Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
Author for correspondence (e-mail:
jacqueli{at}niob.knaw.nl)
Accepted 1 May 2003
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SUMMARY |
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Axial structures that will later express Hox genes are generated in the node region in the period that Hox expression domains arrive there and continue to spread rostrally. However, lineage analysis showed that definitive Hox codes are not fixed at the node, but must be acquired later and anterior to the node in the neurectoderm, and independently in the mesoderm. We conclude that the rostral progression of Hox gene expression must be modulated by gene regulatory influences from early on in the posterior streak, until the time cells have acquired their stable positions along the axis well anterior to the node.
Key words: Mouse, Hox genes, Hoxb1, Hoxb4, Hoxb8, Gastrulation, Clonal analysis, AP patterning
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INTRODUCTION |
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The function of Hox genes in AP patterning in the mouse has been most
clearly demonstrated at levels close to the rostral boundary of their
definitive expression domains. Homeotic transformations of segmented
structures developing from these boundary regions were observed in
loss-of-function mutants (Krumlauf,
1994). Recently, transient abolition of early colinearity, which
is evident during gastrulation, by deletion of a 5' located
Hoxd regulatory region was shown to result in skeletal homeotic
transformations in embryos and neonates
(Kondo and Duboule, 1999
).
These results first demonstrated that the concerted colinear control of Hox
expression from its onset at primitive streak stages is absolutely required
for a correct Hox patterning function.
Most efforts to elucidate the molecular mechanisms that underlie
regionalised Hox gene expression have focussed on relatively late
developmental stages (reviewed by Deschamps
et al., 1999). The few studies addressing the early establishment
of the Hox domains have highlighted the difficulty in correlating these early
patterns with cell behaviour during morphogenetic movements at gastrulation
(Deschamps and Wijgerde, 1993
;
Gaunt and Strachan, 1994
). Hox
genes are activated when the primitive streak is almost fully extended, and
then in the most posterior (caudal) part of the streak which is generating
extra-embryonic mesoderm and not contributing to the embryo proper. The early
transcription domains subsequently spread rostrally to reach the anterior part
of the streak by an unknown, non-lineage-related mechanism
(Deschamps and Wijgerde,
1993
).
After the early Hox expression domains reach the node region, they continue
to spread more rostrally to reach their definitive rostral boundaries in
neurectoderm, mesoderm and endoderm in axial and paraxial structures. Previous
work suggested that mesoderm acquires its positional information when emerging
from the primitive streak (Tam and
Beddington, 1987), and Frohman et al.
(Frohman et al., 1990
)
proposed that differential Hox gene expression is established at that moment.
The fate map of the presumptive neurectoderm at late gastrulation
(Tam, 1989
) similarly
indicates that the epiblast near the anterior end of the streak contain
progenitors of hindbrain and spinal cord; retrospective lineage analysis
indicates that spinal cord is laid down at the node, sequentially from
anterior to posterior (Mathis and Nicolas,
2000
). Analysis of the evolution of the early Hox gene expression
patterns suggested that the successive rostral boundary regions could be fixed
at the node and carried by lineage transmission as the axis was laid down and
the node `regressed' (Deschamps and
Wijgerde, 1993
).
We have investigated the mechanism by which Hox gene expression is initiated and propagated along the streak towards the node, using embryonic explants, and show that the conditions for autonomous Hox expression are already present posteriorly but not anteriorly, at the beginning of gastrulation, more than 12 hours before overt Hox gene expression. We also show that this primed but non expressing posterior tissue can induce Hoxb1 expression in non primed and non expressing anterior streak and epiblast tissue. Second, we examine the possibility that Hox expression boundaries are carried rostral to the node by lineage transmission, and show that this is not so in the neurectoderm, because the precursors that will occupy the future rostral expression boundary region are already anterior to the node when Hox expression reaches the node. No consistent relationship between the Hox gene expression status of cells at the node and the destination of their anteriormost mesoderm descendants was found. We conclude that the rostral progression of Hox gene expression must be modulated by gene regulatory influences from early on in the posterior streak, until the time cells have acquired their stable positions along the axis well anterior to the node.
