MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, UK
Author for correspondence (e-mail:
w.bickmore{at}hgu.mrc.ac.uk)
Accepted 3 March 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Chromatin, Embryonic development, Hox genes, Nuclear organisation, Rhombomere, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously investigated nuclear organisation and chromatin
structure changes at the murine Hoxb cluster using a retinoic acid
(RA) induced ES cell differentiation model
(Chambeyron and Bickmore,
2004). We showed that the progressive transcriptional activation
of Hoxb genes is associated, not only with a visible decondensation
of Hoxb, but also with the choreographed extrusion of the genes out
of their chromosome territory (CT). These observations lend support to a model
in which there is progressive change in large-scale chromatin structure,
initiating at the 3' end, which contributes to the sequential activation
of gene expression from this Hox cluster
(Kmita and Duboule, 2003
;
Roelen et al., 2002
;
Bickmore et al., 2005
).
Although the ES cell system seems to recapitulate the temporal activation
of Hoxb genes (Simeone et al.,
1990), it remains unclear whether this activation mechanism, and
the concomitant chromatin and nuclear re-organisation, reflect the mechanisms
that operate in vivo (Duboule and
Deschamps, 2004
). Co-linear Hox regulation occurs several times
during embryonic development. The first wave of Hoxb expression is
early in gastrulation, initiating in the most posterior (caudal) part of the
primitive streak (PS) (Forlani et al.,
2003
). Later, towards mid-gestation, the establishment of
restricted domains of Hoxb expression in the neural tube also depends
on colinearity.
Here, we have used fluorescence in situ hybridisation (FISH) on mouse embryo tissue sections in order to determine whether the chromatin changes seen at Hoxb during ES cell differentiation also occur during different stages of embryogenesis. We show that at the onset of gastrulation the transcriptional activation of Hoxb1 in the posterior PS is accompanied by chromatin decondensation of Hoxb and extrusion of Hoxb1 from its CT. These events are not seen in the non-expressing extra-embryonic (EE) cells of the same embryos. Decondensation and looping out of Hoxb1 is also seen in cells of rhombomere 4 (r4) at E9.5, but not in rhombomeres 1 or 2, anterior to r4, or in r5. By contrast a more 5' (non-expressed) gene (Hoxb9) does not loop out from the CT in these Hoxb1-expressing cells. We do not detect significant chromatin decondensation of Hoxb in the neural tube at E9.5 posterior to the hindbrain, but there is movement of Hoxb9 out from the CT in the spinal cord, where this gene is expressed. By contrast, Hoxb1 and Hoxb9 both located at the edge of their CT in the spinal cord of the tailbud region, where they are co-expressed.
Last, we show that chromatin decondensation and movement of Hoxb1
does not occur prior to transcriptional activation, either in the cells of the
posterior streak region (PSR) of E6.5 embryos, which can autonomously express
Hoxb1 in explants (Forlani et al,
2003), or the cells of the posterior epiblast (PEP) that can
precociously activate Hoxb1 expression in response to RA
(Roelen et al., 2002
).
We conclude that the programmed events of chromatin decondensation and nuclear re-organisation, seen at the Hoxb complex during ES cell differentiation ex vivo, are reproduced in vivo in at least two distinct stages of development: primitive streak formation and patterning of the neural tube. Nuclear reorganisation is coupled to Hoxb expression, and does not anticipate it during early development. Moreover, there is differential nuclear organisation of Hoxb along the anteroposterior axis of the neural tube, which parallels the co-linear expression of Hoxb genes. These data are consistent with nuclear re-organisation being part of a developmental mechanism involved in the co-linear regulation of Hox genes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
E6.5 and E7.5 embryos in the decidua, and E9.5 embryos, were fixed with 4%
formaldehyde overnight at 4°C, dehydrated through a graded ethanol series,
cleared in xylene, and embedded in paraffin blocks. Adjacent serial sections
were cut at 4 µm and used for FISH, immunohistochemistry or staining with
Haematoxylin and Eosin (HE). HE stained sections from E6.5 and E7.5 embryos
were used to stage embryos according to criteria previously published
(Downs and Davies, 1993).
