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First published online 22 August 2007
doi: 10.1242/dev.02873
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1 Departamento de Biología del Desarrollo Cardiovascular, Centro Nacional
de Investigaciones Cardiovasculares, Instituto de Salud Carlos III, E-28029
Madrid, Spain.
2 Center for Genomic Regulation (CRG), Program on Systems Biology, Barcelona,
Spain.
* Author for correspondence (e-mail: mtorres{at}cnic.es)
Accepted 25 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Clonal analysis, Fate maps, Vertebrate limb, Lineage compartments, Lmx1b
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lawrence, 1973;
Morata and Lawrence, 1975).
Compartment boundaries are therefore unique lines at stereotyped positions in
a developing organ, across which cell intermingling does not take place, even
though it is allowed elsewhere in the primordium. Drosophila
compartment borders do not necessarily coincide with or anticipate any
morphological or cell-type discontinuity even after overt differentiation
(reviewed by Blair, 2003).
Thus, Drosophila compartmental organization is a background
subdivision of embryonic fields that serves to establish positional references
in the primordium and is not necessarily related to anatomical boundaries in
the organism.
Lineage restriction units resembling Drosophila compartments have
also been described in vertebrates. Pioneering work in the chick, involving
labeling of multiple neuroepithelial cells with a lipophilic dye, identified
cell lineage restriction boundaries at the frontiers between rhombomeres
(Fraser et al., 1990).
Similarly, fate mapping of multiple cells identified a cell lineage
restriction border at the dorsoventral (D-V) boundary of the chick limb
ectoderm (Altabef et al.,
1997). In the mouse, the use of inducible site-specific
recombination (Brocard et al.,
1997) has allowed the prospective lineage analysis of groups of
cells defined by the expression of particular genes/enhancers
(Kimmel et al., 2000). These
approaches, collectively known as genetic inducible fate mapping (GIFM), have
been used extensively in the analysis of nervous system development, providing
insightful information on neural tube regionalization, brain morphogenesis,
neural cell-type specification mechanisms and neural stem cell dynamics
(reviewed by Joyner and Zervas,
2006). The use of GIFM in the mouse has also identified several
other lineage restriction borders in the vertebrate neuroepithelium, such as
the midbrain-hindbrain boundary (MHB), the zona limitans intrathalamica (ZLI)
and others (reviewed by Kiecker and
Lumsden, 2005). Similarly, GIFM has revealed the lineage
compartmentalization of the mouse limb ectoderm into dorsal, dorsal apical
ectodermal ridge (AER) and ventral compartments
(Kimmel et al., 2000).
A common characteristic of the lineage compartments described so far in
insects and vertebrates is their occurrence in epithelial structures, so that
each compartment is a two-dimensional epithelial area and the compartment
border is a line between two adjacent compartments (reviewed by
Blair, 2003;
Kiecker and Lumsden, 2005;
Vincent, 1998). Furthermore,
most lineage restriction borders described in both vertebrates and insects are
associated with signaling centers (Kiecker
and Lumsden, 2005), which suggests that a major role of lineage
compartments during embryonic development is signaling-center
stabilization.
In contrast to Drosophila compartments, however, all lineage
restrictions described so far in vertebrates coincide with, or anticipate,
anatomical or cell-type discontinuities. The known restrictions in vertebrates
may thus not be a background subdivision of embryonic fields, but might
instead largely correlate with strategies to allocate cells fated to different
anatomical structures. Systematic searches for cell lineage restrictions
during vertebrate embryonic development, however, have been limited, and
therefore our knowledge of the complete set of restrictions that occur is
likely to be incomplete. Efforts in this direction have made use of the
low-frequency spontaneous recombination of a mutant lacZ transgene
(laacZ) (Bonnerot and Nicolas,
1993). This transgene, driven either by ubiquitous or specific
promoters, has been successfully used to determine fundamental parameters of
tissue growth dynamics and regionalization in the developing heart
(Meilhac et al., 2004;
Meilhac et al., 2003),
paraxial mesoderm (Nicolas et al.,
1996) and CNS (Mathis and
Nicolas, 2000; Mathis et al.,
1999; Wilkie et al.,
2004). This method is constrained, however, by the inability to
control the timing and frequency of recombination events. More recently, GIFM
at low recombination frequency has been successfully used in a random
retrospective analysis of cell lineage at clonal resolution in the hair
follicle (Legue and Nicolas,
2005).
