. Hoxa2flox/flox;Cre-ERT2 homozygous mutant embryos were collected 24 hours or 12 hours (inset in L) after TM treatment. Arrows show Alx4, Bapx1 and Six2 ectopic expression, as well as Msx1 downregulation in the second arch of both Hoxa2–/– and TM-treated Hoxa2flox/flox;Cre-ERT2 mutant embryos. (M-O) Whole-mount in situ hybridization on wild-type (M), Hoxa2–/– (N) and TM-treated Hoxa2flox/flox;Cre-ERT2 (O) 11.5 dpc embryos with a Six2 probe. The Hoxa2flox/flox;Cre-ERT2 mutant embryo in O was treated at 10.5 dpc. Arrows show ectopic expression in the second arch of both Hoxa2–/– and Hoxa2flox/flox;Cre-ERT2 embryos. BA1, first branchial arch; BA2, second branchial arch.