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First published online 8 December 2005
doi: 10.1242/dev.02183
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Department of Molecular and Cell Biology, Division of Genetics, Genomics, and Development, and the Center for Integrative Genomics, University of California, Berkeley, CA 94720-3204, USA.
* Author for correspondence (e-mail: harland{at}socrates.berkeley.edu)
Accepted 25 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hypaxial, Rectus abdominus, Rectus cervicus, Geniohyoideus, lbx1, myoD, myf5, Xenopus laevis, Cell proliferation, Myogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
In amniotes, lbx1 is expressed in a subset of hypaxial myoblasts,
which in the mouse migrate into the limbs, tongue, and diaphragm. By contrast,
lbx1 is completely excluded from inter-limb body wall hypaxial
myoblasts (Dietrich, 1999;
Dietrich et al., 1998). In
addition to the difference in lbx1 expression, these two types of
hypaxial muscles are characterized by different modes of development.
Body-wall muscles form as epithelial extensions of the dermomyotome, whereas
lbx1-positive limb, tongue and diaphragm myoblasts undergo an
epithelial to mesenchymal transition in the dermomyotome before undergoing
long-range migration to their target sites
(Dietrich, 1999). A null
lbx1 mutation in the mouse causes a specific lack of limb musculature
attributed to migration defects, while hypaxial body wall muscles undergo
normal development (Brohmann et al.,
2000; Gross et al.,
2000; Schafer and Braun,
1999).
The pre-metamorphic X. laevis tadpole does not contain limbs and
the entire family of pipid frogs does not develop a tongue
(McDiarmid and Altig, 1999),
suggesting that early tadpoles might not express lbx1. However, these
tadpoles do contain hypaxial muscles, which form the rectus abdominus muscles
(Lynch, 1990;
Martin and Harland, 2001;
Ryke, 1953). These are
homologous to the inter-limb body wall muscles of amniotes
(McDiarmid and Altig, 1999).
The temporal expression of muscle-specific markers in myoblasts that form the
ventral body wall muscles is similar to that of amniote limb myoblasts
(Martin and Harland, 2001).
pax3, which is a marker of proliferative myoblasts, is expressed in
cells that exit the somite and migrate ventrally. Later on,
pax3-positive myoblasts are seen just ventral to differentiated
muscle of the ventral body wall. The expression of myf5, a muscle
regulatory factor (MRF), is similar to that of pax3 in the hypaxial
myoblasts. However, the MRF myoD is absent from hypaxial myoblasts as
they exit the somite. Transcripts of myoD do not appear until cells
begin to differentiate, and they overlap with 12/101 staining, a marker of
differentiated skeletal muscle (Martin and
Harland, 2001).
The expression of myoD has been shown to be associated with the
terminal differentiation of skeletal muscle
(Hopwood et al., 1989;
Hopwood et al., 1992;
Montarras et al., 1989;
Tapscott et al., 1990;
Weintraub et al., 1991).
Indeed, myoD has been demonstrated to be an integral factor in cell
cycle arrest during the terminal differentiation of skeletal muscle
(Halevy et al., 1995).
Although Myf5 and Myod1 have been shown to have partially
redundant roles in mouse skeletal muscle formation, Myf5 is expressed
in proliferative myoblasts that have not exited the cell cycle
(Hopwood et al., 1991;
Martin and Harland, 2001;
Montarras et al., 1991). For
example, the inhibition of muscle differentiation in chick limb myoblasts by
activated Notch results in a downregulation of myoD, but has no
effect on myf5 or pax3 expression
(Delfini et al., 2000).
The precise timing of the development of long-range hypaxial muscles
requires a fine balance between proliferation and differentiation
(Amthor et al., 1999;
Amthor et al., 1998). The
overlying ectoderm of somites provides a proliferative signal for myoblasts.
When ectoderm is removed from chick somites, a transient upregulation of
myoD is observed corresponding to a burst of muscle differentiation,
but further muscle growth is halted. When ectoderm is removed from somites at
limb and tongue levels, the premature differentiation prevents muscle
precursors from migrating, and limb and tongue muscles do not form
(Amthor et al., 1999).
Work on lbx1 in hypaxial myogenesis has focused mainly cell
migration, but lbx1 has also been implicated in cell proliferation.
Forced expression of lbx1 in chick mesoderm and neural tube explants
increases cell proliferation. In chick limb buds, lbx1 increases
myoD expression and the amount of differentiated muscle. This excess
can be rescued by inhibiting cell proliferation
(Mennerich and Braun, 2001).
These results suggest that a role of lbx1 in hypaxial myogenesis may
be to expand the population of myoblast cells.
We have examined the expression pattern of lbx1 in X. laevis tadpoles and found that it is expressed in all of the myoblasts that will populate the rectus abdominus and the geniohyoideus, a hypoglossal muscle. We further demonstrate that lbx1 controls myoblast proliferation through the downregulation of myoD and p27. Thus, muscle defects observed after lbx1 loss of function probably result from the reduced capacity of myoblasts to proliferate.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Embryos were allowed to develop in 0.3x Marc's modified Ringer (MMR)
solution and staged according to the normal table
(Nieuwkoop and Faber,
1967).
