Differential contributions of Mesp1 and Mesp2 to the epithelialization and rostro-caudal patterning of somites
Yu Takahashi1,*,
Satoshi Kitajima1,
Tohru Inoue1,
Jun Kanno1 and
Yumiko Saga2,*
1 Cellular & Molecular Toxicology Division, National Institute of Health
Sciences, 1-18-1 Kamiyoga, Setagayaku, Tokyo 158-8501, Japan
2 Division of Mammalian Development, National Institute of Genetics, Yata 1111,
Mishima 411-8540, Japan
*
Authors for correspondence (e-mail:
yutak{at}nihs.go.jp
and
ysaga{at}lab.nig.ac.jp)
Accepted 29 November 2004
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SUMMARY
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Mesp1 and Mesp2 are homologous basic helix-loop-helix (bHLH) transcription
factors that are co-expressed in the anterior presomitic mesoderm (PSM) just
prior to somite formation. Analysis of possible functional redundancy of Mesp1
and Mesp2 has been prevented by the early developmental arrest of Mesp1/Mesp2
doublenull embryos. Here we performed chimera analysis, using either
Mesp2-null cells or Mesp1/Mesp2 doublenull cells, to clarify (1)
possible functional redundancy and the relative contributions of both Mesp1
and Mesp2 to somitogenesis and (2) the level of cell autonomy of Mesp
functions for several aspects of somitogenesis. Both Mesp2-null and
Mesp1/Mesp2 doublenull cells failed to form initial segment borders or
to acquire rostral properties, confirming that the contribution of Mesp1 is
minor during these events. By contrast, Mesp1/Mesp2 doublenull cells
contributed to neither epithelial somite nor dermomyotome formation, whereas
Mesp2-null cells partially contributed to incomplete somites and the
dermomyotome. This indicates that Mesp1 has a significant role in the
epithelialization of somitic mesoderm. We found that the roles of the Mesp
genes in epithelialization and in the establishment of rostral properties are
cell autonomous. However, we also show that epithelial somite formation, with
normal rostro-caudal patterning, by wild-type cells was severely disrupted by
the presence of Mesp mutant cells, demonstrating non-cell autonomous effects
and supporting our previous hypothesis that Mesp2 is responsible for the
rostro-caudal patterning process itself in the anterior PSM, via cellular
interaction.
Key words: Somitogenesis, Epithelial-mesenchymal conversion, Mesp2, Chimera analysis, Mouse
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Introduction
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Somitogenesis is not only an attractive example of metameric pattern
formation but is also a good model system for the study of morphogenesis,
particularly epithelial-mesenchymal interconversion in vertebrate embryos
(Gossler and Hrabe de Angelis,
1997
; Pourquié,
2001
). The primitive streak, or tailbud mesenchyme, supplies the
unsegmented paraxial mesoderm, known as presomitic mesoderm (PSM). Mesenchymal
cells in the PSM undergo mesenchymal-epithelial conversion to form epithelial
somites in a spatially and temporally coordinated manner. Somites then
differentiate, in accordance with environmental cues from the surrounding
tissues, into dorsal epithelial dermomyotome and ventral mesenchymal
sclerotome (Borycki and Emerson,
2000
; Fan and Tessier Lavigne,
1994
). Hence, the series of events that occur during somitogenesis
provide a valuable example of epithelial-mesenchymal conversion. The
dermomyotome gives rise to both dermis and skeletal muscle, whereas the
sclerotome forms cartilage and bone in both the vertebrae and the ribs. Each
somite is subdivided into two compartments, the rostral (anterior) and caudal
(posterior) halves. This rostro-caudal polarity appears to be established just
prior to somite formation (Saga and
Takeda, 2001
).
Mesp1 and Mesp2 are closely related members of the basic helix-loop-helix
(bHLH) family of transcription factors but share significant sequence homology
only in their bHLH regions (Saga et al.,
1996
; Saga et al.,
1997
). During development of the mouse embryo, both Mesp1
and Mesp2 are specifically expressed in the early mesoderm just after
gastrulation and in the paraxial mesoderm during somitogenesis. Mesp1/Mesp2
double-null embryos show defects in early mesodermal migration and thus fail
to form most of the embryonic mesoderm, leading to developmental arrest
(Kitajima et al., 2000
).
Mesp1-null embryos exhibit defects in single heart tube formation, due to a
delay in mesodermal migration, but survive to the somitogenesis stage
(Saga et al., 1999
),
suggesting that there is some functional redundancy, i.e. compensatory
functions of Mesp2 in early mesoderm. During somitogenesis, both
Mesp1 and Mesp2 are expressed in the anterior PSM just prior
to somite formation. Although we have shown that Mesp2, but not Mesp1, is
essential for somite formation and the rostro-caudal patterning of somites
(Saga et al., 1997
), a
possible functional redundancy between Mesp1 and Mesp2 has not yet been
clearly established.