Immunohistochemistry
Immunohistochemistry on 4 µm paraffin sections was performed with
Vectastain peroxidase staining kit (Vector Laboratories) according to
manufacturer's instruction using a 1:200 dilution of a polyclonal antibody
against Hoxb1 (Covance). For epitope unmasking, sections were treated by
microwave (900 W) for 20 minutes in 100 mM TrisHCl pH 10 and subsequently
blocked in 90% FCS blocking solution. Sections were counterstained with 0.75%
Eosin for 4 minutes and mounted in Histomount.
Whole-mount in situ hybridisation
Antisense probes for Hoxb1 and Hoxb9 were prepared from
cDNAs kindly provided by Robb Krumlauf. The Hoxb1 probe was a T3
transcript from a 0.9 kb EcoRI fragment. The Hoxb9 probe was
a T7 polymerase transcript from a 1.3 kb EcoRI fragment. The probes
were labelled with digoxigenin by in vitro transcription (Roche).
FISH
The protocol for FISH on mouse tissue sections was adapted from
(Newsome et al., 2003).
Briefly, 4 µm sections were laid on Superfrost slides and were heated to
60°C for 20 minutes, washed four times in xylene for 10 minutes each
before dehydration through an ethanol series (100%, 90%, 70%). They were then
microwaved for 20 minutes in 0.1 M citrate buffer, pH 6.0. Slides were cooled
in buffer for 20 minutes, washed and stored in water. Before use, they were
rinsed in 2xSSC, incubated in 2xSSC for 5 minutes at 75°C,
denatured for 3 minutes at 75°C in 70% formamide/2xSSC, plunged into
ice-cold 70% ethanol for 3 minutes, dehydrated through an alcohol series and
air-dried.
Probes were labelled for FISH as described previously
(Mahy et al., 2002b;
Chambeyron and Bickmore, 2004
).
Approximately 200 ng of chromosome paint, and 250 ng BAC or plasmid were used
per slide with 15 µg mouse Cot1 DNA (GibcoBRL) and 5 µg
sonicated salmon sperm DNA (sssDNA).
Image capture and analysis
A focus motor was used to collect image stacks of sections on slides, at
0.5 µm intervals along the z-axis, using a Zeiss Axioplan
fluorescence microscope. Images were captured using a Princeton Instruments
Micromax CCD camera, and deconvolved using Hazebuster software (Scanalytics).
A three-dimensional (3D) image was reconstructed and analysed using IPLab
(Scanalytics).
The distance (d) between two probes in 3D was determined by a more
automated approach than previously described
(Chambeyron and Bickmore,
2004). Previously, a region containing both probe signals was
selected and their segmentation was performed manually. Here, each probe
signal is individually selected and a script then determines the segmentation
levels. Briefly, a maximum pixel projection was made from the deconvolved
image stack and the two probe hybridisation signals were manually delimited.
The xyz coordinates of the weighted signal centroid were determined
for each probe, and d was the distance between the two centroids at opposite
corners of a cuboid (Chambeyron and
Bickmore, 2004
).
Probe localisation relative to the CT in the z stack was
determined visually. When the probe was not in the same z panel as
the CT, it was considered outside the CT, and was attributed an arbitrary
value of 0.5 µm (the interval between two z frames). Where
the probe and the CT were in the same z-plane, the position of the
probe relative to the nearest edge of the CT was determined as previously
described for 2D analyses (Mahy et al.,
2002a; Chambeyron and Bickmore,
2004
).
