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First published online 5 March 2008
doi: 10.1242/dev.017624

Development 135, 1463-1470 (2008)
Published by The Company of Biologists 2008
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Six2 functions redundantly immediately downstream of Hoxa2

Eva Kutejova1, Bettina Engist1, Michelle Self2, Guillermo Oliver2, Pavel Kirilenko3 and Nicoletta Bobola1,3,*

1 Department of Developmental Biology, Max-Planck Institute of Immunobiology, Freiburg, Germany.
2 Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA.
3 Faculty of Human and Medical Sciences, Stopford Building, The University of Manchester, Manchester M13 9PT, UK.


Figure 1
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  		Fig. 1. Hoxa2 binds to the Six2 promoter in vivo. (A,B) Western blot using anti-HA (A) and anti-Hoxa2 polyclonal antibody 43 (B) on whole extracts of human 293 cells transfected with empty vector (control), pCDNA3-Hoxa2-HA (Hoxa2-HA) or pCDNA3-Hoxb2-HA (Hoxb2-HA). Arrows indicate the expected position of Hoxb2-HA. (C) Side view of the facial region of an E11.0 mouse embryo, showing the areas isolated for ChIP (red, maxillary component of first arch and frontonasal mass; blue, second arch). (D) Schematic of the Six2 genomic locus around the transcriptional start site (+1), with red boxes indicating the relative position of the two Hoxa2 binding sites identified in vitro (Kutejova et al., 2005Go) and gray arrows indicating the position of the primers used for PCR amplification. (E) PCR amplification of the immunoprecipitated chromatin from E11.5 second branchial arch (2nd) or from frontonasal mass and first branchial arch (1st) using anti-Hoxa2 polyclonal antibodies 43 and 44 or normal rabbit IgG. (F) Same experiment as in E, using polyclonal anti-polymerase II antibodies to control for the quality of first arch chromatin: first arch chromatin is enriched for the Six2 proximal promoter fragment, as expected for a gene actively transcribed in this area (Oliver et al., 1995Go). (G) PCR amplification with Six2 or IP10 (Cxcl10; control) primers of E10.5 second branchial arch (2nd) chromatin, immunoprecipitated using anti-Hoxa2 antibody 43, or normal rabbit IgG. The number of PCR cycles is indicated in the bottom-right corner. EB, elution buffer. ChIP was performed on three independent pools of samples, and PCRs were performed in duplicate on each pool. Results shown are from a representative set.

 

Figure 2
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  		Fig. 2. Pbx1 binds the Six2 promoter in vivo. (A) PCR amplification (39 cycles) of the immunoprecipitated chromatin from E11.5 frontonasal mass and first branchial arch (1st) or from second branchial arch (2nd) using anti-Pbx1 antibody or normal rabbit IgG. EB, elution buffer. ChIP was performed on two independent pools of samples, and PCRs were performed in duplicate on each pool. Results shown are from a representative set. (B,C) Western blot using anti-Pbx1 on whole extracts of human 293 cells transfected with pCDNA3-Pbx1a (Pbx1a) or pCDNA3-Pbx1b (Pbx1b), or whole extracts from frontonasal mass (FNM), first branchial arch (1st ba) and second branchial arch (2nd ba) of E11.5 embryos. (D) Ponceau staining of the membrane shown in C. Arrowhead, position of Pbx1a; arrow, position of Pbx1b.

 

