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First published online 18 January 2006
doi: 10.1242/dev.02225
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1 Laboratori di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia
e Biochimica, Università di Pisa, Via Carducci 13, Ghezzano, Pisa (PI)
56010, Italy.
2 Dipartimento di Biologia Evolutiva e Funzionale, Università di Parma,
Viale delle Scienze 11/A Parma, (PR) 43100, Italy.
3 Divisione di Oncologia Sperimentale 2, Istituto Nazionale dei Tumori Aviano,
CRO-IRCCS, Via Pedemontana Occidentale, 12, Aviano (PN) 33081, Italy.
* Author for correspondence (e-mail: inardi{at}dfb.unipi.it)
Accepted 28 November 2005
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Has2, CD44, Hyaluronan, Somitogenesis, Myogenesis, Cell migration, Neural crest, ECM, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Turley et al., 2002;
Toole, 2004). Additional cell
regulatory functions of the GAG have been proposed to take place
intracellularly (Lee and Spicer,
2000), but little is known about their significance. Early studies
in the chick embryo strongly suggested that HA is central in creating
migration-permissive microenvironments for the moving precursor cells, such as
those of the peripheral nervous system, muscle and cartilage/bone
(Perris et al., 1991;
Perris and Perissinotto,
2000). Furthermore, the observed HA synthesis and secretion by the
migrating precursor cells themselves suggest that pericellular accumulation of
the GAG could favour the motile behaviour of the cells and potentially
contribute to their correct homing by mediating important interactions with
other ECM components (Perris,
1997; Perissinotto et al.,
2000), or by triggering CD44-mediated signalling cascades
(Ponta et al., 2003).
The vertebrate HA biosynthesis is governed by three catalytic enzymes that
reside at the plasma membrane and are designated Has1, Has2 and Has3. The
catalytic efficiency of these three enzymes seems to differ and the same
applies to the composition of the HA product that they generate
(Itano et al., 1999;
Spicer and McDonald, 1998). In
fact, HA polymers generated by Has1 and Has2 are of a
similar size, reaching molecular weights of up to 2x106 Da,
whereas Has3 polymerizes shorter chains in the range of
2.5x105 Dalton. The precise biological significance of these
diverse catalytic properties of the Has molecules is still not fully
understood, nor it is known how the expression and activity of these enzymes
may be regulated at the molecular and cellular level. Moreover, the three
synthases are expressed in different temporal patterns during mouse
development (Tien and Spicer,
2005) and in particular Has2 has been identified as a
major source of HA during initial organogenesis. Mice with a homozygous
deletion of the Has2 gene manifest severe cardiac and vascular
abnormalities leading to death at midgestation (E9.5-10), underpinning a
pivotal role of Has2 during mammalian embryogenesis
(Camenisch et al., 2000). The
technical limitations in carrying out detailed analyses of the Has2
gene function by transgenic approaches in rodents have incited analogous
investigations in more accessible embryos, such as that of the fish and frog.
Recently, downregulation of the Has2 gene in zebrafish has been found
to perturb the mesodermal cell movements responsible for the gastrulation
process (Bakkers et al.,
2004).
We and others have recently cloned the three known Has genes in
Xenopus laevis and established their spatiotemporal distribution
during early phases of development
(Koprunner et al., 2000;
Vigetti et al., 2003;
Nardini et al., 2004).
XHas1, XHas2 and XHas3 mRNAs are found to exhibit distinct
and non-overlapping spatial expression patterns that differ significantly from
those observed in zebrafish and which suggest an evolutionary divergence in
the control of Has genes expression between tetrapods and teleosts. In
particular, XHas2 is mainly found in mesoderm-derived structures,
such as developing myotomes and heart anlage, and it is expressed by
premigratory trunk neural crest cells (NCC). Interestingly, expression of
XHas2 overlaps that of MyoD in cells committed to a muscle
fate (Nardini et al., 2004).