Here, we apply a novel strategy based exclusively on knock-in alleles of ubiquitous expression, which allows unrestricted clonal analysis of cell lineage from the two-cell stage to the adult mouse. Using this strategy, we have analyzed the topology of cell clone distribution in the developing vertebrate limb, with a particular focus on the mesenchyme. The lineage analysis we present demonstrates that there is no lineage compartmentalization at any position along the proximodistal (P-D) or anteroposterior (A-P) axes, indicating that patterning along these axes does not involve restrictions of cell dispersion at specific axial positions. By contrast, we have identified a D-V lineage restriction boundary in the limb mesenchyme. This is the first example from any organism of a lineage boundary restriction operating within a mesenchymal tissue. The resulting D-V lineage compartments are three-dimensional and the compartment border is a plane that is neither associated with any obvious signaling function nor coincident with any anatomical or cell-type discontinuity.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryos were post-fixed in 4% paraformaldehyde (PFA) and
stored in 80% glycerol, 4% PFA.
Immunohistochemistry
Immunofluorescence for simultaneous detection of Lmx1b and GFP
Embryos were fixed for 30 minutes in 4% PFA, immersed in 30% sucrose until
equilibrated, and frozen in OCT embedding compound. Sections (10 µm) were
cut, air dried for at least 1 hour and washed twice for 5 minutes each in PBS
containing 0.1% Tween 20 (PBT), once for 15 minutes in 0.1% Triton X-100 in
PBT and a further twice for 5 minutes each in PBT. Sections were blocked in 5%
donkey serum in PBT for 1 hour, followed by incubation for 1 hour at room
temperature with goat anti-Lmx1b antibody (Santa Cruz Biotechnology, sc-21231)
diluted 1:100 and anti-GFP antibody (Becton Dickinson, Living Colors, 632460)
diluted 1:100 in 5% donkey serum in PBT. Sections were then washed three times
for 5 minutes each in PBT and incubated with donkey anti-goat Cy3 and donkey
anti-rabbit Alexa-488 secondary antibodies for 30 minutes, washed three times
for 5 minutes each in PBT and mounted in Vectashield (Vector
Laboratories).
Immunohistochemistry for simultaneous detection of ß-gal and Lmx1b
Paraffin sections (8 µm) from whole-mount embryos stained for
lacZ expression were blocked in 3% H2O2 in
methanol for 25 minutes and washed in distilled water. Antigen retrieval was
performed by a 20-minute incubation in a microwave oven at maximum power in
tri-sodium citrate buffer pH 6. Sections were then washed in TBST (TBS plus
0.1% Tween-20) for 5 minutes, blocked in 10% goat serum in TBS (15 mM
Tris-ClH, 4.5 mM Tris-Base, 150 mM NaCl, pH 7.6) for 30 minutes and
subsequently incubated with anti-Lmx1b antibody diluted 1:20 (Abcam, ab-25504)
and a biotinylated goat anti-rabbit secondary antibody. Sections were stained
using the Vectastain ABC Peroxidase Staining Kit. A similar procedure was used
for the simultaneous detection of ß-gal and PECAM, except that it was
performed on cryostat sections and the antigen retrieval step was omitted.
Statistical analysis
To calculate the probability of polyclonal specimens, independence of the
recombination event was assumed. We therefore estimated the expected frequency
of polyclonal specimens affecting adjacent regions by multiplying the observed
frequency of positive cells in each region in isolation.
Optical projection tomography (OPT)
OPT was performed essentially as described
(Sharpe et al., 2002), but
with a reduction in the time of dehydration in methanol and clearing in BABB,
because longer exposure causes the lacZ stain to fade.
Volume rendering and surface mapping were performed using the Visualization Tool Kit software (http://public.kitware.com/VTK/index.php) and the Edinburgh Mouse Atlas software for three-dimensional data processing.
Characterization of an inducible system for clonal analysis in the mouse
To perform retrospective clonal analyses in mouse embryos, we used a
genetic strategy based on the site-specific Cre recombinase
(Sauer and Henderson, 1988).
Labeled cells are produced by the activity of CreERT2, an inducible
recombinase activated by tamoxifen (TM)
(Feil et al., 1997).
Ubiquitous CreERT2 expression is provided by a knock-in insertion of the
CreERT2 cDNA into the 3' UTR of the RNA polymerase II gene
(Guerra et al., 2003), which
yields viable homozygotes with no obvious phenotype. Cre-mediated
recombination is monitored by expression from either the
recombination-activatable R26R or R26R-EYFP knock-in alleles
(Soriano, 1999;
Srinivas et al., 2001).
To achieve lineage tracing at clonal resolution, we established the conditions for low frequency recombination, such that when a group of positive cells is detected, the probability of polyclonal origin remains low. We achieved this by titrating the dose of 4OHTM. We established the optimal induction dose as 2.5 µg 4OHTM/g of body weight, which yielded frequencies of lacZ-positive limbs of 1-20%.
Calculation of the frequency of polyclonal limbs, however, can only be deduced from the frequency of positive limbs if recombination events behave as independent events; that is, if the occurrence of a recombination event does not correlate with the occurrence of a second one in the surrounding cells. To determine this, we induced recombination in embryos carrying both the lacZ and the EYFP Rosa26 recombination reporters. After inducing recombination, we recorded the frequency of limbs positive for EYFP, lacZ, or both reporters. The frequencies we obtained were in complete agreement with the occurrence of independent recombination events (Table 1).