To further clarify the contributions of Mesp1 and Mesp2 to somitogenesis,
analysis of Mesp1/Mesp2 double-null embryos is necessary, but because of the
early mesodermal defects already described, these knockout embryos lack a
paraxial mesoderm, which prevents any analysis of somitogenesis. We therefore
adopted a strategy that utilized chimera analysis. As we have reported
previously, the early embryonic lethality of a Mesp1/Mesp2 double knockout is
rescued by the presence of wild-type cells in a chimeric embryo, but the
double-null cells cannot contribute to the cardiac mesoderm
(Kitajima et al., 2000
). This
analysis, however, focused only on early heart morphogenesis and did not
investigate the behavior of Mesp1/Mesp2 double-null cells in somitogenesis. In
this report, we focus upon somitogenesis and compare two types of chimeras
using either Mesp1/Mesp2 double-null cells or Mesp2-null cells to investigate
Mesp1 function during somitogenesis.
Another purpose of our chimera experiments was to elucidate the cell
autonomy of Mesp functions. In the process of somite formation, mesenchymal
cells in the PSM initially undergo epithelialization at the future segment
boundary, independently of the already epithelialized dorsal or ventral margin
of the PSM (Sato et al.,
2002
). Epithelial somite formation is disrupted in the Mesp2-null
embryo, indicating that Mesp2 is required for epithelialization at the segment
boundary. Although Mesp products are nuclear transcription factors and their
primary functions must therefore be cell autonomous (transcriptional control
of target genes), it is possible that the roles of Mesp2 in epithelialization
are mediated by the non-cell autonomous effects of target genes. We therefore
asked whether the defects in Mesp2-null cells during epithelialization could
be rescued by the presence of surrounding wild-type cells. Additionally, we
would expect to find that the role of Mesp2 in establishing rostro-caudal
polarity is rescued in a similar way.
Our analysis suggests that Mesp1 and Mesp2 have redundant functions and are
both cell-autonomously involved in the epithelialization of somitic mesoderm.
In addition, our results highlight some non-cell autonomous effect of
Mesp2-null and Mesp1/Mesp2-null cells.
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Materials and methods
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Generation of chimeric embryos
As described previously (Kitajima et
al., 2000
), chimeric embryos were generated by aggregating 8-cell
embryos of wild-type mice (ICR) with those of mutant mice that were
genetically marked with the ROSA26 transgene
(Zambrowicz et al., 1997
).
Mesp1/Mesp2 double-null embryos were generated by crossing wko-del
(+/) and
Mesp1(+/)/Mesp2(+/cre) mice as
described previously (Kitajima et al.,
2000
). This strategy enables us to distinguish chimeric embryos
derived from homozygous embryos, which have two different mutant alleles, from
those derived from heterozygous embryos. Likewise, Mesp2-null embryos were
generated by crossing P2v1(+/) mice
(Saga et al., 1997
) and
P2GFP (+/gfp) mice (Y.S. and S.K., unpublished) that were
also labeled with the ROSA26 locus. The genotype of the chimeric
embryos was determined by PCR using yolk sac DNA.
Histology, histochemistry and gene expression analysis
The chimeric embryos were fixed at 11 days postcoitum (dpc) and stained in
X-gal solution for the detection of ß-galactosidase activity, as
described previously (Saga et al.,
1999
). For histology, samples stained by X-gal were postfixed with
4% paraformaldehyde, dehydrated in an ethanol series, embedded in plastic
resin (Technovit 8100, Heraeus Kulzer) and sectioned at 3 µm. The methods
used for gene expression analysis by in-situ hybridization of whole-mount
samples and frozen sections and skeletal preparation by Alcian Blue/Alizarin
Red staining were described previously
(Saga et al., 1997
;
Takahashi et al., 2000
).
Probes for in-situ hybridization for Uncx4.1
(Mansouri et al., 1997
;
Neidhardt et al., 1997
),
Delta-like 1 (Dll1)
(Bettenhausen et al., 1995
) and
Paraxis (Burgess et al.,
1995
) were kindly provided by Drs Peter Gruss, Achim Gossler and
Alan Rawls, respectively. A probe for EphA4
(Nieto et al., 1992
) was
cloned by PCR. For detection of actin filaments, frozen sections were stained
with AlexaFluor 488-conjugated phalloidin (Molecular Probes) according to the
manufacturer's protocol.
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Results
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Possible functional redundancy and different contributions of Mesp1 and Mesp2 in somitogenesis
During somitogenesis, both Mesp1 and Mesp2 are expressed
in the anterior PSM just prior to somite formation and their expression
domains overlap (Fig. 1A).