A two-tailed distribution Student's t-test was used to test the statistical significance of differences in chromatin compaction and probe position with respect to CTs. Where there was clearly a non-normal distribution of data, chi-square analysis was used.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine if there is also a decondensation of Hoxb chromatin
upon the induction of transcription in vivo, we used FISH to determine the
interphase separation between Hoxb1 and Hoxb9 in tissue
sections of E7.5 embryos. According to the published literature,
Hoxb1 expression should be seen from late streak stage onwards,
whereas Hoxb9 expression should not appear until late neural
plate/early head fold stage (Conlon and
Rossant, 1992). To ensure that only embryos expressing
Hoxb1, and not Hoxb9, were analysed, we selected late streak
early bud and neural plate stage embryos. We confirmed the patterns of
Hoxb1 and Hoxb9 expression by RNA in situ hybridisation
(Fig. 1A-C) (Table 1). Hoxb1 protein was
also analysed in sagittal sections of E7.5 embryos using a Hoxb1 antibody
(Fig. 1F). This confirmed the
expression of Hoxb1 in the cells of the primitive streak and the adjacent
mesoderm (PSM) (Forlani et al.,
2003
).
|
|
|
Movement outside of the chromosome territory accompanies Hoxb1 expression in the primitive streak region
Induction of Hoxb expression in ES cells is accompanied, not only
by a decondensation of the locus, but also by a choreographed looping out from
the CT of the expressed genes (Chambeyron
and Bickmore, 2004). To determine if this nuclear re-organisation
also occurs during Hoxb expression in early embryogenesis, we
performed FISH on E7.5 embryo tissue sections, adjacent to those used for
immunohistochemistry, using Hoxb1 or Hoxb9 probes, and a
paint for mouse chromosome 11 (MMU11) (Fig.
3A,B). We measured the distance (µm) between the Hoxb
gene signals and the nearest CT edge in nuclei from extra-embryonic cells and
PSM (Mahy et al., 2002a
;
Chambeyron and Bickmore,
2004
).
|
Therefore, as in ES cells, there is a specific extrusion of the 3' end (Hoxb1) of the Hoxb cluster out of the MMU11 CT when Hoxb1 expression initiates in embryogenesis. Another similarity that we observed with the ES system is that, even in non-expressing tissues (EEM and EEE), Hoxb1 is located closer to the CT surface than Hoxb9 is (Fig. 3D).
Chromatin decondensation and nuclear organisation of Hoxb during hindbrain segmentation
Later in embryogenesis, Hoxb genes show spatially restricted
patterns of gene expression within segments of the developing hindbrain.
Expression of Hoxb1 mRNA (Murphy
et al., 1989; Wilkinson et
al., 1989
) and protein
(Ferretti et al., 1999
;
Arenkiel et al., 2004
) is
restricted to rhombomere 4 (r4) of the hindbrain at E9.5
(Fig. 1D,H). However, these
cells are not descendants of the expressing cells from the E7.5 PS
(Forlani et al., 2003
).
Rather, Hoxb1 expression in r4 is the result of a separate induction
of expression. We therefore determined whether Hoxb1 expression in r4
is accompanied by chromatin and nuclear re-organisation events similar to
those seen in ES cells and in the PSM.
The interphase separation of Hoxb1 and Hoxb9 in rhombomeres 1 or 2 (r1/r2), where neither gene is expressed, is very similar to that in EEM of E7.5 embryos (Fig. 4A). There was decondensation of Hoxb chromatin in r4, but interestingly the distribution of d2 values in these cells did not follow a normal distribution. Instead, 21% of the loci have a highly decondensed chromatin fibre (<d2>=1 µm2) whereas 80% remain more condensed (<d2> 0.02 µm2) (Fig. 4A). This suggests that not all cells of r4 were transcribing Hoxb1 at the time of analysis. A chi-squared test showed that the difference in the distribution of d2 values between r1/r2 and r4 is statistically significant (P<0000). Chromatin decondensation in r4 is also accompanied by a significant (P=0.015) re-localisation of Hoxb1 outside its CT, compared with r1/r2 (Fig. 4B).
|
Therefore, even though separate events lead to the induction of expression of Hoxb1 in the PS early in development (E7.5), and in r4 later on (E9.5), both appear to be accompanied by chromatin decondensation and an extrusion of Hoxb1 out of the CT. By contrast, Hoxb9 is not expressed anywhere in the hindbrain, and we found that in both r4 and r5, most (66%) Hoxb9 hybridisation signals are located within the CT (Fig. 4C).