These findings have prompted us to investigate the possible role of
XHas2 in somitogenesis, myogenic differentiation and trunk NCC
migration, which, thus far, have not been possible to elucidate in
Has2-null mice. To this end, we have also cloned the Xenopus
orthologue of CD44, known to be the principal cell surface receptor of HA
(Wheatley et al., 1993;
Ponta et al., 2003) for which
the precise role during vertebrate development still remains to be
elucidated.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
fixed and stained for ß-galactosidase (200-300 pg injected per embryo) as
previously described (Sive et al.,
2000) using alternatively a red or light-blue substrate for
detection. Whole-mount in situ hybridization was performed on embryos fixed in
MENFA according to standard procedures
(Harland, 1991). Linearized
plasmid from MyoD (HindIII/T7), XCardiac Actin (PvuII/Sp6),
Xp27 (BamHI/T7), XPax3 (BglII/SP6), XGremlin
(SalI/T3) were used to generate digoxigenin-11-UTP-labelled (Roche) antisense
RNA probes from the polymerases indicated. BM purple (Roche) was used as
substrate.
CD44 cloning and tissue distribution
The EST clone IMAGE:7008877 (NCBI GenBank Accession Number BC074228) was
found to correspond to the Xenopus orthologue of CD44
receptor. The EST clone was completely sequenced and the full cDNA sequence
(NCBI GenBank Accession Number DQ143881) aligned with the vertebrate CD44
orthologues using the CLUSTALW
(http://www.ebpac.uk./CLUSTALW)
software. The XCD44 cDNA sequence was found to correspond to the
standard form of CD44. The coding region of XCD44 was subcloned into
the pGEM-T vector (Promega) and used for preparing RNA probe for in situ
hybridization from the linearized plasmid (NotI/T7). RT-PCR analyses
were performed on total RNA from Xenopus embryos isolated at
different developmental stages, ranging from blastula to late tailbud. The
corresponding cDNAs were prepared using ImProm-II Reverse Transcriptase
(Promega). Amplification of the ornithine decarboxylase gene (ODC) has been
used as internal control. The following primers were used: XODC,
5'-AATGGATTTCAGAGACCA-3' (forward) and
5'-CCAAGGCTAAAGTTGCAG-3' (reverse), 25 cycles; XCD44,
5'-CAAGCGCGTACAGTTATCAGC-3' (forward) and
5'-TACAGAGTCTCCCTGGTATCC-3' (reverse), 30 cycles.
Animal cap experiments
Embryos were injected in one blastomere at the two-cell stage with 100 pg
of activin mRNA in vitro transcribed from an activin cDNA
construct kindly provided by Jim Smith (University of Cambridge, UK), using
the SP6 Cap Scribe kit (Roche). Activin-injected embryos were fixed at neurula
stage and processed by whole-mount in situ hybridization to visualize
XHas2 expression. In a second set of experiments, animal caps were
dissected out from stage 8-9 embryos in 1xMBS and, after healing,
cultured in 0.5xMBS. Activin (Sigma) was directly added to the
0.5xMBS medium (100 µg/ml). Caps were incubated for 1-2 hours in
presence of the morphogen, frozen in dry ice and subsequently processed for
RNA extraction. Total RNA was isolated from animal caps of control and
activin-treated embryos and the corresponding cDNAs were prepared using
ImProm-II Reverse Transcriptase (Promega). For PCR amplification, the
following primers were used: XHas2,
5'-ATGCACTGTGAACGGTTTATATGC-3' (forward) and
5'-TCAAGCAAGCACCAAGTCATGTT-3' (reverse), 31 cycles; XBra,
5'-TATATCCACCCAGACTCACCC-3' (forward) and
5'-GATAGAGAGAGAGGTGCCCCG-3' (reverse), 30 cycles; and ODC
(ornithine decarboxylase), 5'-AATGGATTTCAGAGACCA-3' (forward) and
5'-CCAAGGCTAA-AGTTGCAG-3' (reverse), 26 cycles.
mRNA and morpholino injections
The complete coding sequence of XHas2 was obtained by RT-PCR using
the primers described above, on the basis of the sequence reported in NCBI
GenBank (Accession Number AF168465). The PCR products were cloned into pGEM-T
vector (Promega) and subsequently subcloned into the
XhoI/StuI sites of the CS2+ vector. The
full-length clone was completely sequenced and was found to be identical to
the reported sequence for XHas2
(Koprunner et al., 2000).
