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First published online 22 February 2006
doi: 10.1242/dev.02289

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Krox20 hindbrain cis-regulatory landscape: interplay between multiple long-range initiation and autoregulatory elements

¹ INSERM, U784, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France.
² UMR CNRS 7622, Université Pierre et Marie Curie, 9 quai Saint-Bernard, 75252 Paris Cedex 05, France.

View larger version (60K): [in a new window]   Fig. 1. Identification of chick genomic regions containing Krox20 cis-regulatory elements. (A) Schematic representation of the chick Krox20 locus, BAC clones and subfragments tested by mouse transgenesis. Distances are relative to the start site of transcription of the Krox20 gene. The BACs (constructs 1 and 2) and the 65 kb fragment (construct 4) were injected alone and transgenic embryos were analysed by in situ hybridization with a chick Krox20 probe. The 42 kb and 30 kb fragments (constructs 3 and 5), which do not carry the Krox20 gene, were co-injected with a reporter fusion gene, Krox20/lacZ, and transgenic embryos were analysed for ß-galactosidase activity by X-gal staining. For each construct, the table indicates the number of E8.5 transgenic embryos obtained (n) and the number of embryos positive in r3 and r5 (r3/r5), or in the r3 to r5 region (r3-r5). (B-E) Dorsal views of embryos transgenic for the indicated constructs and analysed as indicated in A. Embryos are rostral side upwards. r, rhombomere; ov, otic vesicle.

View larger version (65K): [in a new window]   Fig. 2. Localization of separate elements carrying hindbrain cis-regulatory activities. (A) Schematic representation of the chick Krox20 locus and genomic fragments tested by in ovo electroporation and mouse transgenesis. All fragments, except fragments 10 and 13, were cloned upstream of the lacZ reporter gene driven by a human ß-globin minimal promoter. Fragments 10 and 13 were tested by transgenesis after co-injection with a reporter fusion gene, Krox20/lacZ. In all cases, transcriptional activity of the elements was evaluated by X-gal staining of the embryos. The table indicates the construct number, the presence (+) or absence (–) of activity in r3 and/or r5 in electroporated chick embryos (in ovo), the number of E8.5 transgenic embryos analysed (n) and the number of transgenic embryos expressing in r3 and r5 (r3/r5), the r3 to r5 region (r3-r5) or r5 only (r5). (B-I) Electroporated chick embryos (B-E) and transgenic mouse embryos (F-I), for the indicated constructs. r, rhombomere; ov, otic vesicle; ND, not determined.

View larger version (64K): [in a new window]   Fig. 3. Identification and evolutionary conservation of three Krox20 regulatory elements. (A) Homology plots between chick fragments 7, 12 and 14 and corresponding mouse sequences generated using the VISTA algorithm. The horizontal axis represents the chick sequences with a scale in kilobases and the vertical axis the percentage of homology between mouse and chick sequences in a window of 100 bp with a resolution of 7 bp. Only homology superior to 50% is shown. Genomic chick fragments containing the five conserved elements (A, 12.1, B, 12.3 and C) and indicated by the above bars were cloned upstream of the ß-globin promoter-lacZ reporter. Each construct was tested by in ovo electroporation and transgenesis in the mouse as indicated in Fig. 2. (B) The table indicates the construct names, the presence (+) or absence (–) of activity in r3 and/or r5 in electroporated chick embryos (in ovo), the number of E8.5 transgenic embryos analysed (n) and the number of transgenic embryos expressing in r3 and r5 (r3/r5), the r3 to r5 region (r3-r5) or r5 only (r5). (C-H) Transgenic mouse embryos (C-E) and electroporated chick embryos (F-H), for the indicated constructs. r, rhombomere; ov, otic vesicle.

View larger version (117K): [in a new window]   Fig. 4. Time-course analysis of the activities of elements A, B and C and requirement for Krox20. Transgenic lines carrying chick element A, B or C driving a ß-globin promoter-lacZ reporter were analysed in Krox20 null (A,E,I,M) or wild-type backgrounds (B-D,F-H,J-L,N-P). The embryos were stained with X-gal and the somite stage (ss) or the embryonic age in day post coitum (E) are indicated. (P) Embryo transgenic for construct cC-lacZ analysed by X-gal staining followed by in situ hybridization with a mouse Hoxb1 probe. r, rhombomere; ov, otic vesicle.

View larger version (70K): [in a new window]   Fig. 5. Identification of a functional vHNF1 binding site in element B. (A) Alignment of element B chick and mouse nucleotide sequences showing the presence of a putative vHNF1-binding site (boxed) within a highly conserved region. Conserved residues are indicated with a dash in the mouse sequence. The mutations introduced into the vHNF1 site are indicated above the box. (B) Bandshift analysis of wild-type and mutant chick elements B (cB). Extracts from control (c) or human vHNF1-expressing cells, in the presence or absence of an antibody against vHNF1 were used. The position of the specific complexes is indicated by a black arrow. The supershifted complex is marked by a white arrow. (C,D) Chick embryos analysed by X-gal staining after electroporation with constructs containing the wild-type (C) or mutant versions (D) of chick element B driving the ß-globin promoter-lacZ reporter. FP, free probe; ov, otic vesicle.

View larger version (84K): [in a new window]   Fig. 6. Identification of functional Krox20-binding sites in element A. (A) Alignments of chick and mouse nucleotide sequences of element A showing the presence of seven conserved putative Krox20-binding sites (boxed). Conserved residues are indicated with a dash in the mouse sequence. The mutations introduced by site-directed mutagenesis are indicated above each box. (B,E) Bandshift analysis of the wild-type chick element A (B), and a derivative carrying mutations in the seven Krox20-binding sites (E) using extracts from control (c) or Krox20-expressing bacteria. The positions of specific complexes are indicated with brackets. Specific complexes were identified by the addition of oligonucleotides carrying a high-affinity Krox20-binding site (wt) or a mutated version unable to bind the protein (mt). (C,D,F,G) Chick embryos analysed by X-gal staining after co-electroporation with constructs containing the wild-type (C,D) or mutant versions (F,G) of chick element A driving the ß-globin promoter-lacZ reporter together with the empty expression vector (C,F) or the Krox20 expression vector (D,G). FP, free probe; r, rhombomere; ov, otic vesicle.

View larger version (19K): [in a new window]   Fig. 7. Schematic representation of the different cis-acting elements controlling Krox20 expression in the hindbrain and derived neural crest. Within the Krox20 chick and mouse syntenic regions, the different cis-acting elements show an identical general organisation, although the distances relative to the Krox20 gene vary between species. In this scheme, elements B and C (in orange) are responsible for the initiation of Krox20 expression in r3 and r5, with a likely redundancy in r5, where vHNF1 participates in the activation of element B. This leads to accumulation of Krox20 that then activates element A (in blue), which is responsible for the maintenance of Krox20 expression in the neuroepithelium by positive autoregulation. In addition, the combined action of elements A, B and C is responsible for reaching a threshold level of Krox20 protein in the dorsal part of r5, which together with Sox10 initiates another positive autoregulatory loop, the latter involving the NCE element (in green), that maintains Krox20 expression in migrating r5-derived neural crest cells (Ghislain et al., 2003).