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First published online 5 February 2008
doi: 10.1242/jcs.023234

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Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd

¹ MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh University, Crewe Road, Edinburgh EH4 2XU, UK
² Institut National de la Transfusion Sanguine (INTS), 6 rue Alexandre Cabanel, 75015 Paris, France
³ Laboratory of Genetics and Development, Institut de Recherches Cliniques de Montréal (IRCM), Université de Montréal, 110 avenue des Pins Ouest, H2W 1R7, Montréal, Quebec, Canada
⁴ Department of Zoology and Animal Biology and National Research Centre `Frontiers in Genetics', University of Geneva, Sciences III, and School of Life Science, Ecole Polytechnique Federale, Lausanne, Switzerland

View larger version (60K): [in this window] [in a new window]   Fig. 1. Looping out of Hoxb1-LacZ from the chromosome territory occurs in the primitive streak (PS) of E7.5 transgenic embryos. (A) Map of the Hoxd locus on MMU2 showing the structure and the integration site of the Hoxb1-LacZ transgene. The extent of the homology arms used for recombination is shown in red and the Hoxb1 cis-regulatory retinoic acid response elements (RAREs) and the rhombomere r4 enhancer included in the transgene are depicted (Kmita et al., 2000). Exons of Hoxd genes from Hoxd1 (d1) to Hoxd13 (d13) are shown as unfilled boxes; other genes in the region are shown as black boxes. The orientation of transcription is indicated by arrows underneath the genes. The grey oval locates a region of noncoding sequence conservation that overlaps the global control region (GCR) (Spitz et al., 2003). The locations of the BACs and the Hoxb1-LacZ plasmid used as FISH probes are shown in grey. (B) DAPI staining of a 4 µm E7.5 embryo section used for the FISH analysis showing the nuclei of the PS and extra-embryonic yolk sac mesoderm (EEM). (C) Maximal projection image after deconvolution of 3D FISH using the Hoxb1-LacZ probe (red) hybridised together with a MMU2 chromosome paint (green) on DAPI-counterstained nuclei of the EEM or the PS of E7.5 Hoxb1-LacZ embryos. Bar, 2 µm. (D) Histograms showing the 3D position of hybridisation signals for the Hoxb1-LacZ transgene relative to the MMU2 chromosome territory (CT) edge (black bars), or, for endogenous Hoxb1, relative to the edge of the MMU11 CT (grey bars) in nuclei from the EEM and PS of E7.5 transgenic or wild-type (wt) embryos, respectively. Loci are defined as outside of the CT if the distance measured is > 0.4 µm beyond the visible limits of the CT hybridisation signal.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Hoxb1-LacZ nuclear reorganisation at E7.5 does not spread to flanking regions. Histograms showing the 3D positions of hybridisation signals for (A) the Lnp and (B) the Hoxd probe in nuclei from the EEM and the PS of wild-type (endogenous Hoxd, white bars) or transgenic (black bars) E7.5 embryos.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Chromatin decondensation in E7.5 transgenic embryos. (A) 3D DNA-FISH of: (left panel) Lnp and Hoxd, (middle panel) Hoxb1 and Hoxb9 probes on nuclei from the PS of E7.5 wild-type embryos, and of (right panel) Hoxb1-LacZ and Hoxd probes on nuclei from the PS of an E7.5 Hoxb1-LacZ embryo. Nuclei were counterstained with DAPI. Images are maximal projections of 3D stacks after deconvolution. (B) Distributions of mean-squared interphase distances (d2 in µm2) normalised to genomic separation (in Mb) measured between the Hoxb1 and Hoxb9 probes (dark-grey bars) at endogenous Hoxb and between Lnp and Hoxd probes (white bars) at endogenous Hoxd in EEM and PS nuclei from E7.5 wild-type embryos, or between the Lnp and Hoxb1-LacZ probes (light-grey bars) and between the Hoxb1-LacZ and Hoxd probes (black bars) in EEM and PS nuclei from E7.5 Hoxb1-LacZ embryos.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Nuclear reorganisation and chromatin decondensation in the limb bud of E9.5 Hoxb1-LacZ embryos. (A) Histograms showing the percentage of Lnp, Hoxb1-LacZ or Hoxd hybridisation signals inside, at the edge or outside of the MMU2 CT in the forelimb bud of E9.5 Hoxb1-LacZ embryos (black bars) compared with the endogenous Hoxd (white bars). The cut-offs used for the edge category are 0.4 µm and –0.4 µm from CT edge. (B) Distributions of squared interphase distances (d2 in µm2) standardised to genomic separation (in Mb) measured between Lnp and Hoxd probes (white bars) at endogenous Hoxd in forelimb bud nuclei from E9.5 wild-type embryos or between the Lnp and Hoxb1-LacZ probes (grey bars) and between the Hoxb1-LacZ and Hoxd probes (black bars) in forelimb nuclei from E9.5 Hoxb1-LacZ embryos.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Nuclear reorganisation of the Hoxb1-LacZ transgene and surrounding regions along the antero-posterior axis of E9.5 embryos. (A) Histograms showing the percentage of Hoxb1-LacZ hybridisation signals inside, at the edge or outside of the MMU2 CT in the tail bud, r1/r2 or r4 of E9.5 Hoxb1-LacZ embryos (black bars) compared with endogenous Hoxd (white bars) or endogenous Hoxb1 on MMU11 in wild-type E9.5 embryos (grey bars). (B) Histograms showing the percentage of signals located either inside (red bars), at the edge (orange bars) or outside (yellow bars) of the CT for the 5' flanking, the Lnp and the Hoxd probes in tail bud, r1/r2 or r4 of either wild-type (wt) or Hoxb1-LacZ transgenic (tg) embryos. (C) Percentages of nuclei showing condensed (dark blue), intermediate (blue) or decondensed (pale blue) chromatin structures for the regions between Lnp and Hoxd probe signals (Hoxd wt) and between Hoxb1 and Hoxb9 probe signals (Hoxb wt) in tail bud, r1/r2 or r4 of wild-type embryos or between Lnp and Hoxb1-LacZ probe signals (5' Hoxb1-LacZ) and between Hoxb1-LacZ and Hoxd probe signals (3' Hoxb1-LacZ) in Hoxb1-LacZ embryos.