Capped mRNAs were synthesized in vitro from nuc-ß-gal (NotI/Sp6)
(Chitnis et al., 1995) and from
XHas2-CS2+ (Asp718/Sp6), using the SP6 Cap Scribe kit
(Roche). Sequences of the two non-overlapping XHas2 antisense
morpholino oligos were: XHas2-Mo1 (5' non-translated region of
the gene), 5'-GTTATTGCCTTGGTCCTGTGGTCAC-3'; XHas2-Mo2
(coding region from the start codon),
5'-TGCATATAAACCGTTCACAGTGCAT-3'. Sequences of the two
non-overlapping XCD44 antisense morpholino oligos were:
XCD44-Mo1 (5' non-translated region of the gene),
5'-TGTGCTCCGCAGACAAGAGGCTCCT-3'; XCD44-Mo2 (coding region
from the start codon), 5'-TAACAATCCACAGCATTGAGGCCAT-3'. A control
morpholino was synthesized as a random sequence of the same length (Gene
Tools, Philomath, OR). Embryos were routinely co-injected with morpholino
oligos and ß-gal mRNA into one blastomere at the two-cell stage in
0.1xMMR supplemented with 4% Ficoll. Rescuing experiments were performed
by injecting the optimal effective dose of XHas2-Mo1 (15 ng/embryo)
in conjunction with XHas2 mRNA (800-1000 pg/embryo) and ß-gal
mRNA.
Immunohistochemistry, BrdU incorporation and TUNEL staining
Whole-mount antibody staining was performed following standard procedures
for indirect immunohistochemistry using an anti-muscle ATPase monoclonal
antibody 12/101 (Developmental Studies Hybridoma Bank) at 1:10 dilution and
HRP-conjugated secondary antibodies and DAB for detection. Immunostaining with
the anti-phosphorylated H3 antibody was performed as described by Saka and
Smith (Saka and Smith, 2001).
BrdU incorporation and detection was performed essentially as previously
described (Vernon and Philpott,
2003) and nuclei were counterstained with Hoechst. TUNEL staining
for the identification of apoptotic cells was performed as described by Hensey
and Gautier (Hensey and Gautier,
1998). After whole-mount TUNEL, XHas2-Mo injected neurula
embryos were paraffin wax embedded and sectioned. The obtained slides were
counterstained with Hoechst to allow the simultaneous visualization of normal
and apoptotic nuclei.
Hyaluronan detection on tissue sections
Hyaluronan detection has been carried out using a Neurocan-GFP fusion
protein kindly provided by Professor Uwe Rauch. Wild-type and
XHas2-Mo injected embryos were fixed in 4% paraformaldehyde, paraffin
wax embedded, processed for hyaluronan detection with the Neurocan-GFP probe
essentially as described by Zhang et al.
(Zhang et al., 2004), and
counterstained with Hoechst.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We find here that XHas2 transcription is
ectopically induced in activin mRNA-injected embryos in the
ventrolateral region of the injected side demarcated by the co-injected
ß-gal (100%, n=50; Fig.
1A,A'). This finding indicates that XHas2 is a
downstream target of activin signalling and, hence, may be among the genes
activated through mesoderm induction. In order to further investigate this, we
compared the temporal expression profile of the XBrachyury gene,
known to be a direct target of activin signalling, with that of XHas2
in animal caps. The results reported in
Fig. 1B show that
XHas2 transcription is activated within 1 hour of activin treatment
presenting a time response behaviour similar to XBrachyury. This
observation suggests that XHas2 may be a direct target of activin
signalling.