Mesp1-null embryos form morphologically normal somites and show normal
rostrocaudal patterning within each somite
(Fig. 1B,E-H), indicating that
Mesp1 is not essential for somitogenesis. By contrast, Mesp2 is essential for
both the formation and rostro-caudal patterning of somites, as Mesp2-null
embryos have no epithelial somites and lose rostral half properties, resulting
in caudalization of the entire somitic mesoderm
(Saga et al., 1997
)
(Fig. 1C,D).

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Fig. 1. Mesp1 and Mesp2 are co-expressed in the anterior PSM but
have differing roles in somitogenesis. (A) Overlapping expression of
Mesp1 and Mesp2 is revealed by in-situ hybridization using
the left and right halves of the same embryo. The lines show most recently
formed somite boundaries. (B-C) A Mesp1-null embryo (B) shows the same normal
somite formation as a wild-type embryo (C). Arrowheads indicate somite
boundaries. (D) In Mesp2-null embryos, no somite formation is observed but
Mesp1 is expressed at comparable levels to wild type, although its
expression is anteriorly extended and blurred. (E-H) Mesp1-null embryos show
normal rostro-caudal patterning of somites. (E,F) Expression of a caudal half
marker, Uncx4.1. (G,H) Expression of a rostral half marker,
EphA4. The lines indicate presumptive or formed somite boundaries and
the dotted line indicates approximate position of somite half boundary.
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Although somite formation and rostro-caudal patterning is disrupted in the
Mesp2-null embryo, histological differentiation into dermomyotome and
sclerotome is not affected. It is noteworthy that the Mesp2-null embryo still
forms disorganized dermomyotomes without forming epithelial somites
(Saga et al., 1997
). As
Mesp1 is expressed at normal levels in the PSM of Mesp2-null embryos
(Fig. 1C,D), it is possible
that Mesp1 functions to rescue some aspects of somitogenesis in the Mesp2-null
embryo. In order to further clarify the contributions of both Mesp1 and Mesp2
during somitogenesis, we therefore generated chimeric embryos with either
Mesp2-null cells or Mesp1/Mesp2 double-null cells and compared the behavior of
mutant cells during somitogenesis (Fig.
2).

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Fig. 2. Schematic representation of chimera analysis method. Either Mesp2-null or
Mesp1/Mesp2 double-null embryos, genetically labeled with Rosa locus,
were aggregated with wild-type embryos at the 8-cell stage, and the resulting
chimeras were subjected to analysis at 11.0 dpc.
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Mesp2-null cells tend to be eliminated from the epithelial somite and the dermomyotome, but can partially contribute to both of these structures
We first generated Mesp2-null chimeric embryos
(Mesp2/ with Rosa26: wild) to
analyze cell autonomy of Mesp2 function during somitogenesis. The control
chimeric embryo (Mesp2+/ with Rosa26:
wild) showed normal somitogenesis and a random distribution of X-gal stained
cells (Fig. 3A). The Mesp2-null
chimeric embryos formed abnormal somites that exhibited incomplete
segmentation (Fig. 3B), but
histological differentiation of dermomyotome and sclerotome was observed.
Within the incomplete somite, X-gal-stained Mesp2-null cells were mainly
localized in the rostral and central regions, surrounded by wild-type cells at
the dorsal, ventral and caudal sides (Fig.
3B). The surrounding wild-type cells, however, did not form an
integrated epithelial sheet, but consisted of several epithelial cell
clusters. Such trends were more obviously observed in other sections, where
wild-type cells were found to form multiple small epithelial clusters
(Fig.3C,D). Mesp2-null cells
tended to be eliminated from the epithelial clusters, although they were
partially integrated into these structures (blue arrows in
Fig. 3C,D). Likewise, small
numbers of Mesp2-null cells were found to contribute to the dermomyotome
(Fig. 3E,F). Mesp2-null cells
also appeared to form the major part of the sclerotome.

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Fig. 3. Mesp2-null cells tend to be excluded from the epithelial region of the
somites. (A) The control chimeric embryo undergoes normal somite formation and
shows random distribution of labeled cells. The right panel is a high-power
view of a somite. (B) In the Mesp2-null chimeric embryo, incompletely
segmented somites are formed. Mesp2-null cells tend to be localized at the
rostral and central region of these incomplete segments. Red arrows: wild-type
cell clusters; blue arrows: Mesp2-null cell clusters. (C,D) Other sections
indicating multiple small epithelial cell clusters (arrows). Note that
Mesp2-null cells only partially contribute to the epithelial clusters (blue
arrows). (E,F) A small number of Mesp2-null cells are distributed in the
dermomyotome and are mostly localized at the caudal end. Scale bars: 100
µm.