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Finally, to establish the effective period of clone induction, we determined clone frequency at different times after 4OHTM injection. At the low 4OHTM dose used, we first detected lacZ-positive cells 12 hours after 4OHTM injection at E9.5, but few recombination events were detected at this stage (see Fig. S1C in the supplementary material; Table 2). Most induced clones are already detectable 24 hours after injection, with little increase in clone frequency after this (see Fig. S1D,E in the supplementary material; Table 2). To determine the minimum time of induction, we cultured E9.5 embryos in vitro in a high 4OHTM concentration (50 µM) and found that 6 hours were necessary to observe the first lacZ-positive cells (data not shown). We thus estimate that the majority of recombination events take place between 12 and 18 hours after 4OHTM injection.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the current study, we have analyzed the topological
distribution of cell clones in the limb mesenchyme.
Limb mesenchyme shows D-V compartmentalization
Mice pregnant with embryos at stages E8.5, E9.5 and E10.5 were injected
with 4-hydroxy-tamoxifen (TM), and the distribution of induced clones examined
between E12.5 and E14.5. Analysis of positive cell distribution within the
limb was performed both by direct whole-mount visualization of lacZ
staining and by optical projection tomography (OPT) (n=37)
(Sharpe et al., 2002). At all
injection stages, considerable dispersion of positive cells along the P-D and
A-P axes was observed, indicating a strong tendency for limb mesenchyme cells
to disperse and intermingle with neighbors
(Fig. 2A). Considerable cell
dispersal was also observed along the D-V axis; however, in most specimens
dispersal was restricted to one side of a plane dividing the limb into dorsal
and ventral sectors, and this affected dorsal and ventral clones similarly
(Fig. 2B-H and
Table 3). OPT analysis
confirmed that the distribution of cells at the ventral limits of dorsal
clones (and vice versa) define D-V planes
(Fig. 3).
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To determine whether the frontiers respected by dorsal and ventral clones are consistently located at a reproducible position along the D-V axis, we measured the relative extension of a collection of dorsal and ventral clones at specific positions along the P-D axis of digits 2, 3 and 4 at E13.5 and E14.5. The positions of the boundary measured for each independent dorsal or ventral clone were remarkably consistent, with the majority of boundaries formed at a position displaced dorsally from the midline in 5% of the total D-V extension (Fig. 2I). These results thus identify a single specific boundary at a precise position along the D-V limb axis, which is similarly respected by dorsal and ventral cells.
In a fraction of limbs, however, we detected positive cells, both at dorsal
and at ventral positions, that did not respect this boundary
(Table 3). A statistical
analysis to determine whether these violations might be explained by the
presence of multiple clones predicted that 4.0% of E8.5 injections and 4.4% of
E9.5 injections would contain independent clones in both dorsal and ventral
compartments. However, the observed proportion of limbs showing no D-V
restriction was 15% for E8.5 injections and 14% for E9.5 injections. By
contrast, at E10.5 we found that the proportion of unrestricted clones was
slightly higher but not significantly different from that expected for double
recombination events. These results indicate a strong but not complete D-V
lineage restriction in early limb bud stages. Full D-V lineage restriction
appears to take place only after 
E10.
Vascular cells are insensitive to limb mesenchyme compartmentalization
Limb mesenchyme is composed of resident cells derived from the lateral
plate mesoderm and several other incoming cell populations. Our strategy
labels not only resident limb mesenchymal cells, but also other cell lineages
such as skeletal muscle, endothelium, smooth muscle, melanoblasts and cells of
hematopoietic origin. The observed D-V restriction operates on derivatives of
resident limb cells, as the vast majority of restricted clones colonized
skeletal elements and other derivatives of resident mesenchyme (see below).
However, the incoming cell populations that contribute to the limb could not
be assessed from the lacZ whole-mount stainings alone. An exception
to this limitation was the skeletal muscle clones, which could be identified
by morphology, were found in a low proportion of 1% (3 out of 312) and
were found restricted to either dorsal or ventral muscle masses. This
observation reflects the independent ingression into the limb of muscle
precursors for ventral and dorsal masses (for a review, see
Christ and Brand-Saberi,
2002).
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8% of unrestricted clones (9 out of
109 screened). Unfortunately, we found no histological marker that would allow
simultaneous detection of clone cells and melanoblasts, a cell population that
colonizes the limb bud by E13.5 (Mackenzie
et al., 1997). We were thus unable to determine the contribution
of this population to unrestricted clones.
Lmx1b defines the mesenchymal dorsal compartment
Dorsoventral patterning of the vertebrate limb involves the dorsalizing
action of the secreted molecule Wnt7a, produced by the dorsal ectoderm
(Parr and McMahon, 1995).