Nuclear organisation of Hoxb in the spinal cord
During RA-induced ES cell differentiation, we were able to activate the
more 5' Hoxb9 gene, but at a later time (day 10) than
Hoxb1. At that time point we saw movement of Hoxb9 out of
the interior of the CT to a position at the CT surface, or just beyond it.
However, there was no accompanying visible decondensation of Hoxb
chromatin (Chambeyron and Bickmore,
2004). In the E9.5 embryo, Hoxb9 is strongly expressed
along the neural tube, posterior to the level of somites 7-8
(Chen and Capecchi, 1997
)
(Fig. 1E), but Hoxb1
is not expressed there (Fig.
1D). By contrast, Hoxb1
(Gofflot et al., 1997
) and
Hoxb9 are both expressed in the tailbud region at E9.5
(Fig. 1D,E).
Similar to the situation in Hoxb9-expressing differentiated ES cells, we do not detect gross decondensation of Hoxb chromatin in the spinal cord (SC); the <d2> (0.11 µm2) is not significantly different from that in r5 (P=0.5), which is anterior to the limit of Hoxb9 expression. However, the <d2> measured in the spinal cord of the tail bud region (Tb) (0.15 µm2), was also not significantly larger than that in r5 (P=0.18) (Fig. 5A). This is the first situation in either ES cells or in the embryo, where Hoxb1 expression has not been accompanied by cytological levels of chromatin decondensation.
|
Chromatin organisation in cells capable of autonomous or precocious activation of Hoxb1
More than 12 hours before overt Hoxb1 expression, cells from the
posterior streak region (PSR) of early streak stage embryos (E6.5) can
initiate Hoxb1 expression autonomously in explants
(Forlani et al., 2003). By
contrast, cells from the distal region (DR) alone cannot. This might suggest
some prior opening of Hoxb chromatin structure in PSR cells at the
onset of gastrulation, which anticipates Hoxb expression itself.
Therefore, we analysed chromatin condensation and nuclear organisation of
Hoxb in cells from the PSR (epiblast and mesoderm) and the DR
(epiblast) in early streak stage E6.5 embryos
(Fig. 6A). Examination of
Hoxb1-Hoxb9 d2 values indicated that the locus remains
condensed in both groups of cells (<d2>=0.09±0.02 and
0.10±0.01 µm2, respectively), similar to EEM cells at
E7.5 (<d2>=0.08±0.02 µm2)
(Fig. 6B). Similarly,
Hoxb1 remains within its CT in nuclei from both regions at E6.5
(Fig. 6C). However, as in the
E7.5 EE, at E6.5 Hoxb1 is located closer to the surface of the CT
than Hoxb9 (Fig.
6D).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
However, as in undifferentiated ES cells, Hoxb1 occupies a position closer to the surface of the CT than Hoxb9 in early streak stage embryos (Fig. 6). This aspect of Hoxb nuclear organisation may have a role in the polarised response of the 3' end of the cluster to activation in early embryogenesis
Differences in chromatin structure at Hoxb in the developing hindbrain
It is perhaps not surprising that ES cells, which are derived from the
inner cell mass of the blastocyst, recapitulate the events of Hoxb1
expression that occur at the primitive streak during gastrulation. However,
most of the interest in Hoxb expression has been in the
anteroposterior spatially restricted segmental expression patterns in the
neural tube, which occur later in development. Hoxb1 is expressed in
r4 of the hindbrain of the E9.5 embryo. However, this cannot be the simple
result of lineage transmission from the PS cells that first initiate
Hoxb1 expression, because the r4 precursors are already anterior to
the node when Hoxb1 expression spreads caudally from the PS towards
the node (Forlani et al.,
2003).