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Mesp2 is required for the cell-autonomous acquisition of rostral properties
We have previously demonstrated that suppression by Mesp2 of the caudal
genes Dll1 and Uncx4.1 in presumptive rostral half somites
is a crucial event in the establishment of the rostrocaudal pattern of somites
(Saga et al., 1997
;
Takahashi et al., 2000
). As
Mesp2-null embryos exhibit caudalization of somites, Mesp2-null cells are
predicted to be unable to express rostral properties. Hence, Mesp2-null cells
are expected to distribute to the caudal region of each somite where the
rostrocaudal patterns are rescued by wild-type cells in a chimeric embryo. In
this context, the localization of Mesp2-null cells at the rostral side was an
unexpected finding. We interpret this to mean that the rostral location of
Mesp2-null cells is due to a lack of epithelialization functions (see
Discussion).
To examine rostro-caudal properties in Mesp2-null cells, located in the
rostral side, we analyzed the expression of a caudal half marker gene,
Uncx4.1 (Mansouri et al.,
1997
; Neidhardt et al.,
1997
). Analysis of adjacent sections revealed that
lacZ-expressing Mesp2-null cells, localized at the rostral and
central portion, ectopically expressed Uncx4.1
(Fig. 4A-D). This strongly
suggests that Mesp2-null cells cannot acquire rostral properties even if
surrounded by wild-type cells, and that Mesp2 function is cell-autonomously
required for the acquisition of rostral properties. We also observed that the
small number of Mesp2-null cells distributed mostly to the caudal end of the
dermomyotome (Fig. 3E,F) and
that the expression pattern of Uncx4.1 was normal in the dermomyotome
(Fig. 4E,F). In the sclerotome,
lacZ-expressing Mesp2-null cells often distributed to the rostral
side, where expression of Uncx4.1 was abnormally elevated
(Fig. 4G,H). The vertebrae of
the Mesp2-null chimeric fetus showed a partial fusion of the neural arches,
which was reminiscent of Mesp2-hypomorphic fetuses
(Fig. 4I,J)
(Nomura-Kitabayashi et al.,
2002
). Fusion of proximal rib elements was also observed
(Fig. 4K,L).

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Fig. 4. Mesp2 function is cell autonomously required for rostral properties. (A-D)
Expression of lacZ and Uncx4.1 transcripts at the site of
initial somite formation in control (A,B) and Mesp2-null (C,D) chimeric
embryos. In the control, lacZ-expressing cells are randomly
distributed and Uncx4.1 expression is normal. In the Mesp2-null
chimera, lacZ-expressing Mesp2-null cells at the rostral part of the
incomplete segments (arrows in C) ectopically express Uncx4.1 (arrows
in D). Lines indicate somite boundaries. (E,F) In the dermomyotome, Mesp2-null
cells are mostly localized at the caudal end, and the Uncx4.1
expression pattern is normal. (G,H) In the sclerotome, the distribution of
Mesp2-null cells results in expansion of Uncx4.1 expression (arrows).
(I) The control chimeric fetus shows normal vertebrae. (J) The Mesp2-null
chimeric fetus exhibits partial fusion of the neural arches. (K) The control
chimeric fetus shows normal ribs. (L) The Mesp2-null chimeric fetus shows
proximal rib fusion. Scale bars: 100 µm. C, caudal compartment; R, rostral
compartment.
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Mesp1/Mesp2 double-null cells cannot contribute to the formation of epithelial somites or to the dermomyotome
To address the question of whether Mesp1, in addition to Mesp2, exhibits
any function during somitogenesis, we next generated Mesp1/Mesp2 double-null
chimeric embryos and compared them with the Mesp2-null chimeric embryos
described in the previous sections. We first performed whole-mount X-gal
staining of embryos at 11 dpc. In the control chimeric embryo, the
X-gal-stained Mesp1/Mesp2 double-heterozygous cells distributed randomly
throughout the embryonic body, including the somite region
(Fig. 5A,C). By contrast, the
Mesp1/Mesp2 double-null chimeric embryo displayed a strikingly uneven pattern
of cellular distribution in the somite region. The X-gal stained Mesp1/Mesp2
double-null cells were localized at the medial part of embryonic tail and were
not observed in the lateral part of the somite region
(Fig. 5B,D). Histological
examination of parasagittal sections further revealed obvious differences in
the cellular contribution to somite formation
(Fig. 5E,F). In the control
chimeric embryo, Mesp1/Mesp2 double-heterozygous cells distributed randomly
throughout the different stages of somitogenesis (PSM, somite, dermomyotome
and sclerotome: Fig. 5E). In
the Mesp1/Mesp2 double-null chimeric embryo, neither the initial segment
border nor epithelial somites were formed, but histologically distinguishable
dermomyotome-like and sclerotome-like compartments were generated
(Fig. 5F). In addition,
Mesp1/Mesp2 double-null cells and wild-type cells were randomly mixed in the
PSM, whereas the dermomyotome-like epithelium consisted exclusively of
wild-type cells and the sclerotome-like compartment consisted mostly of
Mesp1/Mesp2 double-null cells. This suggests that either Mesp1 or Mesp2 is
cell-autonomously required for the formation of epithelial somite and
dermomyotome. These results also indicate that PSM cells with different
characteristics are rapidly sorted during somite formation.

