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doi: 10.1242/10.1242/dev.00151

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Evolutionary conserved sequences are required for the insulation of the vertebrate Hoxd complex in neural cells

Marie Kmita, Basile Tarchini, Denis Duboule^* and Yann Hérault

Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland
Present address: Molecular and Experimental Genetics, FRE2358, CNRS, Institut de Transgénose, rue de la Férollerie, 3B, 45071, Orléans cedex 2, France

View larger version (16K): [in a new window]   Fig. 4. Combined deletions in cis. (A) Scheme of the posterior Hoxd complex, with the location of conserved regions X to XII. (B) The first combined deletion in cis was produced by meiotic recombination between the HoxdRXII and HoxdRXI alleles. Recombination (broken line) between the two loxP sites present in these alleles generated the HoxdRXII-del(13) allele (boxed), which carries a deletion of RXII as well as of the Hoxd13 locus containing RXI. (C) The second combined deletion in cis was produced by meiotic recombination between the HoxdRXII and HoxdRX alleles. Recombination (broken line) between the loxP sites present in these alleles generated the HoxdRXII-del(13-12) allele (boxed), which carries a deletion of RXII, as well as of both Hoxd13 and Hoxd12 loci, which contain both RXI and RX.

View larger version (74K): [in a new window]   Fig. 1. (A) Insulating activity within the Hoxd13 to Evx2 intergenic region. The posterior extremity of the Hoxd complex is shown, as well as the position of the Evx2 gene. The expression patterns of both Evx2 and Hoxd13 are depicted above to illustrate enhancer sharing in developing digits (right), whereas expression in the central nervous system (CNS) and spinal cord is detected only for Evx2 (left). The transcriptional orientation of this latter gene is opposite to that of all Hoxd genes (arrows). Enhancer sequences driving Evx2 in various domains of the developing CNS are located downstream the gene, i.e. 5' to the Hoxd cluster; hence, an insulating property is expected to lie between the two promoters (red bar). This was further supported by the relocation of a Hoxd9/lacZ transgene at different positions upstream the cluster (B). When relocated between Evx2 and Hoxd13 (right panel) the transgene was expressed in distal limbs but not in CNS. By contrast, when relocated downstream Evx2 (left panel), the transgene was expressed in both distal limbs and CNS, in a way much related to the Evx2 pattern, demonstrating that Hox promoters can indeed respond to these controls, if placed at an appropriate position.

View larger version (24K): [in a new window]   Fig. 2. Identification and targeted deletion of region XII (RXII). (A) Interspecies conservation within Evx2-Hoxd13 intergenic region. Sequence analyses revealed two stretches of significant conservation, referred to as region XII (RXII), which were found to be located within the insulating area (red bar). The position of these two sequences with respect to both Evx2 and Hoxd13 is schematised below for the mouse (m), chicken (ck) and zebrafish (zf). (B) Sequence alignment of region XII from mouse (m), chicken (ck) and zebrafish (zf) DNA. A high sequence similarity was observed between rodents and avian. The sequence conservation with the zebra fish DNA is less obvious, though significant whenever the respective positions of the two stretches are considered. (C) Strategy to delete region XII through targeted mutagenesis. A targeting vector was engineered lacking region XII and was recombined in ES cells to generate the HoxdRXII-neo mice. The selection cassette was further deleted after crossing these mice with a Cre deleter strain, to produce the HoxdRXII mice. In addition to the deletion of RXII, these mice also carried a loxP site at the exact integration site of the transgene shown under A. This loxP site was used for subsequent meiotic recombination approaches, as described in Fig. 4B,C.

View larger version (11K): [in a new window]   Fig. 3. Nested deficiencies of the posterior Hoxd complex, as produced by targeted meiotic recombination (TAMERE). At the top, the positions of the four regions (RIX to RXII) of high interspecies sequence conservation are show. The three deletions considered in this work are schematised below: Hoxddel(13), a deletion of the Hoxd13 locus including RXI; Hoxddel(13-12), a deletion of both Hoxd13 and Hoxd12 loci, including RXI and RX; and Hoxddel(13-11), a deletion of all three Hoxd13 to Hoxd11 loci, including all conserved sequences but RXII. In each case, a loxP site (red triangle) is left upstream RXII, at the position of the 5' breakpoint of the deletions.

View larger version (115K): [in a new window]   Fig. 5. Expression of Hoxd11 and Hoxd10 in HoxdRXII-del(13-12) animals. (A,B) Lateral and dorsal views, respectively, of an 11.5 dpc foetus analysed for Evx2 transcripts. A strong expression is detected in the developing digits, in a domain that is identical to the distal expression domain of Hoxd11 (D). Evx2 transcripts are also observed in columns of cells with the developing spinal cord, up to the posterior hindbrain (black arrowhead), as well as in a region encompassing the cerebellar anlage (black arrow) up to the isthmus. More rostrally, transcripts are found in the mesencephalon, (white arrow) (Dollé et al., 1994). (C) Control embryo of the same age hybridised with a probe specific for Hoxd11 RNA. None of this CNS domain is observed. (D-F) Ectopic expression of Hoxd11 (D,E) and Hoxd10 (F) in the CNS. In addition to the expected expression patterns in limbs and developing trunk (D), these latter genes show clear ectopic activation in domains virtually identical to those where Evx2 is expressed (D-F, compare with A and B), indicating that they now are under the control of Evx2 neural enhancers.