XHas2 knockdown alters somitogenesis
In order to address the function of the XHas2 during early phases
of development, we used two different non-overlapping antisense morpholino
oligos, XHas2-Mo1 and XHas2-Mo2. These were designed such as
to be complementary to the XHas2 mRNA sequence in a region
surrounding the translational start codon. XHas2 was targeted by
injecting the morpholino oligos in one blastomere of Xenopus embryos
at the two- or four-cell stages. The optimal morpholino concentration was
established by independent pilot experiments and was determined to be in the
range of 10-20 ng/embryo. At higher doses, the survival rate of the embryos
decreased from 90-95% to 5-10% of the total number of injected embryos,
whereas at lower doses we did not observe overt developmental defects. If not
otherwise stated, all the experiments were performed by injecting both the
morpholino oligonucleotides (indicated as XHas2-Mo) and ß-gal
mRNA in order to be able to select properly manipulated embryos on the basis
of Salmon-gal or X-gal staining.
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), impaired heart formation was observed in
Xenopus embryos that were injected with XHas2-Mo in the
marginal zone of a dorsal blastomere at the four-cell stage and processed for
CA in situ hybridization by stage 32. In 79% of these embryos
(n=58), the heart anlage appeared highly abnormal
(Fig. 3E,F). Abrogation of
XHas2 did not seem to cause overt compensatory (or collateral)
effects on the expression pattern of XHas1 and XHas3 (not
shown). The effective abrogation of XHas2 activity in the XHas2-Mo
injected embryos has been verified visualizing the presence of HA on tissue
sections. Recently, a new method has been proposed to detect HA on mice eye
sections by means of a Neurocan-GFP fusion protein that specifically binds HA
and directly visualizes it (Zhang et al.,
2004). We first tested the Neurocan-GFP fusion protein on
Xenopus embryos and then we visualized the presence of HA in
XHas2-Mo-injected embryos at tailbud stage. As shown in
Fig. 3, HA was abundant within
the somitic ECM surrounding the myocytes. By comparing the control and
injected side of the embryo, it was evident that the knockdown of
XHas2 caused a loss of HA in the myocyte pericellular matrix.
In order to identify the developmental time-window within which
XHas2-generated HA was essential for somitogenesis, we analyzed the
expression of early muscle-specific markers, such as XMyoD and
CA at mid-neural plate stage, corresponding to the stage at which
characteristic muscle-specific genes are first detected. In situ hybridization
for XMyoD and CA in XHas2-Mo-injected embryos
demonstrated that loss of XHas2 function had no effect on
XMyoD expression (n=175;
Fig. 4A), whereas it severely
affected the expression of CA in 70% of the embryos (n=111;
Fig. 4B,B'). These
findings suggest that HA synthesized through XHas2 may not be
required for myogenic specification, but is of relevance for the
differentiation of presomitic mesodermal cells into muscle cells, probably by
acting as a vital constituent of their surrounding ECM. To further explore
this possibility, we examined neurula stage XHas2-Mo-injected embryos
for the expression of p27, previously shown to be required for the
cell-cycle exit of myoblasts and their completion of the muscle
differentiation programme (Vernon and
Philpott, 2003). In XHas2-Mo-injected embryos, expression
of p27 was indeed greatly reduced (65%, n=174), in a clear
presomitic mesoderm-restricted manner, whereas it remained unaltered in
primary neurons (Fig. 4C).