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Fig. 5. Mesp1/Mesp2 double-null cells fail to contribute to epithelial somites or
to the dermomyotome. (A-D) Tail regions from X-gal-stained whole-mount
specimens of control (A,C) and double-null (B,D) chimeric embryos. (A,B)
Lateral view. (C,D) Dorsal view. The blue double-heterozygous cells are
randomly distributed in the control embryo, whereas the Mesp1/Mesp2
double-null cells are excluded from the lateral region of the somites
(arrowheads in D). (E,F) Parasagittal sections of tails from chimeric embryos.
(E) The labeled cells are randomly located in the control chimera. (F) The two
types of cells are randomly mixed in the PSM, whereas the dermomyotome-like
epithelium consisted exclusively of wild-type cells and the sclerotome-like
compartment contained mostly Mesp1/Mesp2 double-null cells. Note that normal
epithelial somites are not formed in this chimera. (G,H) Transverse sections
show elimination of Mesp1/Mesp2 double-null cells from the dermomyotome. (I,J)
The dermomyotome-like epithelium in the Mesp1/Mesp2 double-null chimeric
embryo gives rise to dermomyotome, myotome (arrowhead in J) and the dorsal
part of the sclerotome. Red arches indicate the inner surface of dermomyotome.
(K,L) AlexaFluor 488-labeled phalloidin staining shows normal
epithelialization of somites in the control chimera (K) and restriction of
epithelialization in the dermomyotome-like compartment in the Mesp1/Mesp2
double-null chimera (L). dm, dermomyotome; dml, dermomyotome-like epithelium;
dsc, dorsal part of the sclerotome; my, myotome; nt, neural tube; sc,
sclerotome; scl, sclerotome-like compartment; tg, tail gut.
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Subsequent examination of transverse sections confirmed the elimination of
Mesp1/Mesp2 double-null cells from dermomyotome
(Fig. 5G,H). In the mature
somite region, the wild-type dermomyotome-like epithelium was found to form
the myotome (my) (Fig. 5I,J).
Furthermore, the ventral part of this dermomyotome-like epithelium became
mesenchymal and appeared to contribute to the dorsal sclerotome (dsc),
implying that this initial dermomyotome-like epithelium actually corresponds
to the epithelial somite exclusively composed of wild-type cells
(Fig. 5I,J). Fluorescent
phalloidin staining revealed that the apical localization of actin filaments
is limited to the dorsal compartments, which are occupied by wild-type cells
in the Mesp1/Mesp2 double-null chimeric embryo
(Fig. 5K,L), indicating the
Mesp1/Mesp2 double-null cells cannot undergo epithelialization.
It is known that the bHLH transcription factor paraxis (Tcf15 Mouse
Genome Informatics), is required for the epithelialization of somite and
dermomyotome (Burgess et al.,
1995
; Burgess et al.,
1996
). Although Paraxis expression is not affected in
Mesp2-null embryos (data not shown), it is possible that it is influenced by
the loss of both Mesp1 and Mesp2. We therefore examined the expression
patterns of Paraxis in our Mesp1/Mesp2 double-null chimeras. In
wild-type embryos Paraxis is initially expressed throughout the
entire somite region (in both the prospective dermomyotomal and sclerotomal
regions) in the anteriormost PSM and newly forming somites, and then localizes
in the dermomyotomes (Burgess et al.,
1995
). The dorsal dermomyotomal epithelium, composed of
wildtype cells, strongly expressed Paraxis in the chimeric
embryo (Fig. 6A,B). In
addition, adjacent sections revealed that lacZ-expressing Mesp1/Mesp2
double-null cells expressed Paraxis in the medial sclerotomal
compartment (Fig. 6A,B, brackets). This suggests that Paraxis expression in the future
sclerotomal region is independent of Mesp factors. However, at present we
cannot exclude the possibility that the maintenance of Paraxis
expression in the dermomyotome requires the functions of either Mesp1 or
Mesp2.

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Fig. 6. (A,B) Mesp1/Mesp2 double-null cells express Paraxis. Adjacent
parasagittal sections of the Mesp1/Mesp2 double-null chimeric embryo were
stained for either Paraxis (A) or lacZ (B). Note that the
expression domains of the two genes overlap in the medial sclerotomal region
(brackets). (C,D) The rostro-caudal pattern in the dermomyotome is formed in a
partially segregating wild-type cell population. Adjacent sections of the
Mesp1/Mesp2 double-null chimeric embryos were stained for Dll1 (C) or
lacZ (D) mRNA. Red outlines demarcate the dorsal dermomyotome-like
compartments. Note that suppression of Dll1 expression occurs in a
region mostly occupied by wild-type cells (arrows). Scale bar: 100 µm.