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MATERIALS AND METHODS |
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Embryonic explants
Embryos were isolated from the decidua at the desired gestational stage and
Reichert's membrane was removed with tungsten needles
(Hogan et al., 1994). Embryos
were staged according to morphology (Downs
and Davies, 1993
), modified for C57BL/6xCBA embryos
(Edinburgh Mouse Atlas Project;
http://genex.hgu.mrc.ac.uk;
K.A.L., unpublished) and according to size
(Lawson and Pedersen, 1992
).
Explants spanning the length of the primitive streak
(Fig. 2) were excised with a
glass needle while restraining the embryo by the extra-embryonic part with a
tungsten needle. The posterior part of the primitive streak (posterior streak
region, PSR) was separated from the extra-embryonic tissue at the level of the
junction between embryonic and extra-embryonic ectoderm. The anterior part of
the primitive streak designated ASR (anterior streak region, see
Fig. 2) included the distal
part of the embryo, because the anterior part of the streak alone did not grow
well in culture. All explants contained the three germ layers and were about
100x100 µm in size, as measured with a micrometer. An intermediate
piece between the most proximal and distal pieces of the streak was also taken
from older stages ensuring that explants of similar size were taken from
embryos of different age. Explants were transferred individually to small
depression wells made in bacteriological dishes with a darning needle, covered
with 60 µl drops of Dulbecco's modified Eagle's medium (DMEM) plus 15%
fetal calf serum (FCS) under mineral oil and further cultured according to Ang
and Rossant (Ang and Rossant,
1993
). Recombinants were made by aggregating two explants. The
culture period was 24 hours unless otherwise stated. Growth and survival of
the explants were verified by measuring size with a micrometer and viability
with Trypan Blue staining.
|
Identification of the position of labelled cells
HRP-containing cells were identified after culture by staining the embryos
for 1-1.5 hours with Hanker Yates reagent (Polysciences) as described
(Lawson et al., 1991) before
fixing with 2.5% glutaraldehyde in PBS, dehydrating, clearing in 1:2 benzyl
alcohol: benzyl benzoate (BABB), embedding in glycolmethacrylate (Technovit
1700) and cutting 7 µm serial sections followed by staining with Methylene
Blue. The number and position of the labelled cells, in relation to
identifiable landmarks along the AP axis, were recorded in embryos in BABB
before embedding, and crucial embryonic dimensions noted. The embedded embryos
were sectioned in the appropriate orientation to identify the position
orthogonal to the midline of labelled cells i.e. DV position in the
neurectoderm and whether labelled mesoderm was axial, paraxial or lateral
plate.
For comparison of clones in the hindbrain and spinal cord the distance along the midline between the most anterior member of a clone and the boundary between the first and the second somite was measured on a sagittal view of the cleared embryo. The initial axial position of the progenitors was measured along the midline from the anterior junction of epiblast and extra-embryonic ectoderm.
Gene expression
Whole-mount in situ hybridisation was performed as described by Roelen et
al. (Roelen et al., 2002).
Digoxigenin-labelled (Boehringer Mannheim) antisense probes were as
follows. The Hoxb1 probe was a T7 polymerase transcript from a 800
basepairs (bp) EcoRI fragment
(Wilkinson and Krumlauf,
1990). The Hoxb8 probes were a 1:1 mix of a SP6
polymerase transcript from a 350 bp 3' untranslated
SacI-KpnI cDNA fragment and a SP6 polymerase transcript from
a 420 bp SacI fragment containing the first exon of the gene
(Charité et al., 1994
).
The T/Brachyury specific probe was a T7 polymerase transcript from a 2kb
EcoRI fragment (clone pSK75)
(Herrmann et al., 1990
). The
chordin probe is described in Bachiller et al.
(Bachiller et al., 2000
).
Probes were tested on embryos before use on explants.
For ß-galactosidase activity, explants were fixed in 1% formaldehyde,
0.2% glutaraldehyde (Sigma) in phosphate-buffered saline (PBS) for 5 minutes,
rinsed twice in PBS and stained with X-gal as described
(Charité et al.,
1994).
