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First published online 5 January 2005
doi: 10.1242/dev.01536

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Hoxa2 downregulates Six2 in the neural crest-derived mesenchyme

¹ Department of Developmental Biology, Max-Planck Institute of Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany
² Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal

View larger version (60K): [in a new window]   Fig. 6. Hoxa2 binds the Six2 promoter. (A) Schematic representation of the 900Six2 promoter: the black rectangle corresponds to the TATA box. Sequences showing 95% conservation between mouse and human are underlined in red, probes used in bindshift, in blue. (B) Hoxa2-HA binds to BstEII-SmaI probe, and the two retarded complexes (arrows) are supershifted by the anti-HA antibody (arrowhead). (C,D) Hoxa2-HA binds to probes 1 (C) and 2 (D). The addition of cold wild-type double-stranded oligonucleotides (wt1, wt2), but not of mutant oligonucleotides (m1, m2), competes the formation of the complexes (arrows). The sequence of the wild-type and mutant oligonucleotides and their relative position on the promoter are shown; red lowercase letters indicate the introduced nucleotide changes. Cold oligonucleotides were added at 250-fold (3,6) and 500-fold (4,5,7,8,) molar excess. (E) The incubation of Hoxa2-HA with labeled wild-type oligonucleotides results in the formation of the same specific retarded complexes (arrows, 2, 7), recognized by anti-HA antibody (arrowheads, 3, 8). No protein/DNA interaction is observed when Hoxa2-HA is incubated with mutant oligonucleotides (5, 10). (F) Pbx1a and Meis1 cooperate in binding to the proximal Six2 promoter. Pbx1a, Meis1 and Hoxa2 were incubated, separately or in combination, with the BstEII-SspI probe. Hoxa2-HA/DNA complex, black arrows; Pbx1a/DNA complex, arrowhead; Pbx1a/Meis1/DNA complex, red arrow. The position and the sequence of the putative Hoxa2 (blue rectangles) and Pbx/Meis (red rectangle) sites are indicated. The Pbx/Meis site was identified using Patch search at Biobase (www.gene-regulation.com).

View larger version (115K): [in a new window]   Fig. 1. Six2 is upregulated in the second branchial arch of Hoxa2-/- embryos. Whole-mount in-situ hybridization of wild-type (A-C) and Hoxa2-/- (D-F) embryos. (A,D) E10.5 embryos. Six2 is detected in the Hoxa2 mutant second arch (arrow) in a symmetrical location to its first arch counterpart. (B,E) E11.5 embryos. A small area expressing Six2 is visible in the wild-type second arch (arrowhead). The mutant second arch is equivalent to the first arch in terms of Six2 expression; ectopic Six2 expression is also detected close to the developing otic vesicle (white arrow). (C,F) E12.5 embryos. In the mutant, Six2 signal is increased around the otic vesicle (white arrow). (G,H) Expression of Sox9 in E12.5 wild-type (G) and mutant (H) arches marking the position of the cartilaginous condensations. Note the duplicated pattern in the mutant and its position relative to the first branchial cleft (asterisk). (I,L) Sox9 (I) and Six2 (L) probes were hybridized to adjacent sections of E11.5 branchial arches of the Hoxa2 mutant. Arrows indicate the expression domains of Sox9 and Six2 in the first arch and their duplication in the second arch. I, first arch; II, second arch; mx: maxilla; o: otic vesicle; asterisk: first branchial cleft.

View larger version (28K): [in a new window]   Fig. 2. The expression of other Six family members is not affected by Hoxa2. Semi-quantitative RT-PCR on RNA extracted from E10.5 second branchial arches of wild-type (+/+) and Hoxa2-/- (-/-) embryos using specific primers for Six1, Six2, Six4, Six5 and GADPH. The results shown were observed in three independent experiments.