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Mesp1/Mesp2 double-null cells are incapable of acquiring rostral properties
To clarify the rostro-caudal properties of somites in our chimeric embryos,
we examined the expression pattern of Uncx4.1. Control chimeric
embryos exhibited a normal stripe pattern of Uncx4.1 expression
throughout the segmented somite region
(Fig. 7A). By contrast,
Mesp1/Mesp2 double-null chimeric embryos exhibited continuous Uncx4.1
expression in the ventral sclerotomal region
(Fig. 7B). This continuity was
observed in the entire sclerotome-like compartment of the newly formed somite
region and in the ventral sclerotome in the mature somite region. The caudal
localization of Uncx4.1 expression, however, was normal in the
dermomyotome and the dorsal sclerotome, which consisted of wild-type cells
(Fig. 5), even in Mesp1/Mesp2
double-null chimeras. This suggests that, like Mesp2-null cells, Mesp1/Mesp2
double-null cells are incapable of acquiring rostral properties. Since the
mesoderm of Mesp1/Mesp2 double-null embryos lacks the expression of the major
markers of paraxial mesoderm (Kitajima et
al., 2000
), and Mesp1/Mesp2 double-null cells do not exhibit
histological features characteristic of epithelial somites in our current
study, it is possible that Mesp1/Mesp2 double-null cells may lack paraxial
mesoderm properties. However, the analysis of adjacent sections suggests that
lacZ-expressing Mesp1/Mesp2 double-null cells themselves express
Uncx4.1, a somite-specific marker
(Fig. 7C,D), and they had also
been found to have normal expression of Paraxis
(Fig. 6A,B).

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Fig. 7. Rostro-caudal patterning of the sclerotome is disrupted in Mesp1/Mesp2
double-null chimeric embryos. (A) The control chimeric embryos exhibit normal
stripe patterns of Uncx4.1 expression throughout the somite region.
(B) The Mesp1/Mesp2 double-null chimeric embryos exhibit continuous
Uncx4.1 expression in the ventral sclerotomal region. Note that
caudal localization of Uncx4.1 expression is normal in the
dermomyotome and dorsal sclerotome. The insets show a higher magnification of
lumbar somites. (C,D) Adjacent sections showing that lacZ-expressing
Mesp1/Mesp2 double-null cells express Uncx4.1. (E-H) The Mesp1/Mesp2
double-null chimeric fetus exhibits caudalization of the vertebrae and of the
proximal ribs. (E) The control chimeric fetus shows normal metameric
arrangement of the neural arches. (F) The Mesp1/Mesp2 double-null chimeric
fetus shows severe fusion of the pedicles and the laminae of neural arches.
(G) The control chimeric fetus has normal arrangement of ribs. (H) The
double-null chimeric fetus shows severe fusion of the proximal elements of the
ribs. Scale bars: 100 µm.
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It is believed that the rostro-caudal pattern within somites and
dermomyotomes is generated in the PSM and maintained in somites and
dermomyotomes. We observed a normal rostro-caudal pattern in the dermomyotome
(Fig. 7), although wild-type
cells and Mesp1/Mesp2 double-null cells are mixed in the PSM
(Fig. 5), of Mesp1/Mesp2
double-null chimeric embryos. As Mesp products are required for suppression of
Dll1 in the anterior PSM, a normal Dll1 stripe pattern cannot be
formed if Mesp1/Mesp2 double-null cells are randomly distributed in the
anterior PSM. This is because 50% of cells cannot undergo suppression of Dll1
even in the future rostral half region. Therefore, our finding of a normal
rostro-caudal pattern in the dermomyotome of double-null chimeras is
surprising and raises the question of whether wild-type cells can be normally
patterned in the presence of surrounding Mesp1/Mesp2 double-null cells. To
determine how the rostro-caudal pattern in the dermomyotome is formed in the
PSM, we examined the expression pattern of Dll1
(Bettenhausen et al., 1995
),
the stripe expression profile of which is established in the anteriormost PSM
via the function of Mesp2 (Takahashi et
al., 2000
). The lacZ-expressing Mesp1/Mesp2 double-null
cells were subsequently found to be consistently localized in the
sclerotome-like region, where Dll1 expression was abnormally expanded
(Fig. 6C,D). In the
dermomyotome-like region, however, Dll1 expression in the caudal half
was normal. Intriguingly, strong Dll1 expression in the anteriormost
PSM was suppressed in a rostrally adjoining cell population, which is mainly
occupied by wild-type cells (Fig.