Statistics
The anterior limits of clones at different stages or in different germ
layers at the same stage were compared with the Wilcoxon rank test. The
relationship of the anterior limit of clones with progenitor position was
obtained by linear regression analysis
(Snedecor and Cochran,
1967).
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RESULTS |
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We investigated whether Hoxb1 was activated autonomously in embryonic tissues at stages preceding initial gene expression. To do this, we cultured posterior streak region (PSR) and anterior streak region (ASR) (Fig. 2, upper panels) separately at different primitive streak stages, and examined the expression of Hoxb1 and Hoxb1-lacZ. In control experiments, explant culture conditions supported normal Hox and marker gene expression at the different stages, as shown by the maintenance of Hoxb1 and Hoxb8 expression in culture in 100% of PSR and ASR explants from headfold stage embryos (E7.5-7.75): such pieces already express the genes at the time of excision (data not shown). The 24-hour culture period was chosen because this time is sufficient for Hox gene expression to progress from the posterior to the anterior streak region both in vivo and in longitudinally bisected egg cylinders in vitro (Fig. 2, second row and data not shown).
PSR explants excised at different stages between early streak (ES) and late mid streak (LMS) stages expressed Hoxb1 (Fig. 3A-D) and Hoxb1-lacZ (not shown) after culture, with the proportion of positive explants rising from 54% for ES explants to 80-100% from the early mid streak (EMS) to the LMS stages (Fig. 3O). Likewise, 80-100% median streak region (MSR) explants from mid streak (MS) and LMS stages had activated the Hoxb1 gene and transgene (Fig. 3E,F,O) after culture. By contrast, ES ASR explants failed to express Hoxb1 and Hoxb1-lacZ (Fig. 3G,O). The proportion of ASR explants expressing the gene rose with increasing age, from 18% at the EMS stage (with a low number of positive cells in this latter case) (Fig. 3H,O), to 36% at the MS stage (Fig. 3I,O), and to 57% for the LMS embryos (Fig. 3J,O). The absence of Hoxb1 expression in the youngest material was not due to inappropriate culture conditions because the explants expressed the Hox-independent genes brachyury (T) (Fig. 3K,L) and chordin (Fig. 3M,N). Hox gene expression could not be induced by increasing the culture period of ES ASR explants to 30-32 hours (data not shown), whereas 100% MS ASR explants were positive after a similar culture period (versus 36% after 24 hours culture, data not shown).
|
The activation pattern of Hoxb8 in explants was also examined. A similar time period (about 12-16 hours) was found to separate permissiveness to `autonomous' activation in explanted tissues (Fig. 4), and effective activation in the intact embryo in vivo (Fig. 1). PSR explants can autonomously activate Hoxb8 expression after culture from the LMS stage onwards (E7.0) (Fig. 4A,B) but not earlier, and the MSR and ASR explants from the late streak early bud (LSEB) stage on (E7.25) (Fig. 4C-F), although Hoxb8 is only expressed in the embryo from late neural plate/head fold (LNP/HF) stages (E7.75) (Fig. 1G-J). The dynamics of activation of Hoxb1 and Hoxb8 in the explant system suggest that, like Hox expression itself, the process which anticipates this expression in the primitive streak takes place sequentially (for the 3' genes earlier than the 5' genes) in a proximal (posterior) to distal (anterior) sequence (compiled in Fig. 2, bottom panel).
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The spread of Hox expression domains rostral to the node is not
lineage related
Rostral spread of Hox expression continues beyond the node
(Fig. 1) during a period when
the presumptive hindbrain and spinal cord territories are expanding anterior
to the node (Tam, 1989) and
the node `regresses' while generating spinal cord
(Mathis and Nicolas, 2000
).