Isolation of Xenopus laevis lbx1 clone
The primers TLBX-2 (5'-AGACGGCATGACGATTTTTGGC-3') and TLBX-3
(5'-CCGAGATGGTGAGTACGGCTTC-3') were designed from Xenopus
tropicalis genomic sequence and used to screen an arrayed X.
laevis cDNA library. A single clone of lbx1 was found that
contains the majority of the coding region. The primers Xlbx5up
(5'-CCAAGTCAATGTATCAGTCGCTGTG-3') and Xlbx5down
(5'-TCCTGACTGAGGGCTTGTTTAGG-3') were designed from X.
tropicalis genomic sequence and used to amplify the 5' end of the
sequence from X. laevis genomic DNA.
Microinjection of DiI
A 0.25% stock solution of CellTracker CM-DiI (Molecular Probes) was made up
in 100% ethanol. A working solution of 0.1% DiI was diluted in 3 M sucrose.
Micropipettes were pulled and broken to a tip of 
20 µm, backfilled
with DiI solution and pressure injected using a Picospritzer (General Valve).
Tadpoles were immobilized while injecting and for viewing by immersion in
0.05% benzocaine solution.
Whole-mount in situ hybridization and antibody staining
Embryos were allowed to develop until the desired stage and then fixed for
2 hours in MEMFA. In situ hybridization was carried out with RNA probes
labeled with digoxigenin-UTP using a multibasket technique
(Sive et al., 2000).
Differentiated skeletal muscle was visualized with the 12/101 monoclonal
antibody (Kintner and Brockes,
1984). Immunohistochemistry used undiluted monoclonal hybridoma
cell supernatant and a goat anti-mouse IgG conjugated to HRP or fluorescein
(Jackson) as a secondary antibody at a 1:500 or 1:200 dilution, respectively.
In cases where both in situ hybridization and 12/101 staining were carried out
on embryos, in situ staining was performed first, followed immediately by
immunohistochemistry. The anti-phospho-histone H3 antibody (Upstate
Biotechnology) was used at a 1:1000 dilution in 2 mg/ml BSA in PBS plus 0.1%
Triton X100. A goat anti-rabbit IgG secondary conjugated to HRP (BioRad) was
used at a dilution of 1:1000.
Microinjection of MOs
Antisense and control MOs were ordered from Gene Tools. The following
sequences were used: splice blocking,
5'-GAGTGAGGAACTTACCTTCTGCTGC-3'; translation blocking,
5'-TCATCTTTGGAAGTCATAGTGGGAC-3'; control,
5'-CCTCTTACCTCAGTTACAATTTATA-3'; and control zebrafish
lbx1 translation blocking,
5'-TTTAGAGCTGGAGGTCATCTCAGTC-3'. Stock solutions were resuspended
at 50 mg/ml. Initially, 17, 33 and 57 ng of antisense MO were injected into
one cell at the two-cell stage. An optimal dose of 33 ng was determined and
used subsequently.
RT-PCR
RNA was extracted from whole tadpoles at stage 28 and used for RT-PCR
(Wilson and Melton, 1994).
Tadpoles used were untreated, injected with 33 ng of splice-MO into one out of
two cells, or injected with 33 ng of MO into two out of two cells. The
following primers were used: ef1
, U
5'-CAGATTGGTGCTGGATATGC-3' and D
5'-ACTGCCTTGATGACTCCTAG-3', 268 nucleotides; lbx1-intron,
U 5'-TCCTAAACAAGCCCTCAGTCCG-3' and D
5'-CCAACTCATAAATCTGGTGGTTCG-3', 300 nucleotides (properly
spliced). Twenty-one cycles were used to amplify for ef1 and 50 cycles
for lbx1 intron.
mRNA synthesis and microinjection
Synthetic mRNA was made using the mMessage mMachine SP6 kit (Ambion). A
zebrafish lbx1 I.M.A.G.E. EST (GenBank AL831789) was ordered from
RZPD (Germany), subcloned from pSport to cs107, and digested with
AscI for mRNA synthesis. Xenopus myoD was synthesized from
the p3 plasmid (Rupp et al.,
1994). Mouse Myf5 was synthesized from a full-length PCR
insert in cs108 after being digested with AscI. Nuclear
ß-galactosidase mRNA (pCS2-Nls-NlacZ, 100-200 pg) was
co-injected with test mRNAs as a lineage tracer.
The synthesized mRNA was resuspended as a stock solution in DEPC-treated H2O at a concentration of 1 mg/ml. Working solutions of lbx1, myoD, myf5, myoD + lbx1 and myf5 + lbx1 were diluted to 0.1 mg/ml in DEPC-treated H2O. Approximately 4 nl of each mRNA solution was injected into one cell at the two-cell stage. Injections were targeted to the mediolateral region of the embryo.
Mutant lbx1 construction
The 5' end of the zebrafish lbx1 EST (GenBank AL831789) was
amplified with the primers LBXCLAI
(5'-CCATCGATGGCGTATGAGGACTAAAGTTCGGGTG-3') and MLBXR
(5'-GACTTCTTAACGGAGAGAGGCTTGTTGG-3'). The 3' end was
amplified with the primers LBXECORI
(5'-CGGAATTCCGCCTTGCATTTCAAGTTCTTCCGTG-3') and MLBXF
(5'-CCAACAAGCCTCTCTCCGTTAAGAAGTC-3'). The 5' and 3'
amplified fragments were gel purified and mixed together, followed by
amplification using the LBXCLAI and LBXECORI primers. The resulting product
was digested with ClaI and EcoRI and gel purified. This was
then inserted into the ClaI, EcoRI sites of pCS107.
Sectioning and counting nuclei
Tadpoles were embedded in 4% low melt agarose and sectioned using a
vibratome (Oxford). The majority of sections were cut at a thickness of 100
µm, except for sections in which the anti-phosphohistone H3 antibody was
used, which were cut at 200 µm. The anti-phosphohistone H3 stained sections
were cleared and mounted in wells cut into Sylgard coated slides and
visualized using a Zeiss Axioplan microscope. Stained nuclei in the immediate
vicinity of the somite through the entire 200 µm sections were counted
using the microscope, and not from subsequent photographs. Sections at
approximately the level of the 3rd trunk somite were used for counting.
P values were obtained using the paired Student's t-test.
Values less than 0.01 were considered to be statistically significant.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
was used to identify muscles of the X. laevis tadpole. At stage 37
(Fig. 1A), the hypaxial rectus
abdominus and rectus cervicus muscles can be clearly distinguished from those
of the epaxial myotome (McDiarmid and
Altig, 1999; Ryke,
1953). By stage 45 (Fig.
1B), the cranial muscles can clearly be seen. The geniohyoideus
muscles are considered to be of somite origin, and are present as a pair of
muscles extending in an anterior-to-posterior manner near the mouth of the
tadpole (Hammond, 1965;
Hazelton, 1970).
Identification and expression of X. laevis lbx1
PCR primers designed from Xenopus tropicalis genomic sequence were
used to screen an arrayed X. laevis cDNA library. A single clone of
lbx1 was found that contains the majority of the coding region minus
the 5' end. The 5' end of the sequence was determined by PCR of
X. laevis genomic DNA using sequence predictions from the X.
tropicalis genome. X. laevis lbx1 contains two conserved domains
typical of the NK class of transcription factors, including the engrailed
homology domain (eh1) and homeodomain. These domains are identical to the
corresponding regions of X. tropicalis, mouse and zebrafish
lbx1. Overall, the amino acid identities are 94%, 79% and 63% between
X. laevis lbx1 and X. tropicalis, mouse and zebrafish
lbx1, respectively, while the nucleic acid identities are 88%, 66%
and 61%, respectively. The mRNA expression pattern of lbx1
(Fig. 1C-H) is first
established at stage 17 in the neural tissue, and at later stages defines a
dorso-intermediate region of the neural tube, consistent with interneuron
specific expression in the mouse (Gross et
al., 2002). Expression of lbx1 during gastrula and early
neurula stages is not detectable by in situ hybridization (data not shown).
Initiation of mesodermal expression can be seen at stage 26 in the
ventrolateral region of anterior trunk somites
(Fig. 1D, arrowhead). As
development proceeds, expression expands posteriorly and ventrally, consistent
with the expression of other genes that mark the developing hypaxial body wall
(Martin and Harland, 2001). At
stage 37/38, a thin stream of cells appears to be migrating into the head
(Fig. 1G, arrow,
Fig. 3C arrowheads). By stage
42, expression becomes very weak in the ventral domain of the body wall
muscles (Fig. 1H, arrowheads)
before it is eventually lost in this region.
|
|
). At
stage 45, the final location of labeled cells in the head can be seen as a
thin band (Fig. 2C, arrows).
Co-localization of DiI- and 12/101-labeled cells indicates that the migrating
population of cells coming from the anterior trunk somites gives rise to the
geniohyoideus (Fig. 2D-G). The
same population of cells was also found to give rise to the rectus cervicus
muscle (data not shown). DiI-labeled cells only give rise to these muscles on
the side of the tadpole in which the somites were labeled (control side not
shown).
Conserved cell-type of lbx1-positive myoblasts in tadpole hypaxial muscle development
Rectus abdominus and other body wall muscles form as epithelial extensions
of the dermomyotome in amniotes and fail to express lbx1
(Dietrich, 1999). Because
lbx1 is expressed in precursors of the rectus abdominus in X.
laevis, we wanted to determine whether these myoblasts migrate in a
manner similar to lbx1-positive long-range myoblasts of amniotes.
lbx1-positive myoblasts are present at the somite clefts of a stage
37 tadpole, in the posterior-most region of lbx1 expression
(Fig. 3A, arrows). This is also
true for other hypaxial specific markers such as tbx3
(Fig. 3B). In amniotes, the
expression of hypaxial markers span the entire width of the somite in the body
wall region as they extend as epithelia, whereas lbx1-positive
zebrafish fin myoblasts emerge at somite clefts and migrate as mesenchymal
cells (Neyt et al., 2000).
|
). Taken
together, these results suggest conservation in the type of
lbx1-positive myoblast migration in X. laevis, despite
expression of lbx1 in myoblasts that populate the body wall
musculature.
Conserved function of lbx1 in hypaxial muscle development
To determine whether lbx1 is required for hypaxial muscle
development, a splice-blocking antisense MO was used to inhibit lbx1
function. Embryos injected into one cell at the two-cell stage or into both
cells at the two-cell stage with 33 ng of MO into each cell were analyzed by
RT-PCR. The results indicate a clear decrease in properly spliced transcript
(Fig. 4A). Quantification of
the radiolabeled bands showed that the one-cell injection caused a 85%
decrease in radioactive signal from the properly spliced transcript compared
with the control, while the two-cell injection led to a 91% decrease. Embryos
were injected into one cell at the two-cell stage with either 33 ng of the
splice blocking MO or 33 ng of a control MO and then stained with 12/101.
Those injected with the splice blocking MO exhibit a severe decrease in body
wall muscle when compared with the uninjected side at stages 37
(Fig. 4F,G) and 40
(Fig. 4H,I) (87% with defects,
n=197). In addition to body wall muscle defects, anterior ventral
views of stage 40 tadpoles show a loss of the geniohyoideus muscle on the
injected side (Fig. 4O, arrow).
By contrast, the control MO injected tadpoles are largely unaffected on the
injected side at stage 37 and 40 (Fig.
4B-E,N, arrow) (16% with defects, n=85). Apart from the
control MO, which is famously non-toxic, other MOs, such as one directed
against zebrafish lbx1 have never caused hypaxial muscle defects
(data not shown). As a further control, a second MO was designed to block the
translation of lbx1, in order to confirm the knockdown phenotype.
When 33 ng of translation blocking MO was injected into one cell at the
two-cell stage, the same loss of hypaxial muscle phenotype was seen at both
stage 37 and 40 (Fig. 4J-M)
(92% with defects, n=40). Later in development, tadpoles injected
with either the splice or translation blocking MO exhibit some development of
hypaxial muscles (Fig. 4I,M),
but the amount of muscle is always significantly less than on the uninjected
side (compare Fig. 4I,M with
4H,L). These results indicate
that lbx1 is required for the proper development of hypaxial muscles
that are derived from lbx1-positive myoblasts, similar to mouse
lbx1 (Brohmann et al.,
2000; Gross et al.,
2000; Schafer and Braun,
1999).
The lack of hypaxial muscles observed in lbx1 mutant mice is due to aberrant migration of myoblasts. The lack of body wall muscles in Xenopus tadpoles that have been injected with lbx1-splice MO can also be partially linked to abnormal migration of myoblasts. Fig. 4P-S shows stage 37 tadpoles that have been stained with pax3, a marker of proliferative myoblasts, and the 12/101 antibody, which marks differentiated skeletal muscle. Merged images showing both pax3 and 12/101 staining (Fig. 4Q,S) indicate the position of pax3-positive hypaxial myoblasts relative to the somites from which they were derived (12/101, green). In control tadpoles, pax3-positive cells have moved ventrally away from the somites (Fig. 4Q). In MO injected tadpoles (Fig. 4R,S), anterior myoblasts have begun to migrate away from the somites, but more posterior cells remain adjacent to the ventrolateral edge of the somites (80% with migration defect, n=25).
|
;
Denetclaw and Ordahl, 2000).
This expansion is marked by myf5 expression in proliferative
myoblasts at the ventrolateral and dorsomedial lips of the dermomyotome. We
examined myf5 expression in injected tadpoles and found that it is
specifically reduced in the ventrolateral region of anterior somites, but is
expressed at normal levels in dorsal and posterior ventral domains
(Fig. 5D, arrow) (96% with
reduced myf5 expression, n=26). Injection of a control MO
has no effect on myf5 expression
(Fig. 5F) (n=6, one
with slightly reduced myf5 expression). The first eight trunk somites
express lbx1 in their ventrolateral domain
(Fig. 1), which normally
overlaps with myf5 expression in this region
(Fig. 5G-J, arrows). When
lbx1 mRNA is overexpressed in somites, a dramatic increase in
myf5 expression is observed, as well as an enlarged somite
(Fig. 5K) (100% with increased
myf5 expression, n=13). Despite the increased size of the
somite, there is a lack of differentiated muscle, as determined by 12/101
staining (Fig. 5L). The
co-injection of lbx1 mRNA with the lbx1-splice MO rescues
the loss of myf5 expression in the ventrolateral domain of the somite
(Fig. 5M,N, compare with 5D),
in addition to upregulating myf5 in other regions of the somite
targeted by the injection (100% rescue, n=7).
lbx1 controls myoblast proliferation
Because of the enlarged somites with a lack of differentiated muscle in
lbx1-injected tadpoles, we investigated whether lbx1 is
controlling myoblast proliferation rather than directly upregulating
myf5 expression. An antibody to phosphorylated histone H3 (pH3) was
used to visualize mitotic cells (Saka and
Smith, 2001). In normal, unmanipulated tadpoles, an abundance of
mitotic cells are found at the ventrolateral region of developing somites,
where lbx1-positive myoblasts are normally present
(Fig. 6A-D, compare
Fig. 6B with
Fig. 5G).