6C,D, arrows). This implies that wild-type cells and Mesp1/Mesp2
double-null cells rapidly segregate at S1 to S0, after which the
rostro-caudal pattern of Dll1 expression is formed in the partially
segregated wild-type cell population but not in the randomly mixed cell
population. In other words, the separation from Mesp1/Mesp2 double-null cells
enabled normal rostro-caudal patterning of wild-type cells.
Mesp2-null fetuses display caudalized vertebrae with extensive fusion of
the pedicles of neural arches and proximal elements of the ribs
(Saga et al., 1997
). The
Mesp1/Mesp2 double-null chimeric fetuses also exhibited fusion of the pedicles
of neural arches and the proximal ribs
(Fig. 7E-H). Furthermore, the
vertebrae of severe chimeric fetuses were indistinguishable from those of
Mesp2-null fetuses. These observations indicate that Mesp1/Mesp2 double-null
cells can differentiate into caudal sclerotome and possibly contribute to
chondrogenesis.
 |
Discussion
|
---|
Mesp1 and Mesp2 not only exhibit similar expression patterns but also share
common bHLH domains as transcription factors. Previous studies using gene
replacement experiments (Saga,
1998
) (Y.S. and S.K., unpublished) indicate that these genes can
compensate for each other. However, the early lethality of double knockout
mice hampered any further detailed analysis of somitogenesis. An obvious
strategy to further elucidate the functions of Mesp1 and Mesp2 was, therefore,
the generation of a conditional knockout allele for Mesp2 in
Mesp1 disrupted cells in which the Cre gene is specifically activated
in the paraxial mesoderm, which is now underway. Chimera analysis is also a
powerful method as an alternative strategy. Comparisons of chimeras, composed
of either Mesp2-null or Mesp1/Mesp2 double-null cells, made it possible to
determine the contribution of Mesp1 to somitogenesis. Our results indicate
that Mesp1 has redundant functions in the epithelialization of somitic
mesoderm and additionally, by chimeric analysis, we were able to demonstrate
the cell autonomy of Mesp1 and Mesp2 function during some critical steps of
somitogenesis.
The relative contributions of Mesp1 and Mesp2 to somitogenesis
In Mesp1-null mice, epithelial somites with normal rostro-caudal polarity
are generated, whereas Mesp2-null mice exhibit defects in both the generation
of epithelial somites and the establishment of rostro-caudal polarity. Thus,
it seems likely that Mesp2 function is both necessary and sufficient for
somitogenesis. However, dermomyotome formation was observed, without normal
segmentation, even in Mesp2-null mice. In view of the apparent redundant
functions of Mesp1 and Mesp2 in somitogenesis, as demonstrated by our previous
gene replacement study, it was possible that the Mesp1/Mesp2 double-null
embryo would exhibit a much more severe phenotype in relation to
somitogenesis. In our chimera analyses, both Mesp2-null and Mesp1/Mesp2
double-null cells exhibited complete caudalization of somitic mesoderm,
indicating that Mesp1 function is not sufficient to rescue Mesp2 deficiency
and restore rostro-caudal polarity. Likewise, both Mesp2-null and Mesp1/Mesp2
double-null cells were incapable of forming an initial segment boundary,
showing that the contribution of Mesp1 is also minor during this process. By
contrast, whereas Mesp1/Mesp2 double-null cells lacked any ability to
epithelialize, Mesp2-null cells were occasionally integrated into epithelial
somites and dermomyotome, indicating that the contribution of Mesp1 to
epithelialization is significant and that Mesp1 can function in the absence of
Mesp2 (Fig. 8). We therefore
postulate that the epithelialization of dermomyotome, observed in Mesp2-null
embryos, is dependent on Mesp1.

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|
Fig. 8. A schematic summarization of the Mesp1/Mesp2 chimera experiments.
Mesp1/Mesp2 double-null cells can contribute to neither epithelial somite nor
dermomyotome formation, whereas Mesp2-null cells can partially contribute to
both somites and dermomyotome. Red outlines indicate epithelialized tissues
(epithelial somites, dermomyotomes and abnormal small clusters).
|
|
Mesp factors are cell autonomously required for epithelialization of somitic mesoderm but may also be non-cell autonomously required for morphological boundary formation
Conventional interpretations of the results of chimera analysis are
generally based upon the regulative development of the vertebrate embryo and
argue cell autonomy of specific gene functions in embryogenesis
(Ciruna et al., 1997
;
Brown et al., 1999
;
Kitajima et al., 2000
;
Koizumi et al., 2001
).
Mesp1/Mesp2 double-null cells failed to form epithelial somites, even in the
presence of surrounding wild-type cells. In addition, they were incapable of
contributing to dermomyotome, where cell sorting occurs. This strongly
suggests that Mesp factors are cell autonomously required for the
epithelialization of somitic mesoderm. However, we also found striking
non-cell autonomous effects of Mesp mutant cells on wild-type cell behaviors.