Countering this, BMP signaling in the ventral ectoderm promotes ventralization
by activating the transcription factor En1
(Ahn et al., 2001;
Logan et al., 1997;
Pizette and Niswander, 2001).
In the dorsal mesoderm, the transcription factor Lmx1b (Lmx1 in the chick)
responds to Wnt7a signals and specifies the dorsal mesenchymal structures
(Chen et al., 1998;
Riddle et al., 1995;
Vogel et al., 1995).
We noted that the dorsal mesenchymal limb compartment we identified
resembles the expression domain of Lmx1b
(Dreyer et al., 2004;
Schweizer et al., 2004). In
distal undifferentiated limb regions, the Lmx1b-positive dorsal
domain is slightly smaller than the Lmx1b-negative ventral domain
(Fig. 5A,C). At single-cell
resolution, the Lmx1b expression domain border was not completely
straight, showing some interdigitation between Lmx1b-positive and
Lmx1b-negative cells (Fig.
5E,I). To determine the correlation between D-V
compartmentalization and Lmx1b expression, we double-stained limbs
for Lmx1b and clone cells, and found that the dorsal-most cells of
ventral clones were located next to the ventral-most Lmx1b-expressing
cells (n=5) (Fig.
5F-I). Cells of ventral clones did not express Lmx1b,
even when surrounded by Lmx1b-expressing cells
(Fig. 5I). Consistent with the
results obtained for ventral clones, ventral-most cells of dorsal clones
coincided with ventral-most Lmx1b-expressing cells (n=6)
(Fig. 5D,E;
Fig. 6E-J). The Lmx1b
expression domain thus coincides with the dorsal limb mesenchyme compartment,
and Lmx1b expression is excluded from the ventral compartment.
In more-proximal limb regions, chondrogenic condensations take place at the boundary between dorsal and ventral cells, as defined by Lmx1b expression. Within the early prechondrogenic condensation, Lmx1b was expressed in a dorsal domain, showing that chondrogenic cells are recruited from both dorsal and ventral compartments, and therefore that the condensations contain the boundary between dorsal and ventral cells (Fig. 5B; Fig. 6F). As condensations mature, Lmx1b expression was lost in the chondrogenic area but remained in the surrounding tissues, maintaining its relative position with respect to the condensation (Fig. 5A; Fig. 6G).
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There is no compartmentalization along the A-P axis, which is, however, characterized by the temporal and spatial regulation of cell dispersion dynamics
In our analysis, clonal A-P expansion varied according to clone size.
Early-induced large clones colonized up to nearly a half of the total A-P axis
extension, whereas small clones expanded less widely
(Fig. 7A-C, see also
Fig. 3A and
Fig. 8F). Strikingly, despite
extensive mixing of clone cells with their neighbors, most clones displayed
straight A-P boundaries running parallel to the main limb axis, so that the
fraction of the A-P axis colonized by a given clone remained constant along
most of the limb P-D axis. Although this growth pattern suggested possible A-P
lineage restrictions, mapping the boundaries for different clones identified
no specific frontiers respected by limb cells in their expansion across the
A-P axis (not shown).
Clone cell distribution in the autopod showed additional interesting features. We observed two characteristic complementary classes of clones: inter-digital and mid-digital clones (Fig. 7C-F). Inter-digital clone cells were loosely distributed across the whole inter-digital area and the lateral regions of adjacent digits, but stopped abruptly at straight boundaries lateral to the midline of the two neighboring digits. Mid-digital clone cells, conversely, were densely packed and restricted to the central region of a single digit. Mixed clones that partly colonize both areas were also detected and, again, mapping the boundaries for different clones identified no specific respected frontiers. These results indicate there are no A-P compartment restriction boundaries and, in fact, that there is a very notable heterogeneity in cell behavior across the autopod A-P axis. Whereas cells in the inter-digital and lateral digit areas actively disperse and intermingle with neighbors, cells at the central digit regions retain the ordered growth and reduced mixing observed in other P-D limb segments.
Limb cells are not allocated by lineage to proximodistal segments in the early limb bud
Vertebrate limbs develop in a proximal-to-distal sequence, being patterned
along the three axes by the coordinated action of three signaling centers. One
of these centers, the AER, is located at the interface of dorsal and ventral
ectoderm. The AER produces signals that maintain a population of distal
undifferentiated mesenchymal cells in the underlying region. As the limb bud
grows, mesenchymal cells exit the distal region and differentiate to generate
limb structures in a proximal-to-distal sequence
(Rowe and Fallon, 1982;
Saunders, 1948).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We report the topological distribution of clones induced during limb
development. Vertebrate limb development has been extensively used as a
suitable model to understand patterning mechanisms (reviewed by
Tickle, 2003). Detailed fate
maps of the chick limb mesenchyme have been obtained recently by labeling
groups of cells with lipophilic dyes
(Altabef et al., 1997;
Dudley et al., 2002;
Saadi et al., 1993;
Sato et al., 2007;
Vargesson et al., 1997) or by
viral infection (Dudley et al.,
2002). In the mouse, GIFM of Shh-expressing and Shh
signal-receiving cells has been used to determine the fate of ZPA cells
(Harfe et al., 2004;
Scherz et al., 2004) and of
cells receiving Shh signal (Ahn and Joyner,
2004). These approaches have provided important information about
limb growth dynamics and have suggested important modifications to established
models of limb A-P and P-D patterning; however, they were not done at clonal
resolution.
