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First published online 11 September 2008
doi: 10.1242/dev.023614

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Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factors

Michel A. Wassef^1,2,*, Diane Chomette^1,2,*, Marie Pouilhe^1,2,*, Aline Stedman^3,4, Emmanuelle Havis^3,4, Carole Desmarquet-Trin Dinh^1,2, Sylvie Schneider-Maunoury^3,4, Pascale Gilardi-Hebenstreit^1,2, Patrick Charnay^1,2, and Julien Ghislain^1,2

¹ INSERM, U784, Laboratoire de Génétique Moléculaire du Développement and 46 rue d'Ulm, 75230 Paris, France.
² Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris, France.
³ CNRS UMR 7622, Laboratoire de Biologie du Développement, 75252 Paris, France.
⁴ Université Pierre et Marie Curie, 9 quai Saint Bernard, 75005 Paris, France.

View larger version (61K): [in this window] [in a new window]   Fig. 1. Identification of Hox/Pbx- and Meis-binding sites within element C. Part of the chick element C sequence is shown, with the different binding sites (horizontal arrows) and the mutations that have been introduced into each of them (vertical arrows). Gel retardation analyses were performed with the probes schematized underneath the gels and the protein extracts indicated above. A cross within a site indicates that it is mutated. The retarded complexes are indicated by brackets and the specificity of the binding was established by competition with oligonucleotides carrying high-affinity Hox/Pbx- or Meis-binding sites (Competitor) or mutated versions unable to bind these factors (Mut competitor). FP, free probe.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Synergistic activities of Hox, Pbx and Meis factors on element C. Chick embryos were analyzed by X-gal staining after co-electroporation with a lacZ reporter driven by element C and various expression vectors. (A-E) The following expression vectors were used at a concentration of 0.2 µg/µl: (A) empty expression vector, (B) Meis2, (C) Pbx1, (D) Hoxb1, (E) Hoxb9 expression vectors. (F-J) The following expression vectors were used alone or in combination at the indicated concentration: (F) empty expression vector (0.2 µg/µl), (G) Meis2 (0.1 µg/µl), (H) Pbx1 (0.1 µg/µl), (I) Hoxb1 (0.01 µg/µl), (J) Meis2 (0.1 µg/µl), Pbx1 (0.1 µg/µl) and Hoxb1 (0.01 µg/µl) expression vectors. In G-I, total plasmid concentration was brought to 0.2 µg/µl with empty expression vector. ov, otic vesicle.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Hox/Pbx- and Meis-binding sites are essential for element C activity. (A-D) Chick embryos were analyzed by X-gal staining after electroporation with reporter constructs driven by wild-type (A), HP1-site mutant (B), Meis-sites mutant (C) and HP2-site mutant (D) element C (schematized above). (E-I) Constructs driven by wild-type or mutant element C were used to generate mouse transgenic embryos that were analyzed by X-gal staining at around E8.5; the precise somite stage (ss) is indicated. (E) The total number of transgenic embryos obtained with each construct is summarized together with the numbers of embryos showing expression in the r3-r5 region, in r4 and r5 only, or in ectopic locations only. It should be noted that the level of reporter expression in r4/r5 is significantly reduced in the two positive embryos carrying the HP2 mutation. r, rhombomere.

View larger version (91K): [in this window] [in a new window]   Fig. 4. Specificity of Krox20 activation by Hox proteins. (A-D) Flat-mounted chick hindbrains in situ hybridized with a Krox20 probe after electroporation with constructs expressing Hoxa1 (A), Hoxb1 (B), Hoxa2 (C) or Hoxb9 (D), respectively. The hindbrains are shown rostral up and were electroporated on the left side. r, rhombomere.

View larger version (123K): [in this window] [in a new window]   Fig. 5. Overlap between Krox20 and Hoxb1 expression domains at the r3-r4 border. (A-C) Optical sections through the hindbrain of a whole-mounted Hoxb1GFP/+ mouse embryo at the 6-somite stage, immunolabelled for Krox20 (green) and Hoxb1-GFP (red): (A) anti-Krox20 immunofluorescence, (B) anti-GFP immunofluorescence and (C) merge. (D-G) Dorsal views of flat-mounted zebrafish embryos at the tail bud (D,E) or 1-somite (F,G) stages, hybridized with krox20 (red) and hoxb1a (blue). Black arrowheads point to double-labelled cells at the r3-r4 border. White arrowheads point to cells expressing krox20 at the level of prospective r5. E and G are higher magnifications of the outlined regions in D and F, respectively.

View larger version (69K): [in this window] [in a new window]   Fig. 6. Cell-autonomous activation of Krox20 by Hoxb1. (A-C) Flat mount, at the level of the mid-hindbrain boundary, of a chick hindbrain electroporated with a construct expressing HA-tagged Hoxb1, in situ hybridized for Krox20 and immunolabelled for Hoxb1-HA: (A) Krox20 staining, (B) anti-HA immunofluorescence and (C) merge. (D-F) Transverse sections, at the level of the forebrain-midbrain, of 5-somite stage vhnf1-/- embryos co-injected with meis1.1 and hoxb1aMyc RNAs, in situ hybridized for krox20 and immunolabelled for Hoxb1aMyc: (D) krox20 staining, (E) anti-Myc immunofluorescence and (F) merge. (G) ChIP of Hoxb1a-bound element C. Chromatin extracted from zebrafish embryos co-injected with meis1.1 and Myc-tagged hoxb1a RNAs was subjected to the ChIP procedure in the absence (first lane) or presence (second lane) of anti-Myc antibody. Immunoprecipitated DNA was analyzed by PCR using primers designed to amplify the core of element C (297 bp) or an unrelated sequence located -22.4 kb upstream of the transcription start site (422 bp). The third lane shows the PCR products obtained from the input chromatin.

View larger version (13K): [in this window] [in a new window]   Fig. 7. A model for the establishment of Krox20 expression in r3. (A-C) The model shows the evolution of the expression domains of Krox20 and of different established (Pbx, Meis2, Hoxa1/b1) or postulated (X) regulators at different stages of development (A, E7.5; B, E8; C, E8.5). The overlap between the Hoxa1/Hoxb1 and X domains results in a narrow strip of cells activating Krox20 and transiently co-expressing PG 1 genes. These cells then shut down PG 1 gene expression, but maintain Krox20 expression by direct and indirect autoregulatory loops, and r3 extends rostrally by the recruitment of neighbouring cells by non cell-autonomous autoregulation. As the X domain extends posteriorly, the strip of co-expression of Krox20 and PG 1 genes moves caudally, leading to the progressive extension of r3 at the expense of r4. In the rest of r4, Krox20 is repressed by mechanisms downstream of Hoxa1/Hoxb1. See Discussion for further details. The potential role of Hox PG 2 factors in initial activation is not represented. Expression domain extensions are indicated by coloured arrows. Positive and negative regulations are symbolized by arrows and barred lines, respectively. Direct and indirect regulations are indicated by plain or dotted lines, respectively. r, rhombomere; pr, prospective rhombomere.

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