The lbx1-splice MO was injected into one cell at the two-cell stage and stage 31 tadpoles were stained with the pH3 (Fig. 6E) and 12/101 (Fig. 6F) antibodies. A specific loss of mitotic cells is seen in the ventrolateral region of the somite on the injected side, as well as a slight loss of muscle in this region. The opposite is found in lbx1 mRNA-injected tadpoles (Fig. 6G,H), where a dramatic increase in mitotic cells is observed in the somitic region of injected tadpoles (Fig. 6G); this also corresponds to a lack of differentiated muscle (Fig. 6H). Injection of a control MO and ß-galactosidase mRNA has no significant effect on the number of mitotic cells or the amount of differentiated muscle (Fig. 6I,J; results summarized in Fig. 10). At later stages in lbx1 mRNA injected tadpoles, mitosis decreases, corresponding to the terminal differentiation of these cells (Fig. 6K,L). In some cases, the excess differentiated muscle crosses the midline of injected tadpoles (Fig. 6L). By stage 41, the amount of differentiated muscle on the lbx1 mRNA-injected side of the tadpoles greatly exceeds the amount on the uninjected side, though ectopic muscle is never observed outside the somitic region. Fig. 6M,N shows transverse sections through a stage 41 tadpole at a trunk (Fig. 6M) and tail level (Fig. 6N). An increase in differentiated muscle is seen at both levels. This rules out the possibility that the increase in area of muscle in the section is due to distortion of the embryo (due to defective convergence and extension), and thus confirms that that increase in muscle mass is due to increased cell proliferation.
|
;
Halevy et al., 1995;
Hopwood and Gurdon, 1990;
Pownall et al., 2002;
Weintraub et al., 1991). We
therefore investigated whether lbx1 may promote myoblast
proliferation and lack of differentiation through the inhibition of
myoD expression. One cell of each embryo was injected at the two-cell
stage with lbx1 mRNA and analyzed at various stages. At stage 22, a
strong downregulation of myoD expression is observed on the injected
side (Fig. 7A), while
pax3 expression is expanded (Fig.
7F). Transverse sections through stage 24 tadpoles show a complete
loss of myoD expression on the injected side, despite the enlarged
somite (Fig. 7B), again there
is an expansion of pax3 expression on the injected side
(Fig. 7G). As late as stage 33,
the same results are seen. In regions where lbx1 is expressed (marked
by red ß-galactosidase staining), myoD expression is
downregulated (Fig. 7C-E),
while pax3 is expanded (Fig.
7H-J) (100% with reduced myoD expression, n=9;
100% with increased pax3 expression, n=10).
|
If lbx1 normally represses myoD expression, the loss of lbx1 function should result in a transient increase in myoD. This result is observed in stage 26 tadpoles that were injected with the Lbx1-splice MO into one cell at the two-cell stage. A transverse section shows a slight increase of myoD expression on the injected side in the ventrolateral region of the somite (Fig. 7N, arrow) (64% with increased myoD expression on injected side, n=11). In addition, the repression of myoD caused by the ectopic lbx1 expression should coincide with the presence of ectopic lbx1 mRNA. In a stage 31 tadpole, the presence of injected lbx1 mRNA can be detected by in situ hybridization (Fig. 7O). At this stage, myoD is repressed on the injected side (Fig. 7P). By stage 41, ectopic lbx1 mRNA can no longer be detected by in situ hybridization (Fig. 7R). This is also the stage at which a much larger differentiated myotome can be detected on the injected side (Fig. 6M,N). A corresponding increase in myoD expression is observed at this stage (Fig. 7S).
As previously mentioned, the expression of myoD can lead to exit
from the cell cycle and terminal differentiation. In Xenopus, the
cell cycle inhibitor p27 is expressed in developing somites and has
been shown to be required for the proper differentiation of the myotome
(Vernon and Philpott, 2003).
Normal expression is found along the lateral edge of the somite where active
differentiation is occurring. We examined the expression of p27 in
lbx1-injected tadpoles. At stage 31, when injected lbx1 is
present and myoD is repressed, p27 is also repressed
compared to the uninjected side (Fig.
7Q, arrow indicates a region of strong expression on the
uninjected side, which is absent on the injected side). At stage 41, when
injected lbx1 mRNA is no longer present and myoD is
upregulated on the injected side, p27 is also upregulated on the
injected side, indicating that the cells that were overproliferating are now
terminally differentiating (Fig.
7T, right side).
|
Co-injection of myoD with lbx1 represses myoblast proliferation
If lbx1 is controlling myoblast proliferation through the
downregulation of myoD, then the co-injection of myoD with
lbx1 should eliminate the overproliferation of myoblasts. In
Xenopus, the overexpression of myoD or myf5 in the
somitic region causes an expansion of myotome through the recruitment of
non-myogenic cell lineages, rather than an increase in the proliferation of
myoblasts (Ludolph et al.,
1994). In lbx1-injected tadpoles, there is an increase of
mitotic cells (Fig. 9A), a
decrease of differentiated muscle (Fig.
9B) and an expansion of pax3 expression
(Fig. 9C). This is in contrast
to the injection of myoD alone, which causes no change in mitotic
cells (Fig. 9D), a large
increase in differentiated muscle (Fig.
9E), and a slight decrease in pax3 expression
(Fig. 9F). myf5
injection causes a slight expansion of the myotome
(Fig. 9H), but has no effect on
cell proliferation (Fig. 9G) or
pax3 expression (Fig.
9I). When lbx1 is co-injected with myoD, a
phenotype that is the same as myoD alone is observed. There is an
increase in differentiated muscle (Fig.
9K) with no increase in cell proliferation
(Fig. 9J). pax3
expression is also decreased on the injected side of the tadpole
(Fig. 9L). Thus, lbx1
cannot promote myoblast proliferation in the presence of myoD.
However, co-expression of myf5 with lbx1 produces a
phenotype similar to lbx1 alone, where a slightly smaller myotome is
observed (Fig. 9N) but there is
also an increase in cell proliferation
(Fig. 9M) and pax3
expression (Fig. 9O).
The summary of the numbers of phospho-histone H3 nuclei in the immediate somitic region of injected versus uninjected halves of tadpoles is shown in Fig. 10. Significantly more mitotic nuclei are observed on the injected sides of lbx1 and lbx1 + myf5 mRNA-injected tadpoles, while significantly fewer are observed on the lbx1-splice MO injected sides of tadpoles (paired Student's t-test, P<0.01. Fig. 10, indicated with an asterisk). All other injections shown, including the control MO + ß-galactosidase mRNA-injected tadpoles, do not show a significant difference in the amount of mitotic nuclei on the injected side.
Enlarged somites in lbx1-injected tadpoles are not the result of the recruitment of non-myogenic lineages
As previously described, the enlarged myotomes of myoD-injected
tadpoles is the result of the recruitment of non-myogenic lineages, and is not
due to increased cell proliferation
(Ludolph et al., 1994).
Transverse sections of stage 29 tadpoles that have been injected with
myoD (Fig. 11A) or
myoD + lbx1 (Fig.
11B), and stained with the pH3 antibody exhibit larger somites on
the injected side but no increase in cell proliferation. In situ hybridization
for pax8, a pronephric marker, shows that pronephric tubules are lost on the
myoD (91% absent, n=11) or myoD + lbx1
(80% absent, n=10) injected sides of stage 31 tadpoles
(Fig. 11H,J). The pronephric
tubules are present on the uninjected sides
(Fig. 11G,I, arrows). However,
transverse sections through tadpoles injected with lbx1 alone show
normal expression of NCAM in the neural tube on the injected side
(Fig. 11C,arrow), as well as
pax8 in the pronephric tubules (Fig.
11D,arrow) (100% normal, n=8). Similarly, lateral views
of a stage 31 tadpole injected with lbx1 show normal expression of
pax8 in the pronephric tubules on both the uninjected
(Fig. 11E) and injected
(Fig. 11F) sides (arrows).
These results, combined with the cell proliferation results of Figs
6 and
9, indicate that the enlarged
myotomes of lbx1-injected tadpoles are the result of increased
myoblast proliferation and not from the recruitment of non-myogenic
lineages.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This
distinction is based both on the mechanism by which these cells populate
target sites (mesenchymal versus epithelial) and by the genes that they
express. In amniotes, lbx1 was the first molecular marker that was
specific to the long-range type of migratory myoblasts
(Jagla et al., 1995). The
expression of lbx1 in both limb, head and body wall muscles in X.
laevis suggests that the mechanism of epithelial extension to form body
wall muscles has been lost or significantly modified in this animal.
|
;
Gross et al., 2000;
Schafer and Braun, 1999).
Likewise, knockdown of lbx1 function in X. laevis using
antisense MOs depletes body wall and hypoglossal muscles
(Fig. 4). From these data, it
appears that the long-range migration mechanism used in amniote and zebrafish
limbs/fins is also used for body wall muscle development in X. laevis
tadpoles. In support of this idea, in situ hybridization patterns show that
body wall muscle precursors migrate as mesenchymal cells rather than as
epithelial extensions (Fig. 3).
Thus, lbx1-positive myoblasts emerge from the somite clefts before
moving on to populate the body wall muscle
(Fig. 3A,B). This is similar to
what is seen in the migration of zebrafish myoblasts into the fin, suggesting
conservation in the type of migration
(Neyt et al., 2000). Finally,
X. laevis lbx1 is also expressed in hypaxial myoblasts that are
traditionally considered the long-range type, namely limb and hypoglossal
myoblasts (Fig. 3C,E). Taken
together, these data support the idea that body wall myoblasts in X.
laevis are migrating in a manner similar to limb/fin and tongue myoblasts
of amniotes and zebrafish.
