QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 24 January 2007
doi: 10.1242/dev.02779

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morey, C. Articles by Bickmore, W. A. Search for Related Content PubMed PubMed Citation Articles by Morey, C. Articles by Bickmore, W. A. Social Bookmarking                         What's this?

Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation

MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, UK.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Genomic structure of Hoxd. Map of the genomic region surrounding Hoxd on MMU2, showing the position of genes. Map position is in bp. Below this is a detailed view of the Hoxd locus itself. Genes are depicted as arrowed boxes indicating the orientation of transcription. White arrowheaded boxes correspond to the nine Hoxd genes (from 1 to 13) and black arrowheaded boxes represent the flanking genes. The grey oval locates a region of non-coding sequence conservation that includes the GCR. The location of BAC and fosmid clones used as FISH probes are shown (see also Table 2). Data and map positions are taken from NCBI Build 35 of the mouse genome (http://genome.ucsc.edu/cgi-bin/hgGateway) and from ENSEMBL v37, Feb 2006 (http://www.ensembl.org/Mus_musculus/index.html).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Nuclear reorganisation of the Hoxd regulatory domain in the embryo. (A) Histograms showing the 3D position of hybridisation signals for a BAC lying within the 5' flanking region (RP23-288B11, blue), a BAC covering the Lnp gene (RP24-267L11, yellow), or a Hoxd BAC (RP23-15M17, green), relative to the MMU2 CT edge in nuclei from E9.5 control tissues, tailbud or limb bud. Coloured arrowheads indicate the median location for each respective probe. n>100 loci, obtained from three embryos. (B) Mean position±s.e.m. (µm), relative to the edge of the MMU2 CT for the 5' flank (blue), Lnp (yellow) and Hoxd (green) BACs. (C) Three-dimensional DNA FISH using the Hoxd probe (red) hybridised together with a MMU2 chromosome paint (green) on DAPI counterstained nuclei of a 4 µm limb bud section from E9.5 embryo. Raw image before deconvolution. (D) Distribution of 3D interphase distances (d) in µm, measured between the Lnp and Hoxd BACs (black bars), or between the 5' flank and the Lnp BACs (white bars) in control tissues, tailbud or limb bud from E9.5 embryos. Black or white arrowheads indicate the median separation between each probe pair. In total, n>100 loci, obtained from three embryos. (E) Mean±s.e.m. interphase separation (µm), measured between Lnp and Hoxd BACs (black square), or between the 5' flank and the Lnp BAC (white squares) in control tissues, tailbud or limb bud from E9.5 embryos. (F) Three-dimensional DNA FISH with the Lnp (red) and the Hoxd (green) BACs on DAPI counterstained nuclei from E9.5 limb bud, image after deconvolution. Arrowheads point to stretched signals. (G) Histogram showing the percentage of stretched 3D FISH signals detected with the Hoxd, Lnp and 5' flank BAC probes in the control tissues, tailbud and limb bud from E9.5 embryos.

View larger version (77K): [in this window] [in a new window]   Fig. 3 . Expression of Hoxd and flanking genes during ES cell differentiation. RT-PCR analysis of Hoxd and flanking genes (Mtx2, Evx2 and Lnp) in undifferentiated (Un) OS25 ES cells and during 18 days of RA-induced differentiation. Loss of Oct4 expression was used to monitor differentiation and Hprt serves as a constitutively expressed control. +, with RT; -, without RT.

View larger version (67K): [in this window] [in a new window]   Fig. 4. Nuclear reorganisation at and around Hoxd during ES cell differentiation. (A) Histograms showing the distribution of 2D FISH hybridisation signals for the Hoxd (green), Lnp (yellow) and 3' flank (RP23-7D13, red) BACs relative to the edge of the MMU2 CT during differentiation. Arrowheads show the median positions for each probe. (B) Mean±s.e.m. position (µm), relative to the edge of the MMU2 CT for the hybridisation signals from the Hoxd, Lnp and 5' and 3' flanking BACs during the timecourse of differentiation. (C) Distribution of interphase separation (d) in µm, measured between hybridisation signals for the Lnp and Hoxd BACs (black bars), or between the 5' flank and the Lnp BAC (white bars) during the first 8 days of differentiation. Black or white arrowheads indicate the median separation between each probe pair. (D) Mean±s.e.m. interphase separation (d), in µm, measured between four pairs of probe signals (as indicated) during the timecourse of differentiation. For all experiments, n>100 loci.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Nuclear reorganisation within the Hoxd locus. (A) Four-colour DNA FISH using fosmid probes 3' Hoxd (121N10, yellow), 5' Hoxd (469P2, red) and an MMU2 chromosome paint (green), on DAPI (blue) counterstained nuclei from undifferentiated (Un) OS25 ES cells and cells differentiated for 4 and 14 days. Scale bar: 5 µm. (B) Histograms showing the distribution of 3' Hoxd (121N10, yellow) and 5' Hoxd (469P2, red) hybridisation signals relative to the edge of the MMU2 CT during differentiation. Arrowheads show the median values for each probe. (C) Mean position±s.e.m. (µm), relative to the edge of the MMU2 CT, for all three Hoxd probes during the timecourse of differentiation. (D) Distribution of interphase separations (d) in µm, measured between 3' and 5' Hoxd (121N10-469P2) fosmid probe signals in 2D FISH, during the timecourse of differentiation. Arrowheads show the median values. In B, C and D, n>100 territories. (E) Distribution of interphase separations in µm, measured between 121N10 and 860J8 3D FISH signals in undifferentiated ES cells (white bars) and cells differentiated for 4 days (black bars). Arrowheads show the median values. (F) Comparison of median interphase distance (d) between signals for probes 860J8 and 121N10 in undifferentiated cells and after 4 days of differentiation after 3D (solid line) or 2D (dotted line) FISH.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Coordination of looping and decondensation in the Hoxd region. (A) FISH using probes 5' flank (RP23-288B11, yellow), Hoxd (RP23-15M17, red) and MMU2 chromosome paint (green) on DAPI stained nuclei of undifferentiated ES cells or after 8 days of differentiation. (B) Scatter plots of the position of Hoxd (y-axes) and 5' flank (x-axes) BAC probe signals relative to the edge of the MMU2 CT during differentiation. The bottom right quadrant represents territories in which both probes are inside the CT; the top left quadrant represents situations in which both probes were located outside the CT. The other two quadrants represent discordant positions for the two BAC probe signals (one inside and one outside the CT); n>100 territories. (C) Scatter plots of the interphase separation (d) in µm between Hoxd and the 5' flank probe signals (y-axes of both graphs) and the position of each probe relative to the edge of the MMU2 CT (µm) (x-axes) (Hoxd left panels; 5' flank on right). The diagonal lines indicate the best-fit linear regression lines and their corresponding R2 values; n>100 territories. (D) As in A, but with the probes Lnp (RP23-267L11, yellow) and 3' flank (RP23-7D13). At day 8 of differentiation, examples of decondensed (widely spaced probe signals) chromatin outside the CT (upper picture), of condensed chromatin outside the CT (middle picture) or of decondensed chromatin inside the CT (lower picture) are shown. (E) As in B but with BACs Lnp and 3' flank. (F) Scatter plots of the d (µm) between the Lnp (black dots) and 3' flank (red dots) signals (y-axes) and the position (µm) of each probe relative to the edge of the MMU2 CT (x-axes). Scale bars: 5 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter