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First published online 28 May 2008
doi: 10.1242/dev.022020
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1 The Roslin Institute and Royal Dick School of Veterinary Studies, University
of Edinburgh, Roslin, Midlothian, UK.
2 Oxford Biomedica (UK) Ltd, Medawar Centre, Oxford Science Park, Oxford,
UK.
3 Institute for Stem Cell Research, MRC Centre for Regenerative Medicine,
University of Edinburgh, Edinburgh, UK.
Author for correspondence (e-mail:
helen.sang{at}bbsrc.ac.uk)
Accepted 29 April 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Progenitor cell, Tail bud, Chordoneural hinge, Hox genes, Transgenic chicken
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gaertner, 1949;
Seichart and Jelinek, 1968)
(reviewed by Stern et al.,
2006). The primitive streak and tail bud thus supply cells to the
neural tube and mesoderm over the entire post-cranial axis
(Schoenwolf, 1977;
Schoenwolf, 1979a;
Catala et al., 1995;
Psychoyos and Stern, 1996).
Tail bud outgrowth differs morphologically from earlier gastrula stages in
several respects. Formation of the neural tube in the tail bud results from
cavitation of the medullary cord, and is distinct from primary neurulation,
which occurs by the folding and fusion of the dorsal edges of the neural plate
(Criley, 1969;
Kilka and Jelinek, 1969;
Schoenwolf and Delongo, 1980).
The nascent embryonic chick tail subsequently undergoes reduction and
remodeling by selective proliferation, involution and cell death
(Lanot, 1980;
Schoenwolf, 1981;
Sanders et al., 1986;
Uehara and Ueshima, 1988;
Miller and Briglin, 1996).
Despite these differences, similarities in gene expression patterns in the
primitive streak and tail bud (Gont et
al., 1993; Gofflot et al.,
1997; Knezevic et al.,
1998; Cambray and Wilson,
2007), and the analysis of morphogenic movements in the streak and
tail bud (Pasteels, 1937; Gont et al.,
1993; Catala et al.,
1995; Kanki and Ho,
1997; Knezevic et al.,
1998), reveal many commonalities between these two processes in
several vertebrate species. Additionally, mutation studies show that several
genes, including members of the Wnt gene family and brachyury, have a role
during both primitive streak and tail bud outgrowth in mouse
(Herrmann et al., 1990;
Greco et al., 1996;
Yamaguchi et al., 1999) and
zebrafish (Marlow et al.,
2004). This evidence supports the view, as first proposed by
Pasteels (Pasteels, 1943),
that many aspects of tail bud outgrowth are continuations of the gastrulation
process.
Further morphological, gene expression and cell lineage studies have shown
that distinct sub-domains of the primitive streak are analogous to those in
the tail bud in chicken (Catala et al.,
1995; Knezevic et al.,
1998), mouse (Cambray and
Wilson, 2002; Cambray and
Wilson, 2007) and Xenopus
(Gont et al., 1993;
Tucker and Slack, 1995;
Davis and Kirschner, 2000).
The node region (equivalent to the organizer in Xenopus and
zebrafish) contains progenitor cells of the neural tube, notochord and
somites. It also gives rise to cells that contribute to the chordoneural hinge
(CNH) in the tail bud, a region where the posterior end of the notochord abuts
the overlying neural tube (Pasteels,
1943). Like the earlier node, the CNH contributes progeny to the
neural tube, notochord and paraxial mesoderm
(Catala et al., 1995;
Davis and Kirschner, 2000;
Cambray and Wilson, 2002). The
continuous generation of neural tube, notochord and somites by the node region
and CNH, which apparently contain resident cells, is consistent with the
hypothesis that these regions contain multipotent stem cells, i.e. cells that
are capable of giving rise to both further axial progenitor cells in the
streak/tail bud and differentiated cells of multiple lineages in the axis.
A number of reports lend support to this idea. Specifically, single-cell
labeling experiments in the chick and mouse node during gastrulation suggest
that some cells are resident there, contributing descendants over significant
axial stretches to notochord, neural tube and somites
(Selleck and Stern, 1991;
Selleck and Stern, 1992;
Lawson et al., 1991).
Retrospective clonal analysis in the mouse also indicates that the progenitors
of the myotome (a somite derivative) and spinal cord undergo stem cell
divisions (Nicolas et al.,
1996; Mathis and Nicolas,
2000; Eloy-Trinquet and
Nicolas, 2002; Roszko et al.,
2007). Technical issues have so far prevented prospective
single-cell lineage analyses on the node's descendant in the tail bud, the
CNH. However, grafting experiments suggest that this region may contain the
stem cell progenitors of these tissues, as inferred from the retrospective
studies mentioned above. Serial grafts of the CNH to early somite-stage
embryos result in continued retention of graft-derived cells in the CNH, as
well as exit of cells and contribution along the body axis
(Cambray and Wilson, 2002).
Despite the evidence that individual cells reside in the streak and
contribute over long axial distances, these cells, elsewhere termed `stem
cells', do not strictly self-renew (i.e. give rise to exact copies of
themselves) in vivo, as their gene expression changes over time
(Iimura and Pourquié,
2006; Cambray and Wilson,
2007). For this reason, and because the processes of primary and
secondary body development are different, we refer to resident cells as
long-term axial progenitors (LTAPs), and to a population of cells that behaves
in this way as a LTAP population. Short-term axial progenitors (STAPs) are
cells in the streak/tail bud that exit these regions and populate limited
axial regions. It should be noted that the term `LTAP population' is
conceptually equivalent to the neural `stem zone', defined in chick, where
labeled groups of cells give rise to descendants in both the neural tube and
the tail bud; the population as a whole thus behaves as if it contains
resident cells (Brown and Storey,
2000).
When mouse tail bud CNH progenitor cells, which would normally populate
posterior tail somites, were grafted to the node/primitive streak region in
earlier embryos, they populated more anterior axial levels, suggesting that
they were not committed to a particular axial level. Such heterochronic grafts
contributed to more posterior somites than did isochronic grafts placed at the
same site, suggesting that they were not completely equivalent to the earlier
streak progenitors (Tam and Tan,
1992; Cambray and Wilson,
2002). Hox expression provides an important component of the
anteroposterior identity of axial cells (reviewed by
Deschamps and van Nes, 2005),
and can control the timing of ingression of mesodermal precursors from the
epiblast (Iimura and Pourquié,
2006). It is therefore of interest to establish whether Hox gene
expression is determined in axial progenitor cells.
We investigated whether LTAP populations are present in the chicken tail bud during its outgrowth. To facilitate the transplantation of defined populations of cells, we produced transgenic chickens that express the fluorescent protein GFP in every cell of the early embryo. This allowed us to follow transplanted cells temporally and to re-isolate them for transplantation to new host embryos. In a series of grafting experiments, we show that the chicken tail bud contains distinct spatially localised populations of both LTAPs and STAPs. We experimentally confirm the close similarity between the fate maps of mouse and chick tail buds, and we show that tail bud progenitor cells transplanted to earlier primitive streaks can reset their Hox identity to match their new environment.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) with SalI and XhoI (blunt-ended with
T4 DNA polymerase), and ligating the 1.7 kb fragment to a 0.8 kb fragment
containing GFP isolated from pONY8.0G
(Mazarakis et al., 2001) by
digestion with SacII (blunt-ended with T4 DNA polymerase) and
HincII. This was inserted into the 5.4 kb pONY8.45NCZ backbone (F. J.
Wilkes, J. B. Rohll, P.A.R, M. Azzouz, J. E. Miskin, F.M.E., L. E. Walmsley,
N. D. Mazarakis, S. M. Kingsman and K.A.M., unpublished) following excision of
the CMV-lacZ cassette with EcoRI (blunt-ended with T4 DNA
polymerase). Viral vector stocks pseudotyped with vesicular stomatitis virus
glycoprotein envelope (pCMV-VSVG) were prepared using the HEK293T transient
system (Lillico et al., 2007).
The titer of virus [2x107 transducing units (TU)/ml] was
estimated by the transduction of D17 cells.
Production and analysis of transgenic birds
Transgenic chickens were generated as described
(McGrew et al., 2004). Eggs
(39) were injected with viral vector and 16 chicks were hatched. Genomic DNA
samples were obtained from CAM of chicks at hatch, and blood and semen samples
from older birds. PCR analysis was carried out for the presence of proviral
DNA. Eight transgenic founder birds were identified. Two founder cockerels
(1-1 and 3-11) were crossed to wild-type hens and seven transgenic
G1 offspring were generated (7/511 offspring). The number and size
of the proviral insertions in G1 birds was determined by Southern
blot analysis (see Fig. S1 in the supplementary material). Genomic DNA was
extracted from whole blood and digested with EcoRI or StuI.
DNA was resolved on a 0.6% (w/v) agarose gel then transferred to nylon
membrane (Hybond-N, Amersham Pharmacia Biotech). Membranes were hybridised
with 32P-labelled probes for the GFP reporter gene at 65°C, and
labelled DNA detected by autoradiography. Two G1 birds (3-11:205
and 1-1:158) were highly fluorescent, contained single-copy integrants, and
were used to produce G2 transgenic embryos. All experiments, animal
breeding and care procedures were carried out under licence from the UK Home
Office.
GFP expression analysis and flow cytometry
Embryos were staged according to Hamburger and Hamilton
(Hamburger and Hamilton, 1951)
(HH). Embryos were observed using a Leica MZFLIII florescent stereomicroscope
and images captured on a Leica DC300F digital camera. For colocalisation of
Hox gene expression, fluorescence was detected using laser excitation
wavelengths of 488 nm and 543 nm for GFP and Alexa-Fluor 546, respectively,
using an inverted confocal microscope (Nikon eC1; Nikon Instruments). Images
were captured using Nikon EZ-C1 Software v3.40. Flow cytometry was performed
using a Becton Dickinson FACSAria, equipped with a standard filter set, and
Diva analysis software. Stage 11 HH GFP-positive embryos were dissociated
using trypsin, and live cells were gated by propidium iodide exclusion. Cells
from non-transgenic embryos were used to define gating parameters for GFP
fluorescence expression. Data were acquired for 25,000 live events.
Immunohistochemistry and in situ hybridisation of chick embryos
For sections, embryos were isolated and fixed for 30 minutes in 4%
paraformaldehyde/PBS. Tissues were cryo-embedded and sectioned at 14 µm.
Sections were incubated for 30 minutes at 37°C in PBS to remove gelatin.
As this treatment extinguished most of the GFP fluorescence, an Alexa-Fluor
488-conjugated rabbit anti-GFP antibody was used (Molecular Probes; 1:1000
dilution). Basement membranes were stained using rabbit anti-laminin (Sigma;
1:500 dilution). Differentiated neurons were detected with TUJ1/2 (Avance;
1:1000 dilution). Antibodies to chicken Hoxc8 (Abcam, UK) and chicken Hoxc10
were used at 1:150 and 1:20 dilutions, respectively. The antibody to Hoxc10,
developed by T. Jessell, was obtained from the Developmental Studies Hybridoma
Bank, University of Iowa. The secondary antibodies used were Alexa-Fluor
594-conjugated goat anti-rabbit IgG, Alexa-Fluor 594-conjugated goat
anti-rabbit IgG, and Alexa-Fluor 546-conjugated goat anti-mouse IgG (Molecular
Probes). Slides were counterstained with Hoechst 33342, and mounted.
Whole-mount in situ hybridisation was carried out as described previously
(Henrique et al., 1995).
GFP mRNA was detected using a full-length probe to GFP. The
riboprobe to Hoxa10 was described by Burke et al.
(Burke et al., 1995).
Hoxa10 in situ hybridisations to grafted embryos after a 1- or 8-hour
incubation were performed in the same well and stained for the same period of
time.
Chicken grafting experiments
CAG-GFP cockerels (Roslin Greens) were mated to wild-type hens to obtain
transgenic G2 eggs. Fertilised eggs were incubated at 38°C
until the tail bud stage (26 somites, stage 15 HH). GFP-positive embryos were
staged and the caudal end of the embryo was isolated into CM1 Media (DMEM
containing 10% FBS). Two dorsoventral incisions were made using tungsten
needles alongside the neural tube to remove the paraxial mesoderm. A
mediolateral cut was made to remove the endoderm underlying the CNH region,
which was subsequently isolated. A region more caudal to this was isolated
from the posterior ectoderm as ventral tail bud mesoderm (TBM). A region
dorsal to this was dissected from the overlying ectoderm as the dorsal
posterior tail bud (dpTB). The isolated regions were dissected into smaller
pieces that were sized using a graticule. To determine the number of cells
being grafted, control pieces of tissue were dissociated in trypsin and cells
counted. Host embryos were inked and staged. A tungsten needle was inserted
into the CNH, dpTB or ventral tail bud (vTB) regions and the tissue to be
grafted was inserted into this hole. For grafts to stage 8 HH, a small
incision was made just caudal to the node using a tungsten needle and the
tissue to be grafted was inserted into the slit. Grafted embryos were
photographed for GFP fluorescence. Grafted embryos were photographed, usually
after removal of the hind limbs, and processed for in situ hybridisation or
immunohistochemistry.
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) (`GFP transgenic') x MF1 litters were dissected in M2
medium. The whole tail bud was isolated and the dorsoposterior tail bud was
isolated using fine glass needles. First, the end of the tail was excised, and
two dorsoventral longitudinal cuts made to remove the paraxial mesoderm. The
ventral neurectoderm, notochord, hindgut and adjacent ventral TBM were removed
by similar longitudinal cuts in the mediolateral plane. Finally, the dorsal
tail bud was separated by a transverse cut posterior to the dorsal
neurectoderm and the surface ectoderm was removed. Dissection of wild-type MF1
host embryos, grafts and culture were performed as described previously
(Cambray and Wilson, 2002).
After culture, embryo fluorescence was assessed and embryos were processed as
described (Cambray and Wilson,
2007). |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). By
contrast, high-level, widespread expression has been achieved using a compound
CMV enhancer and a chicken β-actin promoter/intron (CAG)
(Niwa et al., 1991) in several
species: Xenopus (Sakamaki et
al., 2005), mouse (Okabe et
al., 1997) and axolotl (Sobkow
et al., 2006). To create chickens carrying a heritable, vital
marker of general utility, we constructed a lentiviral vector containing a
CAG-eGFP reporter (Fig. 1A),
and used this to generate two independent lines of transgenic chickens (see
Materials and methods). Transgenic G1 cockerels were crossed to wild-type hens and the resulting embryos from incubated eggs were observed at several developmental stages. Intense GFP fluorescence was observed in CAG-GFP transgenic embryos, unlike in the previous CMV-GFP transgenic lines (see Fig. S2 in the supplementary material). GFP fluorescence was readily detected throughout the blastodermal disc in freshly laid transgenic eggs (Fig. 1B). Widespread GFP fluorescence was visible in embryos at later stages of development and section analysis confirmed its distribution throughout all tissues (Fig. 1C-G). This ubiquitous expression was verified by flow cytometric analysis of cells from dissociated day 2 (stage 11 HH) embryos. GFP fluorescence was detected in greater than 99% of the cells at this stage of development (Fig. 1I). In situ hybridisation analysis performed on day 2 (stage 14 HH) embryos additionally confirmed that the CAG-GFP transgene was actively transcribed throughout the embryo and extraembryonic regions (Fig. 1H). Together, these observations indicate that the GFP reporter gene is expressed ubiquitously in embryos up to day 5 of development. Cells from these transgenic embryos are useful in grafting experiments to follow cell populations over long periods, and highly suitable for the study of putative stem cell populations. We have therefore used this line to investigate the progenitors of the anteroposterior axis in the chicken primitive streak and tail bud.
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). To
identify the analogous regions in the chicken, a morphological comparison of
the early tail bud in mouse and chicken was carried out
(Fig. 2). The nascent mouse
tail bud at 10.5 days post-coitum (dpc) is morphologically similar to that of
the chicken (Fig. 2B,C). The
chicken tail bud stage begins with the closing of the posterior neuropore at
day 2 of development [stage 13-14 HH; 19-23 somites
(Schoenwolf, 1979b)]. Several
hours later (stage 15 HH, 
26 somites), the posterior-ventral surface of
the tail bud becomes delimited by the cloacal membrane [early tail fold stage
(Schoenwolf, 1977); arrow,
Fig. 2B]. This ventral tail bud
boundary aided the isolation and positioning of transplanted tissues, so this
stage was used for subsequent experiments. The CNH region is the area where
the neural tube and notochord become indistinct from each other and from the
surrounding mesoderm (Fig.
2A-B'). Staining for the extracellular matrix protein
laminin identifies an area just anterior to the CNH in mouse and chicken,
where a basement membrane forms between the nascent notochord and the
floorplate of the neural tube (Fig.
2B',C'). Closer morphological inspection of the
posterior tail bud identifies the dorsal epithelium of the neural tube
(medullary cord), which runs further posteriorly than does the CNH in both
mouse and chick, eventually becoming indistinct from the TBM. This region
comprises the remnant of the stem zone, which contains neural progenitor cells
for the spinal cord of the tail bud (Catala
et al., 1995;
Delfino-Machín et al.,
2005; Diez del Coral et al.,
2004). A striking difference between the chicken and the mouse is
the lack of laminin staining of the posterior surface ectoderm of the chicken
tail bud at these stages (arrowheads, Fig.
2B',C') (Ohta et
al., 2007). Ventrally, the epithelium posterior to the CNH appears
to merge with loose mesenchyme of the TBM in both organisms. Therefore, the
dorsoposterior tail bud (dpTB) has different characteristics from the more
ventral, purely mesenchymal, ventral tail bud (vTB) region, and from the
mainly epithelial CNH. The posterior tail bud has been shown to give rise to
tail somites in chicken (Catala et al.,
1995), but the presence of dorsoventral compartments and their
capacity to act as LTAPs has not been tested in grafting assays in either
organism. To address this question, we performed both isochronic and
heterochronic grafts of the three tail bud regions in the chick (as indicated
in Fig. 2B'): the region
of the CNH where the dorsal basement membrane of the notochord will first
form; a more dorsoposterior region of the tail bud posterior to the medullary
cord (dpTB); and the vTB region.
The tail bud consists of at least three regions of differing fate
We first determined the normal fate of the three regions described above by
homotopic, isochronic grafts in stage 15 HH embryos of GFP-positive
(GFP+) transgenic cells to wild-type hosts. Host embryos were
examined after 48 hours incubation (stage 24 HH). Grafts of the CNH
(approximately 100-150 cells) resulted in GFP+ descendants
extending from the hind limb to the tail bud in the neural tube (most
prominently in the floor plate), and the paraxial mesoderm, and a few cells
located in the notochord (Fig.
3B',B''; Table
1); a population of cells also remained in the CNH region
(n=8/10; Fig.
3A''). Grafts of dpTB containing approximately 100-150 cells
resulted in abnormally formed tail buds (n=3, data not shown). Grafts
of smaller pieces of tissue (approximately 50 cells) resulted in normally
patterned host embryos in which GFP+ cells were located along the
body axis in a region extending from the hind limb to the tail bud
(n=7/7; Fig.
3C'; Table
1), in the paraxial mesoderm
(Fig. 3C''). Grafts of vTB
(approximately 100-150 cells) gave rise to short stretches of paraxial
mesoderm (average of six somites) and GFP+ cells did not remain in
the tail bud (n=0/6; Fig.
3D',D''; Table
1).
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Differently fated regions show different potency on heterotopic grafting
Next, we compared the potency of the three regions by heterotopic,
isochronic grafting to each of the three environments at stage 15 HH, and
incubation for 48 hours, as above. Grafts to the CNH and vTB contained
approximately 100-150 cells, and all grafts to the dpTB contained
approximately 50 cells.
The CNH changed fate on grafting to the dpTB or vTB environment, such that only somitic mesoderm was produced, and GFP+ cells in the mesoderm were negative for the neuronal marker TUJ1 (Fig. 4A,B; Table 1). Cells were retained in the tail bud after grafting CNH to dpTB, but only approximately half of the grafts to vTB (n=4/7) contributed to the tail bud, suggesting that they may partially lose LTAP potency in this environment. The dpTB was not converted to a CNH-like character on grafting to the CNH, as neural descendants were almost completely absent from these grafts. Likewise, these grafts did not produce descendants in the notochord (Fig. 4C; Table 1). When grafted to the vTB, no dpTB cells were retained in the tail bud, showing that these cells lose their LTAP status in this environment (Table 1). vTB cells similarly did not contribute to the neural tube or notochord on grafting to the CNH, and neither the CNH nor the dpTB environment was able to induce their residence in the tail bud (Fig. 4E,F; Table 1). Instead, these grafts gave rise to short stretches of somitic mesoderm. Similarly, grafts of posterior presomitic mesoderm to the CNH region generated short tracts of somitic mesoderm (n=6/6) (data not shown). Together with the fate mapping studies above, these results suggest that: (1) the CNH is adaptable to new environments but is less likely to lose LTAP status than the dpTB; (2) the dpTB is more limited in its potency to produce multiple axial derivatives than the CNH, and readily loses LTAP status in the vTB environment; and (3) the vTB does not have long-term axial progenitor potency.
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;
Psychoyos and Stern, 1996;
Charrier et al., 1999;
Freitas et al., 2001).
Heterochonic grafts of either stage 15 HH CNH (n=11/11) or dpTB
(n=5/5) resulted in a long stretch of GFP+ cells along the
body axis, running from the forelimb level to the tail bud
(Fig. 5D,E). The CNH
contributed cells to neurectoderm and paraxial mesoderm, similar to homotopic
grafts (Fig. 5D'),
whereas descendants of the dpTB were found exclusively in the paraxial
mesoderm (Fig. 5E'). By
contrast, grafts of vTB contributed cells to a short series of somites along
the body axis and did not repopulate the tail bud (n=4/5;
Fig. 5F,F'). These
experiments demonstrate that both the CNH and dpTB appear to act as LTAPs in
heterochronic graft assays. We tested whether the structural equivalent of the
dpTB in the mouse (Fig.
2C') also contains apparent LTAPs with exclusively somitic
potential. GFP+ tissue from this region was grafted to the
node/streak border of E8.5 (3-5 somite) mouse embryos and cultured for 48
hours in vitro. GFP+ cells contributed exclusively to somites, and
in the tail bud were found in the TBM, not the CNH (n=6/6; see Fig.
S3A,B in the supplementary material). Therefore, a similar population to the
chick dpTB exists in mouse.
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|
;
Dubrulle et al., 2001)
(Fig. 7D'). The protein
of a paralogous Hox gene, Hoxc10, is also present in the CNH, dpTB
and TBM of stage 15 HH embryos (Fig.
7A'). No expression was detected immediately anterior to the
CNH in the forming notochord. We investigated whether the progenitor cell populations of the tail bud maintained their Hox gene expression pattern when challenged by heterochronic grafting of stage 15 HH CNH or dpTB to the caudal node region of stage 8 HH embryos, as above (Fig. 5). In situ hybridisation analysis shortly after grafting confirmed that the grafted tissue expressed Hoxa10 (n=7/7; Fig. 7B,B'). However, after an 8-hour incubation, the grafted tissue, like the surrounding host tissue, did not have detectable signal for Hoxa10 (n=9/10; Fig. 7C,C'). After 48 hours, no ectopic anterior Hoxa10 expression was observed in grafted embryos in cells derived from either CNH or dpTB grafts (n=9/9; Fig. 7D,D'). Immunostaining for Hoxc10 protein in similarly grafted embryos after 48 hours revealed that the majority of anterior GFP+ cells were no longer positive for this protein, although a few cells, generally in small clusters, showed some staining (Fig. 7E,E'). All Hoxc10 labeling was lost in anterior GFP+ cells after four days of incubation (data not shown). GFP+ cells in the posterior hindlimb region, as in control isochronic grafted embryos, were Hoxc10 positive (Fig. 7I-J'), like their wild-type neighbours. Therefore, the expression of posterior Hox genes in tail bud progenitor cells is downregulated shortly after transplant to an anterior environment in which these genes are not expressed. Expression is then correctly activated in posterior, but not anterior, graft derivatives.