Nevertheless we do find some aspects of Hoxb nuclear
reorganisation replayed in r4 cells. There is movement of Hoxb1, but
not Hoxb9, out from the CT in nuclei from r4. This did not occur in
more anterior (r1/r2) segments of the hindbrain (Figs
4 and
5). There is also a
decondensation of chromatin at Hoxb that is specific to r4, but
qualitatively different from that seen in PSM at E7.5. In the latter case,
there is a generalised shift in the Hoxb1-Hoxb9
d2 to larger values (Fig.
2B), indicating a relatively uniform response of the cell
population. This is similar to the decondensation seen in RA-induced ES cells
(Fig. 2B). However, the
distribution of d2 values in r4 cells is bimodal
(Fig. 4A). Twenty percent of
loci are hyperdecondensed (<d2>=1.1±0.01
µm2), but the other alleles are as, or more, condensed as those
in r1/r2 (<d2>=0.02±0.02 µm2 and
0.07±0.02 µm2, respectively). We think this means that
only a proportion of cells in r4 will be expressing Hoxb1 at a given
time, perhaps owing to stochastic fluctuation in transcription
(Levsky and Singer, 2003;
Osborne et al., 2004
), or due
to changing levels of Hoxb1 expression in differentiating
subpopulations, e.g. motoneurons (Arenkiel
et al., 2004
). Further investigation of this will necessitate
developing assays (e.g. RNA FISH) that allow both transcription and chromatin
structure to be analysed in the same nucleus of the embryo.
These data suggest that the precise chromatin changes that occur at
Hoxb1 differ between the early phase of activation at E7.5, and the
later segment-restricted phase of expression in the hindbrain. This would be
consistent with the behaviour of a Hoxb1 transgene when transposed to
a 5' position within Hoxd. In this case, there was no
expression of the transposed transgene in r4, but there was still early
mesodermal expression (Kmita et al.,
2000). It will now be interesting to analyse chromatin
condensation and nuclear organisation of the transposed Hoxb1 during
embryogenesis.
Repression of Hoxb1 outside of r4
The maintenance of Hoxb1 expression in r4, and its repression
outside r4, depends in part on auto- and cross-regulatory Hox-mediated
mechanisms (Popperl et al.,
1995; Gavalas et al.,
1998
; Studer et al.,
1998
). Specific cis-acting elements and other trans-acting
regulators also serve to repress Hoxb1 transcription in rhombomeres 3
and 5 (Fox, 2000
;
Giudicelli et al., 2003
). It
is interesting that although in r5 Hoxb1 is closer to the edge, or
just outside, of the CT, compared with its position in r1/2 cells
(Fig. 5D) perhaps
reflecting the history of past Hoxb1 expression in cells destined to
be part of r5 Hoxb chromatin may be more tightly condensed in
r5, than in r1/r2 (Fig. 4A)
(P=0.09). In addition, the chromatin configuration in r5, may relate
to the expression in this hindbrain segment of Hoxb2, located only 12
kb 5' of Hoxb1 (Wilkinson
et al., 1989
).
Nuclear organisation of Hoxb along the anteroposterior axis of the neural tube.
We did not find any nuclear re-organisation of Hoxb9 in
b1-expressing cells at E7.5, and we were careful to restrict our
analysis to pre-late neural plate stage embryos that do not express
Hoxb9 (Fig. 1B).
However, we do see nuclear reorganisation of Hoxb9 later in
development, at E9.5, in the part of the spinal cord that expresses this gene
(Fig. 1E). Compared with r5, in
cells from the SC there is a re-localisation of Hoxb9 loci away from
the interior of the CT, to a position at, or just outside, its edge
(Fig. 5). This is reminiscent
of the situation in ES cells differentiated for 10 days with RA
(Chambeyron and Bickmore,
2004).
The tailbud contains descendants of the node and anterior primitive streak
(Cambray and Wilson, 2002), and
examination of Hoxb nuclear organisation in the spinal cord of this
region at E9.5 allowed us to analyse a situation not seen in our ES
differentiation system Hoxb1 and b9 co-expression.