Given this coincidence, a plausible mechanism for the continued spread of the
expression front along the AP axis is that a cell acquires a Hox code and
positional specification while in the node region and its descendants retain
it after leaving the node region (Deschamps
and Wijgerde, 1993
). Descendants remaining (temporarily) in the
node region would acquire a new Hox code as the more 5' genes are
expressed there. If this hypothesis is valid, predictions about the final
position of the most rostral descendants of cells at the Hox gene expression
front at the node at different stages can be made on the basis of the later
rostral expression limits of different Hox genes in neurectoderm and mesoderm
at E8.5-9.5. Both a general prediction with regard to neurectoderm and
mesoderm descendants, and specific predictions can be made.
The general prediction is that the anterior limit of mesodermal clones will
be several somite lengths posterior to neurectodermal clones generated in the
node region at the same stage (Gaunt et
al., 1988; Frohman et al.,
1990
). Clones labelled with HRP were generated in epiblast at the
node region from LS to HF stages (E7.0-E7.7), and the embryos cultured for 1
day (Fig. 6). Some clones were
also generated in the axial epiblast anterior to the node at LSEB and older
stages. Most (93%) of the clones generated at the node after the LS stage and
contributing to neurectoderm were restricted to the ventral half of the neural
tube; 78% of the clones contributing to non axial mesoderm anterior to the
node were in paraxial mesoderm. The anterior limit of neurectodermal
descendants in clones generated at the node was progressively more posterior
with advancing initial stage (LS versus LSEB, P<0.01; NP versus
HF, P=0.01) with the exception that LSEB and NP did not differ
significantly (Fig. 7, upper
set). A similar progression was seen in the mesoderm (LS stages versus HF,
P<0.01). This trend confirms the sequential addition of neural and
mesodermal material from the node region. At no stage was the anterior limit
reached by mesodermal clones generated at the node posterior to the anterior
limit of neurectodermal clones (Wilkoxon rank test). At the HF stage, mesoderm
clones were even slightly more rostral to neurectoderm ones (P=0.05).
Clones initiated in the mesoderm layer did not differ from those initiated in
the epiblast. Axial mesoderm, which does not express Hox genes in the mouse
(Deschamps and Wijgerde, 1993
),
behaved differently: it remained associated with the node, and therefore
relatively posterior (Fig.7).
Therefore axial extension from the node progresses at similar speed in
neurectoderm and paraxial mesoderm and the general prediction from the
hypothesis was not fulfilled.
|
|
|
Hindbrain and anterior spinal cord elongate both by addition from the
node and by internal growth
Sequential addition of material into the hindbrain and spinal cord from the
node is indicated by the progressively more caudal position of the anterior
limits of clones generated in the node region between LS and HF stages
(Fig. 7, upper set). Of the
clones contributing to neurectoderm, 45% (14/31) extended to the node, whereas
no (0/9) clones generated in the axial region anterior to the node did
(Fig. 7, lower set). This is
supporting evidence of a self maintaining pool of precursors in the node
region for the spinal cord (Mathis and
Nicolas, 2000; Mathis et al.,
2001
) and also for part of the hindbrain.
Comparison of the anterior limits of neurectoderm clones shows a consistency in result in clones generated anterior to the node (Fig. 7, lower set) compared with those generated in the node region (Fig. 7, upper set): the anterior limit is well correlated with the distance from the node of anterior axial progenitors (those anterior to the node and within 55 µm either side of the midline) at all stages. The AP position of the anterior limits of the clones was compared with the position of their anterior axial progenitors by linear regression analysis in order to assess quantitatively whether the neural axis anterior to the node at LSEB to HF stages, representing prospective r3 to spinal cord at the level of S8, is stabilised or is continuing to grow within itself (Fig. 9). The value of the regression coefficient (b) was 2.769 (s.e.=0.463, n=9, P<0.001). This value is also significantly greater than 1 (P<0.01), implying that the part of the axis representing precursors of hindbrain and spinal cord to the level of S8, anterior to the node at LSEB to HF stages, increases in length within 24 hours. The axis is therefore extending within itself anterior to the node, and not only at the node by the sequential insertion of new material. In addition, the posterior displacement of the anterior limits of paraxially generated clones relative to anterior axial ones (LSEB and NP, Fig. 7, lower set) suggests that convergence and extension occur in the more lateral ectoderm until at least the NP stage. These results could explain why labelling epiblast just anterior to the node at the HF stage gives clones with anterior limits in the neurectoderm that are more posterior than might have been expected from the shape of the embryo and the space apparently available for the first somites [compare Fig. 1F with Fig. 7 (HF)]. The results also underline the dynamic nature of the relative AP positions of cells that are leaving, and have recently left, the node during a period when Hox expression domains are traversing rostrally through the region.