To determine whether graft-derived cells in the anterior part of the axis
were capable of expressing any Hox gene correctly, we tested whether these
cells expressed a more anterior Hox gene, Hoxc8. Hoxc8 protein is first
detected in the neural tube and the surrounding paraxial mesoderm in the
brachial region (somite 22) at stage 21-24 HH
(Belting et al., 1998)
(Fig. 7F'). In control
isochronic grafts of GFP+ tissue to the anterior node region,
GFP+ cells located in anterior Hoxc8-expressing regions were Hoxc8
positive (Fig. 7G,G').
Heterochronic grafts, performed as above, were incubated for four days (stage
24 HH) and then GFP+ cells in the paraxial mesoderm at this axial
level were examined for the presence of Hoxc8 protein. GFP+ cells
located in anterior Hoxc8-expressing regions contained detectable Hoxc8
(n=5 embryos, Fig.
7H,H'). Together, these experiments show that tail bud
progenitor cells can reset their Hox gene expression to match that of their
surrounding tissue.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A new tool for avian fate mapping
Seminal research by Le Douarin and colleagues was facilitated by the
development and use of quail-chick chimeras for fate mapping experiments
(Le Douarin, 1969). Detection
of graft derivatives requires fixation of the specimens. Electroporation and
infection using viruses allows transient expression of vital markers, such as
GFP (reviewed by Ishii and Mikawa,
2005), but these techniques are unable to produce long-term
heritability of the marker gene. Lentiviral vectors were recently used to
generate germ line transgenic chickens with high efficiency, including a line
incorporating a CMV-GFP transgene, which showed very limited expression in
embryos (McGrew et al., 2004)
(see Fig. S2 in the supplementary material). The CAG-GFP transgene described
here drives ubiquitous expression of GFP during chicken embryogenesis, at
higher levels than in a previously described line of transgenic chickens
(Chapman et al., 2005). This
CAG-GFP line will be a powerful resource for investigations in the chicken
requiring tracking of cells in vivo over long periods.
Progenitor populations in the tail bud
Our data support and extend previous research demonstrating that a resident
population of LTAPs is located in the CNH of the mouse tail bud using
heterochronic grafts to early somite stage embryos
(Cambray and Wilson, 2002). By
comparing chick and mouse dpTB in this study, we complete these data to show a
highly similar organisation of progenitors, despite some differences in tail
bud morphology. Only heterochronic grafts were possible in the mouse to test
the capacity of tail bud regions to act as long-term progenitors, because
mouse embryos can be manipulated and cultured for only a limited period. In
the chick it has been possible to perform both heterochronic and isochronic
grafts, and thus to extend the findings from mouse. The fact that the pattern
of axial tissue and tail bud contribution is very similar whether grafts are
isochronic or heterochronic suggests a continuity of cell functions over long
periods of axis elongation. This correlates well with the expression of
primitive streak markers in tail bud regions, and indeed in several organisms
it has already been noted that the gene expression patterns highlight a
structural continuity between the primitive streak, or its equivalent, and the
tail bud (Delfino-Machín et al.,
2005; Cambray and Wilson,
2007). These expression studies also show that the dpTB expresses
markers characteristic of the primitive streak. As primitive streak and dpTB
share the property that they produce mesoderm exclusively, even when
transplanted to a region that produces neural and notochord derivatives
(Cambray and Wilson, 2007)
(Fig. 5,
Table 1), this molecular
similarity between the streak and tail bud also correlates with a functional
continuity.
At least early on, the node region can give rise to cells in the primitive
streak (Forlani et al., 2003).
We have observed that the CNH can give rise to cells in the dpTB after
homotopic grafting (data not shown), but the converse was not seen. Therefore,
at least some dpTB cells may derive from the CNH, raising the possibility that
CNH cells are a more primitive progenitor type than are the dpTB cells. This
is supported by the low potential of cells of the dpTB to remain in the tail
bud following multiple passages. Our experiments, because they deal with
populations rather than single cells, are not informative about the
progression of lineage restriction in these tail bud domains, and, in
particular, cannot distinguish between multipotent progenitors in the CNH and
several lineage-restricted populations.
Retrospective lineage analysis in the mouse suggests that axial progenitor
cells are restricted in their contribution to the somite along the
mediolateral axis (Eloy-Trinquet and
Nicolas, 2002). In our experiments, homotopic grafts in the tail
bud produced GFP+ descendants bilaterally in both the medial and
lateral compartments of the somite. However, homotopic grafts of the CNH and
dpTB gave rise to a greater proportion of GFP+ cells in medial
somitic derivatives than did grafts to the vTB (Figs
3,
4). Cells placed
heterotopically in the CNH or heterochronically in the node generally
contributed to the medial somitic compartment, demonstrating that cells
destined for lateral somitic regions (i.e. vTB cells) are constrained by the
CNH to exit to medial locations (see Fig.
5C'-F'; see Fig. S3 in the supplementary material). As
the CNH is mostly composed of node/streak border derivatives that exit this
region to medial somite regions (Selleck
and Stern, 1991; Selleck and
Stern, 1992; Catala et al.,
1996; Psychoyos and Stern,
1996; Charrier et al.,
1999; Freitas et al.,
2001; Cambray and Wilson,
2007; Iimura and
Pourquié, 2007), this pattern of exit is also strikingly
conserved through axis elongation. It is not certain whether the progenitors
of lateral somitic regions are also LTAPs in an, as yet, untested region, or
whether the short-term contributing cells in the vTB make up the bulk of
lateral somite progenitors.
Hox identity is plastic in tail bud progenitor cells
We demonstrate that Hox gene expression is not determined in progenitor
cells in the tail bud. Hox identity in the paraxial mesoderm is known to
become fixed before somite formation
(Kieny et al., 1972;
Nowicki and Burke, 2000;
Omelchenko and Lance-Jones,
2003). Similarly, Iimura and Pourquié observed the
maintenance of Hoxb9 expression in primitive streak cells at a 6-hour
timepoint after transplant to a younger streak not expressing Hoxb9
(Iimura and Pourquié,
2006), suggesting that these cells retain their anteroposterior
identity. It is possible that our timepoint (8 hours) just bypasses this
period, or that the size of the graft is crucial for downregulation owing to
cell contact with non-expressing neighbours. The latter phenomenon has been
observed with Hox-expressing cells in the hindbrain, where clumps of
heterotopically grafted cells maintained their Hox expression profile, while
those that became isolated from the main clump and were surrounded with
non-expressing cells downregulated Hox expression
(Trainor and Krumlauf, 2000).
We observed some residual Hoxc10 expression several days after
transplantation, associated with clumps of GFP+ cells rather than
dispersed cells (Fig. 7D),
indicating that cell-cell interactions may be important in maintaining or
modulating Hox gene expression. Interestingly, Iimura and Pourquié
proposed that activation of sequentially more posterior Hox genes in subsets
of epiblast cells retards their ingression, providing a mechanism to control
the timing of cell ingression through the streak
(Iimura and Pourquié,
2006). If such a mechanism is used, it is unlikely to act by
activating Hox expression cell-autonomously, as our data show that the Hox
identity of axial progenitor cells is influenced by neighbouring cells.
We have provided molecular evidence that LTAP populations at tail bud stages have more posterior identities than at earlier stages, confirming that in vivo these cells do not `self-renew', i.e. give rise to exact copies of themselves. However, their interpretation of anterior cues by changes in Hox gene expression suggests that under certain conditions they have the potential to self-renew.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/13/2289/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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LTR), and contains the
virus packaging site (
) and a neomycin resistance open reading frame
5' to the CAG-eGFP transgene. (B-E) GFP fluorescence in
transgenic embryos at (B) new laid egg stage, (C) stage 4 HH, (D) stage 15 HH
and (E) 5 days (transgenic on left). (F,G) Transverse sections
of (F) stage 15 HH GFP+ and control embryos, and (G) day 5
GFP+ and control embryos. (H) Stage 13 HH embryos hybridised
for GFP (transgenic on left). (I) Flow cytometric analysis of GFP
fluorescence (stage 11 HH embryos). Black line, non-transgenic embryo; blue
and green lines, transgenic lines 158 and 205, respectively. Scale bars: 0.5
mm.