We next asked whether the downregulation of p27 could be related to an altered proliferation, or to augmented apoptosis rate, in the presomitic mesoderm depleted of HA. To this end, we performed BrdU incorporation assays, immunohistochemistry with an anti-PH3 antibody and TUNEL assays to determine the relative levels of proliferating and apoptotic cells. We found that the amount of proliferating cells in the injected side of the neurula stage embryos was not significantly altered (n=80, Fig. 4E,F), whereas at the same stage, programmed cell death was greatly increased (70%, n=84, Fig. 4G,G'). Assessment of the relative percentage of apoptotic cells in paraxial mesoderm sections at neurula stage revealed that 30-40% of the presomitic cells were apoptotic in the injected side of the XHas2-Mo embryos (data not shown). In accordance, at early tailbud (stage 22), a clear reduction in the size of the forming somites was evident (Fig. 4D). When we carefully examined XHas2-Mo injected embryos at tailbud stages by TUNEL assay, we did not find any significant increase in the apoptotic rate in muscle cells, neither at stage 24 (n=52, Fig. 4H,H') nor at stage 30 embryos (n=45; Fig. 4I,I'). In these embryos, a high level of apoptosis was normally found in the telencephalon with no differences between the injected and the uninjected side of the embryo (Fig. 4I''). It may therefore be concluded that XHas2 activity plays a crucial role in preventing cells from apoptosis during a limited developmental time window that seems to concur with the early steps of myogenic differentiation.
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). The transcriptional profile of XCD44 determined by RT-PCR analysis and whole-mount in situ hybridization showed that XCD44 was first transcribed at early neurula stage (stage 13) and persisted until tadpole stage 45 (Fig. 5A). At late neurula stage, the XCD44 transcript was detected in the presomitic mesoderm where it showed a segmental pattern that preceded the subsequent mesoderm segmentation and somite formation (Fig. 5B). By stage 24, XCD44 was strongly expressed in the somites (Fig. 5C,C') and in the cement gland, and at lower levels in the migrating cranial NCC (Fig. 5C). In tailbud embryos (stage 32), XCD44 was transcribed in the somites, in the otic vesicle and in the branchial arches (Fig. 5D), whereas later on, it was found to be downregulated in the somitic tissue, except for the dorsal somite tips, and de novo expressed in the central nervous system, notochord and migrating hypaxial muscle cell precursors (Fig. 5E). Overall, the spatiotemporal gene expression pattern of XCD44 largely overlapped that of XHas2, suggesting that the receptor could, at least in part, mediate the hyaluronan function during these stages of development.
XCD44 activity is not necessary for the early muscle differentiation and somitogenesis
Two non-overlapping morpholino oligos, XCD44-Mo1 and
XCD44-Mo2, complementary to the 5'-end region of
XCD44, were injected into one blastomere of Xenopus embryos
at the two- or four-cell stages (15-20 ng/embryo). The downregulation
experiments were performed by co-injecting a mixture of the two morpholino
oligos (XCD44-Mo) that was found to yield a stronger effect and was
consistently used throughout the study. ß-Gal mRNA was injected with the
XCD44-Mo in order to select properly manipulated embryos on the basis
of the Salmon-gal or the X-gal staining. Morphological analysis of the
injected embryos at tailbud stage did not reveal evident developmental
abnormalities and in particular somite formation and segmentation seemed to be
unaffected (Fig. 6D,D').
To detect possible alterations at cellular and molecular level in the injected
side of the embryos, we further analyzed the expression of the genes involved
in the early phases of myogenic commitment MyoD (n=56,
Fig. 5F) and p27
(n=53; Fig. 5G), and
the specific differentiation marker CA (n=60;
Fig. 5H). The expression
pattern of these genes at neurula stage appeared unaffected. The TUNEL assay,
performed at the same stage, did not reveal any increase in the rate of
apoptotic cells in the presomitic region of the injected side (n=68;
Fig. 5I). These observations
suggested that XCD44 was not required for either early myogenesis or
somite formation.
|
). In
XHas2-Mo-injected embryos, XPax3 expression persisted in the
ventral part of somites and migrating hypaxial cells expressing XPax3
were not detectable in the injected side of the embryo (68%, n=90;
Fig. 6C,C'). These
observations suggested that, when deprived of XHas2, hypaxial muscle cells
were unable to commence their proper migration. A summary of the data obtained
in XHas2 and XCD44 loss-of-function experiments during
muscle development is reported in Table
1.
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XHas2 knock down impairs trunk neural crest cell migration
As we have previously found that XHas2 is expressed in
premigratory trunk NCC (Nardini et al.,
2004), we investigated here whether downregulation of the enzyme
could also interfere with the migratory process of these precursor cells.
XHas2-Mo-injected embryos were therefore hybridized with
XGremlin to visualize migrating NCC
(Hsu et al., 1998). In 70%
(n=125) of these embryos, NCC failed to progress along their normal
migratory pathways, resulting in randomly dispersed cells along the flank of
the embryo, when compared with the control side
(Fig. 7A-C). This observation
did not allow us to discern if the detected phenotype was a direct consequence
of the loss of XHas2 activity in NCC, or a secondary alteration
resulting from the abnormal development of somites in
XHas2-Mo-injected embryos. In order to approach this aspect, we
performed double in situ labelling of the somites, using the muscle-specific
antibody 12/101 and the XGremlin probe to detect migrating NCC.
Through this analysis, we could assert that the perturbation of NCC migration
was consistently associated with the somitic mass reduction and segmentation
abnormalities (n=120; Fig.
7D,D'). In accordance, XHas2-Mo-injected embryos
exhibiting a mild muscle phenotype did not show an altered NCC migration.
Although XCD44 is implicated in cell migration and mobility,
XCD44-Mo-injected embryos did not show disturbed migration of trunk
NCC (Fig. 7E,E').
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). On
one hand, this finding asserts the indispensable role of HA during embryonic
development but, on the other hand, it leaves the precise role of the
glycosaminoglycan during embryogenesis unresolved. To address this problem, we
made use of a different vertebrate model in which functional abrogation of
selected genes may be effectively performed in a more accessible embryo. Thus,
after having accomplished the cloning and characterization of the expression
pattern of the three known Has genes during Xenopus development
(Vigetti et al., 2003;
Nardini et al., 2004), we have
addressed here the function of XHas2 in the early developmental
processes by exploiting a targeted knockdown approach at the
post-transcriptional level. Our data highlight a crucial role of
HA/XHas2 in promoting survival and differentiation of myoblast cells,
as well as in the somite formation. Our findings also assert a previously
proposed key role for HA in mediating precursor cell migration with particular
reference to muscle cell precursors of the ventral body wall and trunk NCC.
Recently, a similar loss-of-function study performed in zebrafish embryos
showed that Has2 knockdown affected gastrulation
(Bakkers et al., 2004).
However, no gastrulation defects have been detected following XHas2
knockdown in Xenopus, or deletion of the murine gene. This could in
part be due to a complementary activity of Has1, which is found to be
an early transcribed HA synthase both in Xenopus and mammals, but
absent in the early zebrafish embryo. It may also be further speculated that
such a developmentally related discrepancy in Has gene expression between
tetrapods and teleosts may underlie a possible evolutionary divergence in the
specific requirement of HA in different tissues and in specific time-windows
of development.
|
). To
better understand the possible mechanisms involved in the HA function during
Xenopus somitogenesis and cell migration events, we cloned and
analyzed the role of CD44 during Xenopus development. So
far, the localization of CD44 transcripts during vertebrate
development has not been reported and its role during embryogenesis is still
unclear.
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XHas2 but not XCD44 activity is required during early somitogenesis
Tailbud Xenopus embryos that received a unilateral injection of
XHas2 morpholino at the two-cell stage failed to correctly organize
their flank musculature. In fact, in a mild version of the observed phenotype,
embryos had smaller somites with a morphology deviating from the typical
chevron-like arrangement observed in their normal counterparts. In severely
affected phenotypes, the segmental organization of the myotomes appeared
disrupted, with the individual somites remaining unsegregated and deprived of
intersomitic boundaries. These observations raised the possibility that
XHas2 abrogation, could affect the structural integrity of the inter-
and intra-somitic ECM, which in turn could have interfered with the
establishment of correct cell-cell contacts between the forming myoblasts
(Gurdon et al., 1993;
Cossu et al., 1995). In situ
analyses confirmed the decreased HA accumulation within the myocyte
pericellular matrix as a consequence of the XHas2 abrogation. It is
also likely that this loss might has perturbed the intercellular connections
that are thought to be crucial for proper formation of the somites, as cells
normally undergo a coordinate 90° rotation and realign along the
anteroposterior axis of the embryo
(Keller, 2000).