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DISCUSSION |
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Neurectoderm cells acquire their Hox code after they have left the
node region
The regions of the neural tube that will be positionally specified by the
Hox genes are formed from the LS stage onwards by a combination of the
sequential addition of cells from the node region
(Fig. 7), convergence and
extension of paraxial ectoderm anterior to the node
(Fig. 7 and data not shown) and
subsequent longitudinal extension within the neurectoderm
(Fig. 9). Comparison of the
clonal behaviour of neurectoderm generated at the node and the spread of Hox
gene expression past the node showed that nascent neurectoderm does not
acquire and fix its Hox code at the node: cells whose descendants will
contribute to definitive anterior boundary regions of the Hox domains (both
3' and 5' genes) are already anterior to the node when the waves
of Hox expression arrive there. Therefore the early Hox domains have to `catch
up' with the cells that will later occupy the rostral boundary domains. In
addition, the relative AP positional values in the neurectoderm are changing
anterior to the node as the axis elongates and while Hox expression domains
are spreading rostrally through this region, implying that positional
specification in terms of a stable Hox code must be acquired later. The delay
before Hox codes are fixed may correspond to the time required for
stabilisation of the relative AP positions of cells in the neurectoderm and
mesoderm, when clonal growth in the neurectoderm changes from an AP to a DV
mode (Mathis and Nicolas,
2000), and when cell mixing stops in unsegmented paraxial mesoderm
(Tam, 1988
).
Clonal expansion and Hox gene expression in paraxial mesoderm versus
neurectoderm
Although the rostralmost neurectoderm descendants of cells at the node when
the expression domains arrived there ultimately occupy more posterior
positions than the anterior expression boundary of the genes considered, it is
not so for the mesoderm. The most anterior mesodermal descendants of epiblast
cells near the node when the Hoxb1 expression domain arrived there
did, in some embryos, contribute to paraxial positions at or near the anterior
boundary region of this Hox gene, and in others were more posterior. By
contrast, the most anterior mesodermal descendants of cells near the node when
the expression domain of Hoxb8 reached the node were found at
positions much more rostral than the expression boundary of this gene. A
conclusion from these data is that the regulatory interactions responsible for
generating the sequential 3' to 5' Hox expression boundaries are
different in the neurectoderm and in the mesoderm and must involve down
regulation in the mesoderm at least of Hoxb8. This is unsurprising in
the light of the recent findings about mesoderm-specific modulation of Hox
gene expression in the segmental plate
(Zakany et al., 2001). The Hox
codes may well be reset by the oscillatory mechanism in the mesoderm
descendants of cells near or anterior to the node, after their ingression
through the streak.
Axial extension anterior to the node progresses at similar speed in
neurectoderm and mesoderm. The offset of rostral expression boundaries of Hox
gene expression in the mesoderm compared with the neurectoderm can not be
accounted for by germ layer specific clonal distribution of descendants from
progenitors around or anterior to the node, but must also result from
differential gene regulation including transcriptional induction in the
neurectoderm and downregulation in the mesoderm. The anterior progression of
the Hox expression domains in both neurectoderm and mesoderm appears to be
modulated by gene regulation from early on until at least early somite stages.
Transcriptional regulation of Hox genes, although usually studied at later
stages, has indeed been shown, in several cases, to depend on germ
layer-specific regulatory elements
(Gilthorpe et al., 2002;
Gould et al., 1998
;
Marshall et al., 1994
;
Sharpe et al., 1998
).