View larger version (73K): [in a new window]   Fig. 3. Middle ear skeletal phenotype of a2-Six2 transgenics. (A) Schematic representation of the second (brown background) and first-arch-derived skeleton. (B) In the absence of Hoxa2, duplicated first arch elements derive from the second arch (brown). (C) Expression of Six2 in E10.5 wild-type embryos. Six2 mRNA is almost excluded from the second (black arrow) and more posterior arches. (D) Six2 is ectopically expressed in the second (white arrow) and more posterior arches and in the somitic mesoderm of a2-Six2 embryos. (E) Middle ear skeleton of an E18.5 wild-type embryo. The stapes is shown in the oval window (*) and after dissection. (F) Middle ear skeleton of a transgenic littermate. An ectopic cartilage, connected to the styloid process, extends to face the incus (black arrow); ventral view of the dissected styloid process, with the ectopic cartilage delimited by arrows, is shown on the right. The styloid process is thicker (arrowhead) and the manubrium of the malleus is curved (white arrow). The stapes, dissected and shown on the right, is incomplete. Malformation of the tympanic ring was observed only once. (G) Mirror image cartilages in the Hoxa2 mutant. Duplicated elements are marked with an asterisk. (H) Lateral view of a wild-type skull: orange arrow indicates the distal extremity of the lesser horn of the hyoid bone; the end of the styloid process is marked by a white arrow, the greater horn is also indicated. (I) In transgenic embryos, the lesser horn elongates and fuses to the styloid process, generating a continuous structure resembling a Meckel-like cartilage (white arrow). (J) Overexpression of Six2 does not affect Hoxa2 levels. Semi-quantitative RT-PCR on RNA extracted from E10.5 second arches of wild-type and a2-Six2 (tg) embryos, using specific primers for Six2, Hoxa2 and GADPH. g, greater horn; i, incus; l, lesser horn; m, malleus; s, styloid process; st, stapes; t, tympanic ring.

View larger version (81K): [in a new window]   Fig. 4. Hoxa2 is sufficient to repress Six2 in the facial mesenchyme. (A,B) In-situ hybridization with Six2 probe, in E10.5 wild-type embryo (A) and in E10.5 Msx2-Hoxa2 transgenic embryo (B). Six2 expression is downregulated in the maxilla (black arrow) and periocular mesenchyme (white arrow) of Msx2-Hoxa2 embryos. The corresponding embryo halves hybridized with the Hoxa2 probe are shown in the insets.

View larger version (52K): [in a new window]   Fig. 5. Identification of a promoter fragment responsive to Hoxa2. (A) The most proximal 900 bp of the Six2 promoter (-893; +37) are sufficient to drive lacZ expression in the proximal area of the first branchial arch, similarly to Six2 endogenous expression. (B) In the absence of Hoxa2, an equivalent staining to the first arch was observed in the second branchial arch (arrowheads) and in the mesenchyme proximal to the branchial arches (arrows), reproducing the Six2 expression pattern observed in Hoxa2 mutant embryos (see Fig. 1; white asterisk: first cleft). Higher expression of the transgene was also observed in the caudal area of the second branchial arch, where Six2 is not differentially regulated (white arrow). I, first branchial arch; II, second branchial arch.

View larger version (91K): [in a new window]   Fig. 7. Skeletal phenotype of transgenic embryos. Wild-type (A,D,F) and Six2 transgenic embryos (B,C,E,G) are shown. (A) Hyoid bone (black arrow), greater and lesser horn, and thyroid and cricoid cartilage of wild-type embryos. (B) The hyoid bone (black arrow) is malformed and fused to the thyroid cartilage. The greater horn is fused to the thyroid cartilage (arrowhead) and to the lesser horn. The thyroid and cricoid cartilages are thickened and the cricoid cartilage elongates ventrally (asterisk). (C) The hyoid bone (arrow) and the laringeal cartilages have fused in a disorganized structure. The rings of the trachea are also fused to each other. (D) The exoccipital bone and the atlas are separated by cartilage (arrow). (E) The corresponding area in transgenic embryos (arrow) displays fusion of the atlas to the exoccipital bone. (F) Ossification is almost completed in the supraoccipital bone (arrow). (G) In transgenic embryos the supraoccipital bone shows very reduced ossification (arrow points to the expected position for the supraoccipital bone). at, atlas; cr, cricoid cartilage; ex, exoccipital bone; g, greater horn; l, lesser horn; su, supraoccipital bone; th, thyroid cartilage.