As XHas2 is expressed in the pre-somitic mesoderm, we asserted
whether altered somitogenesis caused by loss of XHas2 activity was connected
to an interference with the early phases of myogenic differentiation. When
examined at neurula stage, XHas2-deprived embryos showed a
significant loss of CA expression and a substantial increase in the
apoptosis confined to the region where XHas2 has been abrogated.
Conversely, in the same area of the embryo, myoblast proliferation and the
expression of the precocious muscle marker MyoD appeared unaffected.
At the same developmental stage, we also detected a downregulation of
p27, known to be a hallmark of the cell-cycle exit of myoblasts and
their subsequent differentiation (Vernon
and Philpott, 2003). These data strongly suggested that
XHas2 activity was not essential for the early myogenic commitment,
or for myoblasts proliferation, but that it was required for the survival and
differentiation of the early muscle precursors. Indeed, the role of
XHas2 in preventing these cells from undergoing apoptosis seems to be
restricted to a specific developmental period, which coincides with the early
steps of myogenic differentiation in the presomitic mesoderm. The effect of HA
on survival and differentiation of presomitic mesodermal cells could have been
exerted through a structural supportive effect of the ECM, or alternatively HA
produced by the myoblast cells could activate intracellular signals via
binding to specific receptors such as CD44.
The pre-somitic expression of XCD44 supported the above idea and
was consistent with findings in other cellular systems showing that
HA-mediated activation of CD44 exerts an anti-apoptotic effect through
phosphorylation of FAK, activation of RhoB
(Fujita et al., 2002) and
upregulation of specific anti-apoptotic genes
(Marhaba et al., 2003).
However, abrogation of XCD44 through morpholino injection revealed an
unaffected early myogenesis and undisturbed formation of the trunk musculature
at tailbud stages. Moreover, in XCD44 morpholino-injected neurula
embryos, there was no increase in apoptotic rate, suggesting that the
XHas2 generated HA prevented early myoblast death by a different
mechanism than through CD44 signalling. However, proper assembly of the
mesodermal ECM is likely to be required for bringing about community effects,
for proper diffusion/gradient formation of morphogens, and/or for a normal
action of other instructive signals responsible for the differentiation and
survival of myogenic precursors. For example, it has been demonstrated that
members of the TGFß superfamily are more stable if complexed with HA,
whereas they are rapidly degraded in matrices depleted of HA
(Locci et al., 1995).
Comprehensively, our results suggest two possible scenarios for the
HA/XHas2 function during muscle development. First, HA/XHas2
seem to have an early key role in the presomitic mesoderm where they support
myoblast survival. XHas2 depletion alters the muscle differentiation
programme leading, in turn, to the development of embryos with strongly
compromised somite arrangement. Second, the mild muscle phenotype observed in
some of these embryos suggested an additional later role of XHas2 in
contributing to the formation of a hydrated ECM necessary for cell-cell
contacts and morphogenetic movements. In this regard, it is interesting to
note that the XHas2 knockdown phenotype observed at tailbud stages
was highly reminiscent of the one obtained by functional abrogation of type I
cadherins (Giacomello et al.,
2002), or paraxial proto-cadherins
(Kim et al., 2000), which are
cell adhesion molecules known to be essential for somitogenesis. The
possibility to abrogate gene function in a time-regulated manner in
Xenopus, or through conditional gene deletion in mice should allow
the clarification of this aspect.