A single continuous phase of induction of the Hox expression domains
between initial transcription and establishment of the rostral expression
boundaries
The data suggest that the Hox expression domains are established gradually
from the posterior streak to their definitive rostral boundaries anterior to
the node, by cell-cell signalling driving transcriptional modulation. Induced
by genetic interactions occurring at the time of primitive streak formation
and extension, Hox gene expression may continue to spread anteriorly beyond
the node under the influence of the same or a similar gene regulation
mechanism, and does not rely on proliferative expansion of Hox-expressing
cells. The explant and lineage experiments therefore suggest that a single
continuing process drives the rostral extension of the Hox domains both
posterior and anterior to the node. The node region itself, around which the
laying down of tissues along the extending axis is coordinated, would not be
specifically involved in instructing newly generated cells as to their AP
identity and Hox code, but seems to be passively traversed by the progressing
Hox domains.
Although some of the candidate Hox-inducing molecules, such as Wnt3, Wnt3a
and Fgf8, could be acting early in the primitive streak, as already discussed,
additional inducers might come into action around and anterior to the node
during the axial extension phase which we studied. Such possible inducers are
Wnt8 (Bouillet et al., 1996),
the Cdx transcription factors (van den
Akker et al., 2002
) and effectors of the oscillatory mechanism of
the Notch pathway in the paraxial mesoderm
(Zakany et al., 2001
). During
this period and later, retinoic acid signalling has been shown to sequentially
shift the definitive expression boundaries of 3' to 5' Hox genes
rostrally in the neurectoderm (Marshall et
al., 1994
; Studer et al.,
1994
; Gould et al.,
1998
; Oosterveen et al.,
2003
). Stabilisation of the Hox expression domains would only take
place subsequently, possibly by the epigenetic polycomb and trithorax
maintenance system taking over the control of the restricted Hox expression
domains, thus putting an end to the rostral spreading of gene expression in
the neurectoderm and mesoderm (Yu et al.,
1998
; Tomotsune et al.,
2000
; Akasaka et al.,
2001
). Disruption of the regulatory interactions would, at any
stage, lead to altered Hox expression and patterning defects; for example,
precocious Hox expression in the primitive streak
(Kondo and Duboule, 1999
)
would result in the disruption of the sequential arrival of the Hox expression
domains at the level of the cells to be instructed in the prospective axial
and paraxial structures, causing aberrant AP instruction and patterning
alteration that cannot be corrected by subsequent regulation.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akasaka, T., van Lohuizen, M., van der Lugt, N.,
Mizutani-Koseki, Y., Kanno, M., Taniguchi, M., Vidal, M., Alkema, M., Berns,
A. and Koseki, H. (2001). Mice doubly deficient for the
Polycomb Group genes Mel18 and Bmi1 reveal synergy and
requirement for maintenance but not initiation of Hox gene expression.
Development 128,1587
-1597.
Ang, S. L. and Rossant, J. (1993). Anterior
mesendoderm induces mouse Engrailed genes in explant cultures.
Development 118,139
-149.
Bachiller, D., Klingensmith, J., Kemp, C., Belo, J. A., Anderson, R. M., May, S. R., McMahon, J. A., McMahon, A. P., Harland, R. M., Rossant, J. et al. (2000). The organizer factors Chordin and Noggin are required for mouse forebrain development. Nature 403,658 -661.[CrossRef][Medline]
Beddington, R. S. P. and Lawson, K. A. (1990). Clonal analysis of cell lineages. In Postimplantation Mammalian Embryos A Practical Approach (ed. A. J. Copp. and D. L. Cockroft), pp. 267-291. Oxford: Oxford University Press.