Migration defects in lbx1 loss of function may be secondary to reduced myoblast proliferation
Several results support a role for lbx1 in mouse hypaxial myoblast
migration (Brohmann et al.,
2000; Gross et al.,
2000; Schafer and Braun,
1999; Uchiyama et al.,
2000). Using antisense MOs to knockdown lbx1, we have
observed a similar effect in Xenopus. However, we also found that
lbx1 plays a central role in myoblast proliferation, and that this
may be its primary role.
|
; Amthor et al.,
1998). In the chick, reducing the proliferative capacity of
myoblasts by removing the ectoderm at limb and tongue levels causes a lack of
limb and tongue musculature (Amthor et al.,
1999). Furthermore, in mouse lbx1 mutants, some limb
muscles form, in addition to tongue and diaphragm muscles
(Brohmann et al., 2000;
Gross et al., 2000;
Schafer and Braun, 1999). The
precursors to all of these muscles are normally lbx1 positive.
Although they form, these muscles are reduced in size, suggesting that
myoblasts have not proliferated to the proper extent. If lbx1 is only
involved in the migration of myoblasts, one would expect there to be more
severe defects in tongue and diaphragm muscles. If the starting population of
hypaxial myoblasts has a reduced ability to proliferate, and some myoblasts
differentiate at various points along the migratory path, than those sites
farthest from the somite would exhibit the most severe defects. This is what
it seen in mouse mutants, where the distal regions of limbs completely lack
musculature, but the more proximal regions, as well as the diaphragm and
tongue, have musculature, except in reduced sizes. This is also consistent
with the lack of restriction of myoblast fates, such that muscle compartments
are specified at the periphery and filled progressively by migrating myoblasts
(Kardon et al., 2003).
|
|
;
Gross et al., 2000;
Schafer and Braun, 1999). Finally, evidence that lbx1 is not directly involved in myoblast migration comes from the overexpression of lbx1. When lbx1 is overexpressed, there is no increase in number of migratory cells. In fact, there are fewer hypaxial muscles in these tadpoles (data not shown).
lbx1-positive myoblasts contribute to epaxial musculature
At early tadpole stages, Xenopus contain two types of skeletal
muscle in the trunk. In the dorsal region, there is the dorsalis trunci,
representing the epaxial domain. The ventral region contains the rectus
abdominus, which is the hypaxial component of the trunk musculature
(McDiarmid and Altig, 1999).
In lbx1-splice MO-injected tadpoles, the ventral component of
anterior epaxial muscle is reduced, with a corresponding loss of myf5
staining in this region (Fig.
5B,D). This indicates that lbx1-positive myoblasts
contribute to the formation of epaxial musculature. This is the first example
of a case where lbx1-positive myoblasts contribute to both epaxial
and hypaxial muscle, suggesting that the developmental programs of these two
muscle types are more similar than previously suggested. The role of
lbx1 in both types of muscle development also supports earlier
arguments that the primary role of lbx1 is to promote myoblast
proliferation, and not migration.
In vivo evidence that lbx1 controls myoblast proliferation
Previous work has shown that lbx1 can promote the proliferation of
myoblasts in tissue explants (Mennerich
and Braun, 2001). Here, we show that this is also true in the
context of the whole embryo. When ectopic lbx1 mRNA was targeted to
the somitic region, enlarged somites were formed. Initially, these enlarged
somites contained a smaller amount of differentiated muscle
(Fig. 6H). Later on, after the
injected lbx1 had been depleted, the enlarged somites differentiated
into a large amount of muscle (Fig.
6M,N). This was the first indication that lbx1 was
controlling cell proliferation in the somite, as myoblasts can divide or
differentiate, but not do both. However, lbx1 lacks the ability to
induce myoblasts, as injections targeted outside the somite did not result in
the formation of ectopic myoblasts or muscle (data not shown). The presence of
expanded pax3 expression as well as a higher number of mitotic cells
in lbx1-injected tadpoles indicates that lbx1 is a major
regulator of cell proliferation (Fig.
9A-C).
lbx1 controls myoblast proliferation by downregulating myoD
In the chick, lbx1 overexpression in the limb region leads to an
increase in myoD expression and limb muscle formation. This process
requires cell proliferation, indicating that lbx1 may be expanding
the population of myoblasts (Mennerich and
Braun, 2001). We have found that the increase in myoD
expression in these experiments is the end result of a period of myoblast
proliferation in which myoD is not expressed. The overexpression of
lbx1 in the somitic region strongly inhibits the expression of
myoD, so we suggest that the increase in myoD expression in
the chick experiments may follow a period of myoblast proliferation in which
myoD is not expressed. This would fit with the putative role of Lbx1
protein as a transcriptional repressor. In addition to having a homeobox
DNA-binding domain, it also has an engrailed homology region, which has been
shown to mediate transcriptional repression
(Jagla et al., 2001). Indeed,
our results demonstrate that after the injected lbx1 can no longer be
detected by in situ hybridization, the intensity of myoD staining on
the injected side is actually greater than the amount on the uninjected side
(Fig. 7S), similar to what was
seen in the chick experiments.