Clonal distribution along the limb A-P axis
We found that early-induced large clones derived from single cells colonize
considerable A-P extensions by mixing extensively with neighboring cells. At
the same time, these clones have remarkably straight A-P borders, so that
their relative contribution to the total A-P limb extension is maintained at
constant size and position along the P-D axis. These borders do not represent
lineage restrictions, but rather indicate that limb cell precursors keep their
relative positions along the A-P axis as they contribute progressively to the
P-D axis. These results suggest that during an early phase, cells disperse
vigorously across the limb primordium, so that descendants of single cells can
colonize large sectors of the total initial limb bud. A second phase, likely
to start when the limb bud starts to elongate, would be characterized by the
cessation of cell mixing and therefore the maintenance of the relative A-P
positions of cells in the undifferentiated distal mesenchyme. During autopod
generation, ordered growth is maintained at the mid-digital region, while
intense cell mixing behavior is regained in the inter-digital areas. Multiple
cell-labeling experiments in the chick autopod also suggested extensive mixing
in the inter-digital areas versus restricted cell movement in the mid-digital
regions (Omi et al., 2000). As
a result of this restricted movement, mid-digital areas constitute barriers
for the dispersion of inter-digital cells, a phenomenon that might be relevant
in the light of the recent model proposing that inter-digital regions are
responsible for specifying digit identity
(Dahn and Fallon, 2000).
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). The more recent early allocation model proposes instead
that cells in the early limb bud are first simultaneously specified to the
main limb P-D segments in the early limb bud and then expand, get irreversibly
determined, and differentiate in a proximal-to-distal sequence
(Dudley et al., 2002;
Sun et al., 2002). The results
of our clonal analysis indicate that there are no lineage restrictions at any
position along the P-D limb axis at the stages examined. Lineage tracing,
however, does not assess cell specification status and therefore does not
exclude the existence of a pre-pattern of P-D regional specification in the
early limb bud. The results obtained nonetheless argue against a strong
formulation of the early allocation model, in which individual cells would be
assigned to specific P-D fates early in limb bud development. Similar results
have recently been reported in the chick
(Pearse et al., 2007;
Sato et al., 2007).
D-V compartmentalization of the vertebrate limb mesenchyme
The most significant result from our analysis is the detection of a lineage
boundary that subdivides the resident limb mesenchyme into dorsal and ventral
compartments. The compartmentalization observed may extend to some incoming
cell lineages that enter the limb from external sources, such as the skeletal
muscle precursors, whose dorsal mass cells acquire Lmx1b expression
as they enter the limb (Schweizer et al.,
2004), but not to others, such as endothelial cells, which we
found to be insensitive to the D-V border. A D-V lineage restriction has also
been detected in parallel studies by retroviral clonal analysis in the chick
(Pearse et al., 2007) and GIFM
from the Lmx1b locus in the mouse (R. Johnson, personal
communication).
This is the first example in metazoans of a lineage compartment operating in a mesenchymal structure. As such, these compartments are organized in three dimensions and their borders are not lines, as is the case in epithelial compartments, but rather a plane between mesenchymal cells. This finding suggests that compartmentalization strategies during embryonic development might be more widely used than previously thought, especially in vertebrates, in which patterning of three-dimensional mesenchymal structures is a frequent process.
The identification of lineage compartment borders in Drosophila
was achieved through the use of cell clones that have a growth advantage with
respect to the cells of the background [wild-type cells versus Minute
mutant cells] (Garcia-Bellido et al.,
1973). Despite the reduced tendency of imaginal disc cells to
disperse, this growth advantage allowed wild-type clones to colonize enough
territory to delineate the compartment borders, demonstrating the robustness
of the mechanisms restricting cell mixing between compartments. In the case of
the vertebrate limb, the mesenchymal cells exhibit a strong tendency to
disperse during organ growth, resulting in the occupation of large volumes by
clones composed of a relatively small number of cells, such that the
compartment border is effectively delineated by the majority of the clones in
the absence of any growth advantage. It remains to be explored, however,
whether the restriction mechanisms operating in the vertebrate limb mesenchyme
are sufficiently robust to avoid cell mixing in the case of clones that do
have a growth advantage.
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).
We found that the territory corresponding to the limb dorsal compartment
correlates with the expression domain of the transcription factor Lmx1b.