Bouillet, P., Oulad-Abdelghani, M., Ward, S. J., Bronner, S., Chambon, P. and Dollé, P. (1996). A new mouse member of the Wnt gene family, mWnt-8, is expressed during early embryogenesis and is ectopically induced by retinoic acid. Mech. Dev. 58,141 -152.[CrossRef][Medline]
Charité, J., de Graaff, W., Shen, S. and Deschamps, J. (1994). Ectopic expression of Hoxb8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures. Cell 78,589 -601.[Medline]
Deschamps, J. and Wijgerde, M. (1993). Two phases in the establishment of HOX expression domains. Dev. Biol. 156,473 -480.[CrossRef][Medline]
Deschamps, J., van den Akker, E., Forlani, S., de Graaff, W., Oosterveen, T., Roelen, B. and Roelfsema, J. (1999). Initiation, establishment and maintenance of Hox gene expression patterns in the mouse. Int. J. Dev. Biol. 43,635 -650.[Medline]
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 Morata, G. (1994). Colinearity and functional hierarchy among genes of the homeotic complexes. Trends Genet. 10,358 -364.[CrossRef][Medline]
Frohman, M. A., Boyle, M. and Martin, G. R. (1990). Isolation of the mouse Hox-2.9 gene; analysis of embryonic expression suggests that positional information along the anterior-posterior axis is specified by mesoderm. Development 110,589 -607.[Abstract]
Gaunt, S. J. (2001). Gradients and forward spreading of vertebrate Hox gene expression detected by using a Hox/lacZ transgene. Dev. Dyn. 221, 26-36.[CrossRef][Medline]
Gaunt, S. J., Sharpe, P. T. and Duboule, D. (1988). Spatially restricted domains of homeo-gene transcripts in mouse embryos: relation to a segmented body plan. Development 104,169 -179.
Gaunt, S. J. and Strachan, L. (1994). Forward spreading in the establishment of a vertebrate Hox expression boundary: the expression domain separates into anterior and posterior zones, and the spread occurs across implanted glass barriers. Dev. Dyn. 199,229 -240.[Medline]
Gellon, G. and McGinnis, W. (1998). Shaping animal body plans in development and evolution by modulation of Hox expression patterns. BioEssays 20,116 -125.[CrossRef][Medline]
Gilthorpe, J., Vandromme, M., Brend, T., Gutman, A., Summerbell, D., Totty, N. and Rigby, P. W. (2002). Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY. Development 129,3887 -3899.[Medline]
Gould, A., Itasaki, N. and Krumlauf, R. (1998). Initiation of rhombomeric Hoxb4 expression requires induction by somites and a retinoid pathway. Neuron 21, 39-51.[Medline]
Herrmann, B. G., Labeit, S., Poustka, A., King, T. R. and Lehrach, H. (1990). Cloning of the T gene required in mesoderm formation in the mouse. Nature 343,617 -622.[CrossRef][Medline]
Hogan, B., Beddington, R. and Constantini, F. (1994). Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Ikeya, M. and Takada, S. (2001). Wnt-3a is required for somite specification along the anteroposterior axis of the mouse embryo and for regulation of cdx-1 expression. Mech. Dev. 103,27 -33.[CrossRef][Medline]
Kondo, T. and Duboule, D. (1999). Breaking colinearity in the mouse HoxD complex. Cell 97,407 -417.[Medline]
Krumlauf, R. (1994). Hox genes in vertebrate development. Cell 78,191 -201.[Medline]
Lawson, K. A., Meneses, J. J. and Pedersen, R. A. (1991). Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development 113,891 -911.[Abstract]
Lawson, K. A. and Pedersen, R. A. (1992). Early mesoderm formation in the mouse embryo. In Formation and Differentiation of Early Embryonic Mesoderm (ed. R. Bellairs, E. J. Sanders and J. W. Lash), pp. 33-46. New York: Plenum Press.
Liu, P., Wakamiya, M., Shea, M. J., Albrecht, U., Behringer, R. R. and Bradley, A. (1999). Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22, 361-365[CrossRef][Medline]
Maloof, J. N., Whangbo, J., Harris, J. M., Jongeward, G. D. and
Kenyon, C. (1999). A Wnt signaling pathway controls hox gene
expression and neuroblast migration in C. elegans.
Development 126,37
-49.
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]
Mathis, L. and Nicolas, J. F. (2000). Different
clonal dispersion in the rostral and caudal mouse central nervous system.
Development 127,1277
-1290.