Hypaxial muscle cells migration is controlled by XHas2 and CD44 activity
In addition to its importance in trunk muscle cell development,
XHas2 appears to be involved in Pax3-positive hypaxial
muscle cell migration which in Xenopus is crucial for the ventral
body wall musculature formation (Martin
and Harland, 2001). Pre-migratory expression of XHas2 in
these precursor cells suggests that the motility impairment, observed in the
XHas-Mo-injected embryos, may, at least in part, be determined by the
failure of these cells to produce HA during their locomotory process. This
disturbed migratory pattern could be reconciled with a HA-mediated modulation
of the cytoskeleton involved in cell motility and attributed to the engagement
of CD44 linking to actin filaments through the ERM (ezrin, radixin and moesin)
family proteins (Legg and Isacke,
1998). In breast cancer cell lines, HA has recently been shown to
be capable of serving as a chemoattractant for directional migration in a CD44
activity-dependent manner probably via the intracellular association with ERM
proteins (Tzircotis et al.,
2005). As we find that the consensus binding motif for ERM
proteins is conserved in the XCD44 orthologue and both this receptor and
XHas2 are co-expressed in hypaxial muscle precursor cells, it is
likely that involvement of cytoskeletal components may be of relevance for the
HA-XCD44 controlled migration of these precursor cells. The restricted
alteration in cell motility in hypaxial muscle precursors following
XCD44 knockdown, raises an intriguing specific requirement of the
receptor activity in a defined subpopulation of muscle cell precursors. In
accordance, although the presence of CD44 has been detected in the mouse
somites during development (Wheatley et
al., 1993), neither the CD44 gene deletion in mice
(Protin et al., 1999) nor our
post-transcriptional abrogation in Xenopus led to developmental
abnormalities in the trunk musculature. The activity of alternative hyaluronan
receptors during somites formation may be a possible explanation for this
paradox.
Hyaluronan is crucial for proper trunk NCC migration
XHas2-deprived embryos showed altered trunk NCC migration,
corroborating a proposed pivotal role of HA during NCC development
(Perris et al., 1996;
Perris, 1997;
Perris and Perissinotto, 2000;
Epperlein et al., 2000). In
fact, previous experimental evidence from our group have indicated that
different local accumulations of HA-versican complexes may define
migration-permissive and non-permissive environments for NCC migrating in the
trunk region of the embryo (Perris et al.,
1991; Perissinotto et al.,
2000). It is noteworthy that Xenopus migrating trunk NCC
do not express XCD44 and that depletion of the receptor in somitic
cells does not influence the NCC movements. Although the presence of HA in the
extracellular environment seems to be crucial for allowing proper NCC
migration, it is not clear if the impaired movement of trunk NCC observed in
XHas2-depleted embryos was due to a failure of these cells to
synthesize and secrete HA, or to a structural deficit caused by its loss from
the ECM of the NCC migratory pathways. An alternative possibility could be
that the altered NCC migration could result from a morphogenetic loss of the
structural integrity of the somites through which the cells migrate.
As a locally and temporally controlled tissue-specific knockdown of a gene is technically difficult to accomplish in Xenopus, we performed a double in situ labelling to simultaneously visualize muscle cell and migrating trunk NCC. Although further studies are needed to fully clarify this issue, the results suggest that defective NCC migration consistently coincides with areas of severe somitic malformation. This latter result may suggest that both a correct structure of somite and a HA enrichment of the ECM, contributed by NCC and myocytes, are necessary to create a specific environmental condition necessary to allow NCC migration. It is also plausible that there may be a different threshold requirement of XHas2 activity in muscle cell precursors and NCC, whereby a partial loss of HA production may be sufficient to alter muscle formation (mild phenotype), but only a virtually complete depletion of matrix HA may interfere with both muscle development and NCC migration.
On the basis of our data, we suggest two different modes through which HA may govern cell migratory phenomena during embryogenesis: by directly influencing the migration process in cells expressing the HA receptor CD44, such as in the case of hypaxial cells; and by serving a structural function in the ECM through its linkage to different proteoglycans that may participate in the elaboration of permissive and non-permissive routes for NCC movement.
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ACKNOWLEDGMENTS |
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