Eliminating Lmx1b function in the mouse results in double-ventral
limbs (Chen et al., 1998), and
overexpression of the ortholog gene Lmx1 in chicken results in
double-dorsal limbs (Riddle et al.,
1995; Vogel et al.,
1995), demonstrating this factor's essential role in dorsal
specification. Qiu and co-workers (R. Johnson, personal communication) report
that at least some of the cellular properties involved in maintaining the
dorsal compartment are encoded by Lmx1b and that after an initial
unstable period, Lmx1b expression is transmitted stably to daughter
cells. This is supported by results with chicken explants and recombinant
limbs, which suggest that after an initial phase of activation by Wnt7a,
Lmx1 expression is stably maintained independently of external cues
(Piedra et al., 2000;
Riddle et al., 1995). However,
expression of Lmx1b is not dorsally restricted in the early mouse
limb bud (Loomis et al.,
1998), a result which suggests that the mesenchymal D-V
compartment might not be established in the early limb bud and might appear
only after Lmx1b expression becomes restricted to dorsal regions. In
fact, our results suggest that D-V restriction is incomplete in 4OHTM
injections at or before E9.5. Previous studies in the chick failed to identify
any D-V lineage restriction in limb mesenchyme before HH16
(Altabef et al., 1997),
indicating that compartmentalization in the chick may take place after this
stage and after the initial Lmx1 activation at stage HH15+
(Altabef and Tickle, 2002).
Indeed, the initial D-V specification of limb mesoderm depends on external
signals, first from paraxial mesoderm and lateral somatopleura, and later from
ectoderm (Michaud et al.,
1997). Similar to classical compartments in Drosophila and others described later in vertebrates, the dorsal limb mesenchymal compartment therefore appears to constitute an independent unit of genetic regulation, and Lmx1b appears to be a major regulator of its properties.
Besides the Lmx1b expression transition, there was no other feature that we could associate with the mesenchymal D-V boundary, even at electron microscope resolution (data not shown). A barrier mechanism for the maintenance of the lineage restriction border is therefore unlikely. The compartment boundary might instead be maintained by a differential adhesion mechanism that restricts the strong tendency of mesenchymal limb cells to disperse and mix with neighbors. Such a mechanism would fit well with the observed irregular shape of the border between Lmx1b-positive and Lmx1b-negative cells.
Functional significance of limb mesenchyme D-V compartmentalization
One of the most widely accepted ideas about compartments is that lineage
restriction serves to stabilize signaling centers at compartment borders,
which constitute organizers for adjacent compartments (reviewed by
Vincent, 1998). Classical
insect compartment borders, as well as the AER and the MHB in vertebrates, are
signaling centers with organizer properties. Compartments at both sides of
these borders are independent developmental fields that respond to the
organizer activity of the signaling center. Lineage boundaries with no
organizer activity, such as rhombomeric limits, also express signaling
molecules with essential patterning functions. By contrast, in the case of the
D-V limb mesenchymal border, there is no evidence for any organizer or
specific signaling activity, as none of the known signaling molecules is
specifically expressed at the D-V interface. The subdivision of the
developmental field into independent units of patterning and growth might thus
be a more fundamental characteristic of lineage compartments than their
association with signaling regions. In the case of mesenchymal compartments,
in addition to defining independent patterning units, compartmentalization
might be relevant to the complex morphogenetic movements that
three-dimensional mesenchymal structures such as limb buds have to go through
(for example, D-V flattening and bending). Exploring the existence of further
compartments of this sort will determine how widely this mechanism is used to
pattern mesenchymal fields during metazoan development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/20/3713/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ahn, K., Mishina, Y., Hanks, M. C., Behringer, R. R. and Crenshaw, E. B., 3rd (2001). BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. Development 128,4449 -4461.[Medline]
Ahn, S. and Joyner, A. L. (2004). Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell 118,505 -516.[CrossRef][Medline]
Altabef, M. and Tickle, C. (2002). Initiation of dorso-ventral axis during chick limb development. Mech. Dev. 116,19 -27.[CrossRef][Medline]
Altabef, M., Clarke, J. D. and Tickle, C. (1997). Dorso-ventral ectodermal compartments and origin of apical ectodermal ridge in developing chick limb. Development 124,4547 -4556.[Abstract]
Blair, S. S. (2003). Lineage compartments in Drosophila. Curr. Biol. 13,R548 -R551.[CrossRef][Medline]
Bonnerot, C. and Nicolas, J. F. (1993). Clonal analysis in the intact mouse embryo by intragenic homologous recombination. C. R. Acad. Sci. III 316,1207 -1217.[Medline]
Brocard, J., Warot, X., Wendling, O., Messaddeq, N., Vonesch, J.
L., Chambon, P. and Metzger, D. (1997). Spatio-temporally
controlled site-specific somatic mutagenesis in the mouse. Proc.