Mathis, L., Kulesa, P. M. and Fraser, S. E. (2001). Fgf receptor signalling is required to maintain neural progenitors during Hensen's node progression. Nat. Cell Biol. 3,559 -566.[CrossRef][Medline]
Meier, S. and Tam, P. P. (1982). Metameric pattern development in the embryonic axis of the mouse. I. Differentiation of the cranial segments. Differentiation 21, 95-108.[Medline]
Murphy, P. and Hill, R. E. (1991). Expression of the mouse labial-like homeobox-containing genes, Hox 2.9 and Hox 1.6, during segmentation of the hindbrain. Development 111, 61-74.[Abstract]
Oosterveen, T., Niederreither, K., Dolle, P., Chambon, P.,
Meijlink, F. and Deschamps, J. (2003). Retinoids regulate the
anterior expression boundaries of 5' Hoxb genes in posterior hindbrain.
EMBO J. 22,262
-269.
Partanen, J., Schwartz, L. and Rossant, J.
(1998). Opposite phenotypes of hypomorphic and Y766
phosphorylation site mutations reveal a function for Fgfr1 in anteroposterior
patterning of mouse embryos. Genes Dev.
12,2332
-2344.
Perea-Gomez, A., Lawson, K. A., Rhinn, M., Zakin, L., Brulet,
P., Mazan, S. and Ang, S. L. (2001). Otx2 is required for
visceral endoderm movement and for the restriction of posterior signals in the
epiblast of the mouse embryo. Development
128,753
-765.
Riese, J., Yu, X., Munnerlyn, A., Eresh, S., Hsu, S. C., Grosschedl, R. and Bienz, M. (1997). LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic. Cell 88,777 -787.[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 .[CrossRef][Medline]
Sharpe, J., Nonchev, S., Gould, A., Whiting, J. and Krumlauf,
R. (1998). Selectivity, sharing and competitive interactions
in the regulation of Hoxb genes. EMBO J.
17,1788
-1798.
Snedecor, G. W. and Cochran, W. G. (1967). Statistical Methods, 6th edn. Iowa: Iowa State University Press.
Studer, M., Popperl, H., Marshall, H., Kuroiwa, A. and Krumlauf, R. (1994). Role of a conserved retinoic acid response element in rhombomere restriction of Hoxb-1. Science 265,1728 -1732.[Medline]
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R.
(1999). Targeted disruption of Fgf8 causes failure of cell
migration in the gastrulating mouse embryo. Genes Dev.
13,1834
-1846.
Tam, P. P. L. (1988). The allocation of cells in the presomitic mesoderm during somite segmentation in the mouse embryo. Development 103,379 -390.[Abstract]
Tam, P. P. L. (1989). Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation. Development 107, 55-67.[Abstract]
Tam, P. P. L. and Beddington, R. S. P. (1987). The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development 99,109 -126.[Abstract]
Tomotsune, D., Shirai, M., Takihara, Y. and Shimada, K. (2000). Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the Polycomb group of genes. Mech. Dev. 98,165 -169.[CrossRef][Medline]
van den Akker, E., Forlani, S., Chawengsakshophak, K., de
Graaff, W., Beck, F., Meyer, B. and Deschamps, J. (2002).
Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and
posterior axis elongation. Development
129,2181
-2193.
Wilkinson, D. G., Bhatt, S., Cook, M., Boncinelli, E. and Krumlauf, R. (1989). Segmental expression of Hox-2 homoeobox-containing genes in the developing mouse hindbrain. Nature 341,405 -409.[CrossRef][Medline]
Wilkinson, D. G. and Krumlauf, R. (1990). Molecular approaches to the segmentation of the hindbrain. Trends Neurosci. 13,335 -339.[CrossRef][Medline]
Yu, B. D., Hanson, R. D., Hess, J. L., Horning, S. E. and
Korsmeyer, S. J. (1998). MLL, a mammalian
trithorax-group gene, functions as a transcriptional maintenance
factor in morphogenesis. Proc. Natl. Acad. Sci. USA
95,10632
-10636.
Zakany, J., Kmita, M., Alarcon, P., de la Pompa, J. L. and Duboule, D. (2001). Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock. Cell 106,207 -217.[Medline]