We found that while lbx1 represses myoD, and causes
proliferation; the proliferative effect can be reversed by restoring
myoD expression with injected mRNA (Figs
9,
10). Thus the enlarged somites
caused by lbx1 expression are different in origin from enlarged
somites caused by ectopic expression of myoD or myf5. The
overexpression of myoD and myf5 in Xenopus embryos
has previously been shown to produce enlarged myotomes, as the result of the
induction of myogenesis in cells that would otherwise have a different fate
(Ludolph et al., 1994). We
confirmed this observation by showing that overexpressed myoD causes
no change in the number of mitotic cells, a slight decrease in pax3
expression, and an increase in differentiated muscle
(Fig. 9D-F). When we
co-expressed myoD and lbx1, the same result was obtained,
indicating that lbx1 does not have the ability to control cell
proliferation independent of myoD levels
(Fig. 9J-L). This indicates
that the control of cell proliferation by lbx1 is achieved through
the inactivation of myoD expression.
Further evidence that lbx1 represses myoD
In addition to the gain-of-function experiments showing that lbx1
inhibits myoD expression, we also find evidence in MO
loss-of-function experiments that links lbx1 to the inhibition of
myoD transcription. If lbx1 has a direct role in repressing
myoD, loss of lbx1 function should result in a transient
upregulation of myoD. Stage 26 tadpoles that have been injected with
the lbx1-splice MO exhibit an increase in myoD expression on
the injected side (Fig. 7N),
suggesting that the repression by endogenous lbx1 has been
relieved.
We have also shown that the putative repressor domain of lbx1 is essential for its function. The removal of a 12 amino acid stretch that encompasses the entire eh1 domain changes the function of lbx1. When overexpressed, the mutant construct no longer inhibits myoD. Instead, a weak increase in myoD expression is seen on the injected side (Fig. 8A). Furthermore, rather than pax3 being upregulated on the injected side, it is inhibited (Fig. 8B). These results indicate that lbx1 normally functions as a repressor and that the removal of the eh1 domain produces a weak dominant-negative effect.
The nature of the dominant negative activity of lbx1eh-
The opposite activity of lbx1eh- relative to wild-type
lbx1 indicates that it is acting as dominant negative. Contrasting
with the lbx1-splice MO, the lbx1eh- injections
also have an effect outside of the normal lbx1 expression domain.
This result suggests that lbx1eh- interferes with the
normal function of a protein other than lbx1. This may include
another NK-class transcription factor, or perhaps even pax3, which is
expressed throughout the entire dorsoventral axis of the somite. Pax3
contains both a homeobox DNA-binding domain as well as an eh1 repressor
domain, similar to lbx1. The Lbx1eh- protein may bind to
homeobox-binding sites and prevent the binding of endogenous homeobox
containing proteins. Owing to the lack of the eh1 domain, it would not be able
to repress the target gene. A similar phenotype to lbx1eh-
overexpression is observed when the bagpipe homologue koza is
overexpressed in Xenopus (Newman
and Krieg, 2002). Bagpipe is a member of the NK-class of
transcription factors. Importantly though, koza contains a
non-conservative amino acid substitution in the eh1 domain. This may in effect
cause it to be a natural dominant-negative protein.
A molecular link between lbx1 and cell cycle control
The cell cycle inhibitor p27 is expressed in developing
Xenopus somites. When its function is reduced using antisense MOs,
enlarged undifferentiated somites result, similar to lbx1-injected
tadpoles (Vernon and Philpott,
2003). These results indicate that p27 is a central
player in the precise timing of myotome differentiation and that lbx1
may inhibit its expression. We tested this by examining p27
expression in lbx1-injected tadpoles. At stage 31, when injected
lbx1 mRNA is still present, p27 expression is inhibited on
the injected side (Fig. 7Q). At
stage 41, when injected lbx1 mRNA is no longer detectable,
p27 expression is enhanced on the injected side
(Fig. 7T). These results are
similar to myoD expression in the same tadpoles and indicate that
cell-cycle inhibition is disrupted in lbx1-injected tadpoles.
Recruitment of non-myogenic lineages is not responsible for enlarged myotomes in lbx1-injected tadpoles
The enhanced cell proliferation seen in lbx1-injected tadpoles
suggests that this is the cause of enlarged somites, but there may also be a
recruitment of non-myogenic lineages similar to what is seen in
myoD-injected tadpoles (Ludolph
et al., 1994). We examined tissues surrounding the somite in
lbx1-injected tadpoles and found that both the neural tube and
pronephros form normally (Fig.
11C-F). However, the pronephros is absent in both myoD
and myoD + lbx1-injected tadpoles
(Fig. 11G-J). These results
demonstrate that the enlarged somites in lbx1-injected tadpoles are
the result of enhanced cell proliferation and not from the recruitment of
non-myogenic lineages.
|
ACKNOWLEDGMENTS |
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|
REFERENCES |
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