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First published online April 13, 2005
doi: 10.1242/10.1242/dev.01813

Development 132, 2215-2223 (2005)
Published by The Company of Biologists 2005
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Nuclear re-organisation of the Hoxb complex during mouse embryonic development

Séverine Chambeyron*, Nelly R. Da Silva*, Kirstie A. Lawson and Wendy A. Bickmore{dagger}

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



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  		Fig. 1. Expression of Hoxb1 and Hoxb9 during mouse embryogenesis. (A-C) Whole-mount in situ hybridisation to detect Hoxb1 (A) or Hoxb9 (B,C) expression in E7.5 embryos. Hoxb1 expression in the primitive streak and adjacent mesoderm (PSM) is seen in a late streak/early bud embryo (A). No Hoxb9 expression is detected in late neural plate stage embryos (B), but is detected in PSM of headfold stage E7.5 embryos (C). (D,E) Whole-mount in situ hybridisation to detect Hoxb1 (D) or Hoxb9 (E) expression in E9.5 embryos. Hoxb1 expression is seen in the tailbud (Tb) and in a segment of the hindbrain (D). A sagittal section of this embryo shows that this latter region is rhombomere 4 (r4) (shown in inset at higher magnification). There is no Hoxb1 expression in the anterior region of the spinal cord (SC). Hoxb9 expression (E) is seen in the SC and Tb, but not in the hindbrain. (F) Immunohistochemistry staining of a near sagittal section from an E7.5 (neural plate stage) embryo, with antibody that recognises Hoxb1, and counterstained with Eosin. Hoxb1 is seen in the PSM. There is no staining in extra-embryonic mesoderm (EEM). (G) DAPI staining of an adjacent E7.5 section that was used for FISH analysis. (H) Immunohistochemistry staining for Hoxb1 on a sagittal section from an E9.5 embryo. The positions of rhombomeres 1 to 4 (r1-4) are indicated. (I) DAPI staining of an adjacent E9.5 section that was used for FISH analysis. Scale bars: 200 µm.

 


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  		Fig. 2. Chromatin decondensation at Hoxb during early embryogenesis. (A) FISH of Hoxb1 (red) and Hoxb9 (green) on nuclei from extra embryonic mesoderm (EEM) and primitive streak/adjacent mesoderm (PSM) of one E7.5 embryo. Nuclei were counterstained with DAPI (blue). Scale bar: 2 µm. (B) Distribution of squared interphase distances (d2) in µm2 measured between probes for Hoxb1 and Hoxb9, on nuclei from EEM and PSM of one E7.5 embryo, and in ES cells differentiated with RA for 2 days.

 


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  		Fig. 3. Nuclear re-organisation of Hoxb1 during early embryogenesis. (A) FISH with MMU11 chromosome paint (green) and Hoxb1 probe (red) on a field of nuclei from E7.5 primitive streak regions. The image is a maximal pixel intensity projection from a 3D image stack. (B) Single z-plane images from FISH as in A, of single nuclei from E7.5 EEM and PSM. Nuclei were counterstained with DAPI (blue). Scale bars: 2 µm. (C) Histograms showing the position of Hoxb1 and Hoxb9 hybridisation signals, relative to the inside, edge or outside of the MMU11 territory, in nuclei from E7.5 EE cells (black bars) and PSM (white bars). The EE and PSM analysed were from two and three embryos from different litters in the case of Hoxb1, and from one embryo for Hoxb9. Negative distances indicate signals localised beyond the visible limits of the detectable CT. For Hoxb1, data from EEM and EEE have been pooled together (n≥60). (D) Position (mean±s.e.m.) of Hoxb1 (black squares) and Hoxb9 (white circles) relative to the inside, edge or outside of the MMU11 territory in nuclei from EE and PSM at E7.5 (n≥60).

 


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  		Fig. 4. Chromatin decondensation and nuclear re-organisation of Hoxb1 in rhombomere 4. (A) The distribution of squared interphase distances (d2) in µm2 measured between probes for Hoxb1 and Hoxb9, in rhombomeres 1 and 2 (r1/r2), r4 and r5 from E9.5 embryos. (B) Histogram showing the position (µm) of Hoxb1 hybridisation signals, relative to the inside, edge or outside of the MMU11 territory, in nuclei from r1/r2 (black bars), r4 (white bars) and r5 (grey bars) of two E9.5 embryos from different litters. Negative distances indicate signals localised beyond the visible limits of the detectable CT (n≥60). (C) Histogram showing the position of Hoxb9 hybridisation signals, relative to the MMU11 territory edge, in nuclei from r4 (white bars) and r5 (grey bars) of two E9.5 embryos from different litters. Negative distances indicate signals localised beyond the visible limits of the detectable CT (n≥60).

 


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  		Fig. 5. Nuclear re-organisation of Hoxb in the spinal cord. (A) The distribution of squared interphase distances (d2) in µm2 measured between probes for Hoxb1 and Hoxb9, in rhombomeres 5 (r5), spinal cord (SC), and tailbud (Tb) from E9.5 embryos. (B,C) Histogram showing the position of Hoxb9 (B) or Hoxb1 (C) hybridisation signals, relative to the inside, edge or outside of the MMU11 territory, in nuclei from r5 (grey bars), SC (hatched bars) and Tb (black bars) of two E9.5 embryos from different litters. Negative distances indicate signals localised beyond the visible limits of the detectable CT (n ≥60). (D) Positions (mean±s.e.m.) of Hoxb1 (black squares) and Hoxb9 (white circles) relative to the inside, edge or outside of the MMU11 territory in nuclei located along the anteroposterior axis of the neural tube at E9.5, from r1/r2 down to the tailbud (Tb) (n≥60).

 


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  		Fig. 6. Chromatin condensation and nuclear re-organisation of Hoxb in the E6.5 early streak embryo. (A) HE staining (left) and DAPI counterstaining (right) of serial transverse sections from an E6.5 embryo. The position of the posterior streak region (PSR), and the posterior (PEP) and anterior (AEP) epiblast are indicated. Scale bar: 200 µm. (B) The distribution of squared interphase distances (d2) measured between probes for Hoxb1 and Hoxb9, in PSR, DR, PEP and AEP of E6.5 embryos. Measurements from E7.5 EEM are shown for comparison. (C) Histogram showing the position of Hoxb1 hybridisation signals, relative to the MMU11 territory edge, in nuclei from E7.5 EE (black bars), E6.5 DR (white bars) and E6.5 PSR (grey bars). Negative distances indicate signals localised beyond the visible limits of the detectable CT (n≥60). (D) Position (mean±s.e.m.) of Hoxb1 (squares) and Hoxb9 (circles) relative to the MMU11 territory edge in nuclei from E7.5 EE and E6.5 PSR (n≥60).

 

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