Natl. Acad. Sci. USA 94,14559
-14563.
Chen, H., Lun, Y., Ovchinnikov, D., Kokubo, H., Oberg, K. C., Pepicelli, C. V., Gan, L., Lee, B. and Johnson, R. L. (1998). Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome. Nat. Genet. 19, 51-55.[CrossRef][Medline]
Christ, B. and Brand-Saberi, B. (2002). Limb muscle development. Int. J. Dev. Biol. 46,905 -914.[Medline]
Dahn, R. D. and Fallon, J. F. (2000).
Interdigital regulation of digit identity and homeotic transformation by
modulated BMP signaling. Science
289,438
-441.
Dreyer, S. D., Naruse, T., Morello, R., Zabel, B., Winterpacht, A., Johnson, R. L., Lee, B. and Oberg, K. C. (2004). Lmx1b expression during joint and tendon formation: localization and evaluation of potential downstream targets. Gene Expr. Patterns 4, 397-405.[CrossRef][Medline]
Dudley, A. T., Ros, M. A. and Tabin, C. J. (2002). A re-examination of proximodistal patterning during vertebrate limb development. Nature 418,539 -544.[CrossRef][Medline]
Feil, R., Wagner, J., Metzger, D. and Chambon, P. (1997). Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237,752 -757.[CrossRef][Medline]
Fraser, S., Keynes, R. and Lumsden, A. (1990). Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions. Nature 344,431 -435.[CrossRef][Medline]
Garcia-Bellido, A., Ripoll, P. and Morata, G. (1973). Developmental compartmentalisation of the wing disk of Drosophila. Nat. New Biol. 245,251 -253.[CrossRef][Medline]
Guerra, C., Mijimolle, N., Dhawahir, A., Dubus, P., Barradas, M., Serrano, M., Campuzano, V. and Barbacid, M. (2003). Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4,111 -120.[CrossRef][Medline]
Harfe, B. D., Scherz, P. J., Nissim, S., Tian, H., McMahon, A. P. and Tabin, C. J. (2004). Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118,517 -528.[CrossRef][Medline]
Joyner, A. L. and Zervas, M. (2006). Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system. Dev. Dyn. 235,2376 -2385.[CrossRef][Medline]
Kiecker, C. and Lumsden, A. (2005). Compartments and their boundaries in vertebrate brain development. Nat. Rev. Neurosci. 6,553 -564.[CrossRef][Medline]
Kimmel, R. A., Turnbull, D. H., Blanquet, V., Wurst, W., Loomis,
C. A. and Joyner, A. L. (2000). Two lineage boundaries
coordinate vertebrate apical ectodermal ridge formation. Genes
Dev. 14,1377
-1389.
Lawrence, P. A. (1973). A clonal analysis of segment development in Oncopeltus (Hemiptera). J. Embryol. Exp. Morphol. 30,681 -699.[Medline]
Legue, E. and Nicolas, J. F. (2005). Hair
follicle renewal: organization of stem cells in the matrix and the role of
stereotyped lineages and behaviors. Development
132,4143
-4154.
Logan, C., Hornbruch, A., Campbell, I. and Lumsden, A. (1997). The role of Engrailed in establishing the dorsoventral axis of the chick limb. Development 124,2317 -2324.[Abstract]
Loomis, C. A., Kimmel, R. A., Tong, C. X., Michaud, J. and Joyner, A. L. (1998). Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs. Development 125,1137 -1148.[Abstract]
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]
Mathis, L. and Nicolas, J. F. (2000). Different clonal dispersion in the rostral and caudal mouse central nervous system. Development 127,1277 -1290.[Abstract]
Mathis, L., Sieur, J., Voiculescu, O., Charnay, P. and Nicolas, J. F. (1999). Successive patterns of clonal cell dispersion in relation to neuromeric subdivision in the mouse neuroepithelium. Development 126,4095 -4106.[Abstract]
Meilhac, S. M., Kelly, R. G., Rocancourt, D., Eloy-Trinquet, S.,
Nicolas, J. F. and Buckingham, M. E. (2003). A retrospective
clonal analysis of the myocardium reveals two phases of clonal growth in the
developing mouse heart. Development
130,3877
-3889.