Figure 3
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  		Fig. 3. Regulation of the Six2 promoter by Six proteins. (A,C) E11.5 and E12.5 wild-type mouse embryos show Six2 promoter (-893 to +37)-driven lacZ expression in the maxilla (m), first branchial arch (arrows), second branchial arch (arrowhead) and limbs (l), recapitulating Six2 endogenous expression. (B,D) E11.5 and E12.5 Six2 mutant embryos show unchanged staining in these embryonic areas in the absence of Six2. (E) Nucleotide sequence of the proximal Six2 promoter, with Six-binding sites (highlighted in gray) and the sequence of Hoxa2 binding sites (red) indicated. The identification of Six-binding sites is based for site 1 on the Six-binding consensus and footprinting analysis (Spitz et al., 1998Go) (N.B. and E.K., unpublished) and for site 2 on the Six-binding consensus, in vitro binding and functional analysis (Spitz et al., 1998Go; Brodbeck et al., 2004Go) (N.B. and E.K., unpublished). Numbers indicate nucleotide positions relative to the transcriptional start site (+1). (F) Unchanged Six2 binding in the presence of Hoxa2. Six2 was translated in rabbit reticulocytes in vitro and incubated with a labeled Six2 promoter fragment (-181 to -48). Increasing amounts of pcDNA3-Hoxa2 programmed reticulocytes were added to the binding reaction mix, keeping the total amount of extract constant in each binding mix by adding unprogrammed reticulocytes. Arrow, the Six2-probe complex; arrowhead, the Hoxa2-probe complex. No ternary complex was observed; however, addition of Hoxa2 did not perturb Six2 binding to the probe.

 

Figure 4
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  		Fig. 4. Middle-ear skeletal phenotype of Hoxa2-/-; Six2-/- and Hoxa2-/-;Six2+/- mutant mice. (A-E) Dissected middle-ear skeleton from wild-type (A), Hoxa2-/- (B), Hoxa2-/-; Six2-/- (C,D) and Hoxa2-/-; Six2+/- (E) E18.5 embryos. (A) Malleus (m), incus (i), gonial bone (g) and tympanic ring (t) are derived from first arch; stapes (s) is second arch-derived. (B-E) In the absence of Hoxa2, the stapes disappears and duplicated first arch elements form in the second arch (asterisks). The gonial bone abnormally extends to connect the wild-type tympanic ring with its duplicated counterpart. (C) Removal of Six2 rescues the ectopic growth of the gonial bone. The malleal duplication is also reduced (compare double-ended arrows in B and C). (D) The extent of the rescue of the ectopic growth of the gonial bone in Hoxa2-/-; Six2-/- was variable, but the ectopic gonial bone, when present, never extended to reach the duplicated tympanic ring (arrowhead), as invariably observed in Hoxa2-/- embryos (arrowhead in B). (E) Complete reversal to a wild-type gonial bone was observed also in Hoxa2-/-; Six2+/- embryos.

 

Figure 5
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  		Fig. 5. Expression of Igf1 and Igfbp5 in wild-type and mutant mouse embryos. (A-F) In situ hybridization on whole-mount E11.5 wild-type (A,C,E), Hoxa2 mutant (B,D) and a2-Six2 transgenic embryos (F) using Igf1 (A,B) and Igfbp5 (C-F) probes. The arrow in A and B indicates the embryonic area where Igf1 is upregulated in the mutant. The arrow in C and D indicates the domain where Igfbp5 expression in the Hoxa2 mutant is lost. In E and F, arrows and arrowheads indicate areas of Igfbp5 expression in the second branchial arch; note the reduced Igfbp5 expression in the second branchial arch of embryos overexpressing Six2. I, first branchial arch; II, second branchial arch. (G) Semi-quantitative RT-PCR on RNA extracted from E10.5 second branchial arches of wild-type (wt) and a2-Six2 (tg) embryos using primers specific for Six2 and Gapdh.

 

Figure 6
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  		Fig. 6. Six2 binds the Six5 binding site identified in the mouse Igfbp5 promoter. (A) Incubation of the wild-type oligonucleotide probe in the presence of unprogrammed reticulocytes does not result in the formation of a specific retarded complex. Incubation of the probe with Six2-programmed reticulocytes gives rise to a retarded complex (arrow), which is competed by the addition of cold double-stranded wild-type oligonucleotide (wt), but not by oligonucleotide with a nucleotide substitution in the Six5 binding site (m). Cold oligonucleotides were added at 100- and 500-fold molar excess. (B) Incubation of the labeled mutant oligonucleotide does not result in the formation of a specific complex in the presence of Six2-programmed reticulocytes. (C) Sequence of wild-type and mutant oligonucleotides. The nucleotide changed in the mutant oligonucleotide is highlighted in red.

 

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© The Company of Biologists Ltd 2008