We do not see Hoxb chromatin decondensation in these cells
(Fig. 5A) and both
Hoxb1 and b9 have a similar mean position at the edge of the
CT (Fig. 5D). Therefore, there
seems to be a different relationship between Hoxb expression and
nuclear organisation in this part of the neural tube, compared with more
anterior regions. In this regard it is interesting to note that the expression
of Hoxb13, the most 5' member of the Hoxb cluster, and
located
100 kb from b9, is restricted to the tailbud region
(Zeltser et al., 1996).
We conclude that there is differential nuclear organisation of Hoxb along the anteroposterior axis of the neural tube, in a pattern that parallels the spatial pattern of gene expression. To our knowledge, this analysis represents one of the first studies of nuclear organisation in situ within solid tissues of the developing mouse embryo. Combined with the use of mutant embryos it may now provide the opportunity to dissect the mechanisms that bring about chromatin decondensation and nuclear gene movement during mammalian development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arenkiel, B. R., Tvrdik, P., Gaufo, G. O. and Capecchi,
M. R. (2004). Hoxb1 functions in both motoneurons
and in tissues of the periphery to establish and maintain the proper neuronal
circuitry. Genes Dev.
18,1539
-1552.
Bickmore, W. A., Mahy, N. L. and Chambeyron, S. (2005). Do higher-order chromatin structure and nuclear re-organization play a role in regulating Hox gene expression during development? Cold Spring Harbor Symp. Quant. Biol. (in press).
Cambray, N. and Wilson, V. (2002). Axial progenitors with extensive potency are localised to the mouse chordoneural hinge. Development 129,4855 -4866.[Medline]
Chambeyron, S. and Bickmore, W. A. (2004).
Chromatin decondensation and nuclear reorganization of the HoxB locus upon
induction of transcription. Genes Dev.
18,1119
-1130.
Chen, F. and Capecchi, M. R. (1997). Targeted mutations in hoxa-9 and hoxb-9 reveal synergistic interactions. Dev. Biol. 181,186 -196.[CrossRef][Medline]
Conlon, R. A. and Rossant, J. (1992). Exogenous
retinoic acid rapidly induces anterior ectopic expression of murine
Hox-2 genes in vivo. Development
116,357
-368.
Downs, K. M. and Davies, T. (1993). Staging of
gastrulating mouse embryos by morphological landmarks in the dissecting
microscope. Development
118,1255
-1266.
Duboule, D. and Deschamps, J. (2004). Colinearity loops out. Dev. Cell, 6, 738-740.[CrossRef][Medline]
Ferretti, E., Schulz, H., Talarico, D., Blasi, F. and Berthelsen, J. (1999). The PBX-regulating protein PREP1 is present in different PBX-complexed forms in mouse. Mech. Dev. 83,53 -64.[CrossRef][Medline]
Forlani, S., Lawson, K. A. and Deschamps, J.
(2003). Acquisition of Hox codes during gastrulation and axial
elongation in the mouse embryo. Development
130,3807
-3819.
Fox, E. A. (2000). The previously identified r3/r5 repressor may require the cooperation of additional negative elements for rhombomere restriction of Hoxb1. Brain Res. Dev. Brain Res. 120,151 -164.[Medline]
Gavalas, A., Studer, M., Lumsden, A., Rijli, F. M., Krumlauf, R.
and Chambon, P. (1998). Hoxa1 and Hoxb1
synergize in patterning the hindbrain, cranial nerves and second pharyngeal
arch. Development 125,1123
-1136.
Giudicelli, F., Gilardi-Hebenstreit, P., Mechta-Grigoriou, F., Poquet, C. and Charnay, P. (2003). Novel activities of Mafb underlie its dual role in hindbrain segmentation and regional specification. Dev. Biol. 253,150 -162.[CrossRef][Medline]
Gofflot, F., Hall, M. and Morriss-Kay, G. M. (1997). Genetic patterning of the developing mouse tail at the time of posterior neuropore closure. Dev. Dyn. 210,431 -445.[CrossRef][Medline]
Kmita, M. and Duboule, D. (2003). Organizing
axes in time and space; 25 years of colinear tinkering.
Science 301,331
-333.