Meilhac, S. M., Esner, M., Kelly, R. G., Nicolas, J. F. and Buckingham, M. E. (2004). The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell 6,685 -698.[CrossRef][Medline]
Michaud, J. L., Lapointe, F. and Le Douarin, N. M. (1997). The dorsoventral polarity of the presumptive limb is determined by signals produced by the somites and by the lateral somatopleure. Development 124,1453 -1463.[Abstract]
Morata, G. and Lawrence, P. A. (1975). Control of compartment development by the engrailed gene in Drosophila. Nature 255,614 -617.[CrossRef][Medline]
Nicolas, J. F., Mathis, L., Bonnerot, C. and Saurin, W. (1996). Evidence in the mouse for self-renewing stem cells in the formation of a segmented longitudinal structure, the myotome. Development 122,2933 -2946.[Abstract]
Omi, M., Sato-Maeda, M. and Ide, H. (2000). Role of chondrogenic tissue in programmed cell death and BMP expression in chick limb buds. Int. J. Dev. Biol. 44,381 -388.[Medline]
Parr, B. A. and McMahon, A. P. (1995). Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature 374,350 -353.[CrossRef][Medline]
Pearse, R. V., II, Scherz, P. J., Campbell, J. K. and Tabin, C. J. (2007). A cellular lineage analysis of the chick limb bud. Dev. Biol. (in press).
Piedra, M., Rivero, F., Fernandez-Teran, M. and Ros, M. A. (2000). Pattern formation and regulation of gene expressions in chick recombinant limbs. Mech. Dev. 90,167 -179.[CrossRef][Medline]
Pizette, S. and Niswander, L. (2001). Early steps in limb patterning and chondrogenesis. Novartis Found. Symp. 232,23 -36; discussion 36-46.[Medline]
Riddle, R. D., Ensini, M., Nelson, C., Tsuchida, T., Jessell, T. M. and Tabin, C. (1995). Induction of the LIM homeobox gene Lmx1 by WNT7a establishes dorsoventral pattern in the vertebrate limb. Cell 83,631 -640.[CrossRef][Medline]
Rowe, D. A. and Fallon, J. F. (1982). The proximodistal determination of skeletal parts in the developing chick leg. J. Embryol. Exp. Morphol. 68, 1-7.[Medline]
Saadi, A., Gallien, C. L., Guyot-Lenfant, M. and Chanoine, C. (1993). A new approach of urodele amphibian limb regeneration: study of myosin isoforms and their control by thyroid hormone. Mech. Dev. 43,49 -56.[CrossRef][Medline]
Sato, K., Koizumi, Y., Takahashi, M., Kuroiwa, A. and Tamura,
K. (2007). Specification of cell fate along the
proximal-distal axis in the developing chick limb bud.
Development 134,1397
-1406.
Sauer, B. and Henderson, N. (1988).
Site-specific DNA recombination in mammalian cells by the Cre recombinase of
bacteriophage P1. Proc. Natl. Acad. Sci. USA
85,5166
-5170.
Saunders, J. W. (1948). The proximodistal sequence of origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 108,363 -403.[Medline]
Scherz, P. J., Harfe, B. D., McMahon, A. P. and Tabin, C. J.
(2004). The limb bud Shh-Fgf feedback loop is terminated by
expansion of former ZPA cells. Science
305,396
-399.
Schweizer, H., Johnson, R. L. and Brand-Saberi, B. (2004). Characterization of migration behavior of myogenic precursor cells in the limb bud with respect to Lmx1b expression. Anat. Embryol. 208,7 -18.[CrossRef][Medline]
Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A.,
Hecksher-Sorensen, J., Baldock, R. and Davidson, D. (2002).
Optical projection tomography as a tool for 3D microscopy and gene expression
studies. Science 296,541
-545.
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Srinivas, S., Watanabe, T., Lin, C. S., William, C. M., Tanabe, Y., Jessell, T. M. and Costantini, F. (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1,4 .[CrossRef][Medline]
Summerbell, D., Lewis, J. and Wolpert, L. (1973). Positional information in chick limb morphogenesis. Nature 224,492 -496.
Sun, X., Mariani, F. V. and Martin, G. R. (2002). Functions of FGF signalling from the apical ectodermal ridge in limb development. Nature 418,501 -508.[CrossRef][Medline]
Tickle, C. (2003). Patterning systems - from one end of the limb to the other. Dev. Cell 4, 449-458.[CrossRef][Medline]
Torres, M. (1998). The use of embryonic stem cells for the genetic manipulation of the mouse. Curr. Top. Dev. Biol. 36,99 -114.[Medline]
Vargesson, N., Clarke, J. D., Vincent, K., Coles, C., Wolpert, L. and Tickle, C. (1997). Cell fate in the chick limb bud and relationship to gene expression. Development 124,1909 -1918.[Abstract]
Vincent, J. P. (1998). Compartment boundaries: where, why and how? Int. J. Dev. Biol. 42,311 -315.[Medline]
Vogel, A., Rodriguez, C., Warnken, W. and Izpisua Belmonte, J. C. (1995). Dorsal cell fate specified by chick Lmx1 during vertebrate limb development. Nature 378,716 -720.[CrossRef][Medline]
Wilkie, A. L., Jordan, S. A., Sharpe, J. A., Price, D. J. and Jackson, I. J. (2004). Widespread tangential dispersion and extensive cell death during early neurogenesis in the mouse neocortex. Dev. Biol. 267,109 -118.[CrossRef][Medline]
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