Kmita, M., van Der, H. F., Zakany, J., Krumlauf, R. and Duboule,
D. (2000). Mechanisms of Hox gene colinearity: transposition
of the anterior Hoxb1 gene into the posterior HoxD complex.
Genes Dev. 14,198
-211.
Levsky, J. M. and Singer, R. H. (2003). Gene expression and the myth of the average cell. Trends Cell Biol. 13,4 -6.[CrossRef][Medline]
MacKenzie, M. A., Jordan, S. A., Budd, P. S. and Jackson, I. J. (1997). Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo. Dev. Biol. 192,99 -107.[CrossRef][Medline]
Mahy, N. L., Perry, P. E. and Bickmore, W. A.
(2002a). Gene density and transcription influence the
localization of chromatin outside of chromosome territories detectable by
FISH. J. Cell Biol. 159,753
-763.
Mahy, N. L., Perry, P. E., Gilchrist, S., Baldock, R. A. and
Bickmore, W. A. (2002b). Spatial organization of active and
inactive genes and noncoding DNA within chromosome territories. J.
Cell Biol. 157,579
-589.
Marshall, H., Studer, M., Popperl, H., Aparicio, S., Kuroiwa, A., Brenner, S. and Krumlauf, R. (1994). A conserved retinoic acid response element required for early expression of the homeobox gene Hoxb-1. Nature 370,567 -571.[CrossRef][Medline]
Murphy, P., Davidson, D. R. and Hill, R. E. (1989). Segment-specific expression of a homoeobox-containing gene in the mouse hindbrain. Nature 341,156 -159.[CrossRef][Medline]
Newsome, P. N., Johannessen, I., Boyle, S., Dalakas, E., McAulay, K. A., Samuel, K., Rae, F., Forrester, L., Turner, M. L., Hayes, P. C. et al. (2003). Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology 124,1891 -1900.[CrossRef][Medline]
Osborne, C. S., Chakalova, L., Brown, K. E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J. A., Lopes, S., Reik, W. et al. (2004). Active genes dynamically colocalize to shared sites of ongoing transcription. Nat. Genet. 36,1065 -1071.[CrossRef][Medline]
Popperl, H., Bienz, M., Studer, M., Chan, S. K., Aparicio, S., Brenner, S., Mann, R. S. and Krumlauf, R. (1995). Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell 81,1031 -1042.[Medline]
Roelen, B. A., de Graaff, W., Forlani, S. and Deschamps, J. (2002). Hox cluster polarity in early transcriptional availability: a high order regulatory level of clustered Hox genes in the mouse. Mech. Dev. 119, 81-90.[CrossRef][Medline]
Simeone, A., Acampora, D., Arcioni, L., Andrews, P. W., Boncinelli, E. and Mavilio, F. (1990). Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature 346,763 -766.[CrossRef][Medline]
Spitz, F., Gonzalez, F. and Duboule, D. (2003). A global control region defines a chromosomal regulatory landscape containing the HoxD cluster. Cell 113,405 -417.[CrossRef][Medline]
Studer, M., Gavalas, A., Marshall, H., Ariza-McNaughton, L.,
Rijli, F. M., Chambon, P. and Krumlauf, R. (1998). Genetic
interactions between Hoxa1 and Hoxb1 reveal new roles in
regulation of early hindbrain patterning. Development
125,1025
-1036.
van den Engh, E. G., Sachs, R. and Trask, B. J. (1992). Estimating genomic distance from DNA sequence location in cell nuclei by a random walk model. Science 257,1410 -1412.[Medline]
Wilkinson, D. G., Bhatt, S., Cook, M., Boncinelli, E. and Krumlauf, R. (1989). Segmental expression of Hox-2 homeobox-containing genes in the developing mouse hindbrain. Nature 341,405 -409.[CrossRef][Medline]
Zeltzer, L., Desplan, C. and Heintz, N. (1996).
Hoxb-13: a new Hox gene in a distant region of the
HOXB cluster maintains colinearity.
Development 122,2475
-2484.