That is, both types of Mesp mutant cell not only failed to undergo normal
somitogenesis, but also inhibited the normal morphogenesis of wild-type cells.
This implies that there are non-cell autonomous roles for Mesp factors in the
establishment of the future somite boundary, as we will discuss further.
Initial epithelial somite formation is achieved by the
mesenchymal-epithelial transition of cells located in the anterior PSM. A
future somite boundary is established at a specific position in the PSM,
followed by gap formation between the mesenchymal cell populations.
Subsequently, cells located anterior to the boundary are epithelialized. This
process is known to be mediated by an inductive signal from cells posterior to
the boundary (Sato et al.,
2002
). Therefore, defects in epithelial somite formation can be
explained in two principal ways: a lack of cellular ability to epithelialize
(cell autonomous) and a lack of an inducing signal, which is produced in the
anterior PSM by a mechanism mediated by Notch signaling (thus non-cell
autonomous). In the case of chimeras of Mesp1/Mesp2 double-null cells, no
local boundary formed by locally distributed wild-type cells was observed,
i.e. even a gap between wild-type cells was never observed in the mixture of
Mesp1/Mesp2 double-null cells and wild-type cells. It is likely, therefore,
that the wild-type cell population can form a boundary only after separation
from Mesp1/Mesp2 double-null cells (Fig.
8). By contrast, some local boundaries between epithelial
wild-type cell clusters were occasionally observed in chimeras with Mesp2-null
cells. Considering that there is functional redundancy between these
transcription factors, it is possible that either Mesp1 or Mesp2 is necessary
for the formation of a signaling center or source of the putative inductive
signal. Hence, we cannot exclude the possibility that the lack of Mesp
function may affect non-cell autonomous generation of the inductive signal in
the anterior PSM.
Formation of epithelial somites requires paraxis, which is a transcription
factor (Burgess et al., 1996
;
Nakaya et al., 2004
). We
observed that Mesp1/Mesp2 double-null cells at the medial sclerotomal region
expressed Paraxis, indicating that Mesp factors are not absolutely
required for Paraxis expression. Defects in epithelial somite
formation in paraxis-null embryos, with normal Mesp2 expression
(Johnson et al., 2001
), and in
Mesp2-null embryos, with normal Paraxis expression, imply that
epithelial somite formation independently requires both gene functions.
Mesp2 is cell autonomously required for the acquisition of rostral properties
The distribution of Mesp2-null cells in the Mesp2-null chimeric embryos may
appear somewhat paradoxical, as they are localized at the rostral side in the
incomplete somites but at the caudal side in the dermomyotome. Initial
localization at the rostral and central region, however, is likely to be due
to the relative lack of epithelialization functions. In mammalian and avian
embryos, mesenchymal-to-epithelial conversion of the PSM commences from the
rostral side of the future somite boundary, i.e. the caudal margin of the
presumptive somite (Duband et al.,
1987
). Epithelialization then proceeds anteriorly in the dorsal
and ventral faces and in such a process, Mesp2-null cells, which are less able
to participate in epithelialization, may therefore be pushed to the central
and rostral sides. Thus, the majority of the Mesp2-null cells localize to the
central, prospective sclerotomal region and a small number of them are
integrated in the future dermomyotomal region. The incomplete somites then
undergo reorganization into dermomyotome and sclerotome, and small numbers of
Mesp2-null cells in the dermomyotome may be sorted out to the caudal end.
Therefore, the apparently complex distribution pattern of Mesp2-null cells is
likely to reflect a combination of defects in epithelialization and
rostro-caudal patterning. In the incomplete segments of Mesp2-null chimeric
embryos, the Mesp2-null cells fail to acquire rostral properties even when
localized at the rostral side. Moreover, in the dermomyotome, where
rostro-caudal patterning is rescued, Mesp2-null cells are mostly localized in
the caudal region. These observations suggested that the requirement of Mesp2
for the acquisition of rostral properties is cell autonomous. Similarly, it
has been reported that presenilin 1 (Psen1) is required for acquisition of
caudal half properties (Takahashi et al.,
2000
; Koizumi et al.,
2001
) and that Psen1-null cells cannot contribute to the caudal
half of somites in chimeric embryos, showing cell autonomous roles for Psen1
(Koizumi et al., 2001
).
Mesp mutant cells affect the rostro-caudal patterning of somites due to the lack of cellular interaction with wild-type cells
In a previous study, we have shown that the rostro-caudal patterning of
somites is generated by complex cellular interactions involved in positive and
negative feedback pathways of Dll1-Notch and Dll3-Notch signaling, and
regulation by Mesp2 in the PSM (Takahashi
et al., 2003
). In chimeras with either Mesp2-null or Mesp1/Mesp2
double-null cells, the mutant cells were distributed evenly and did not show
any sorting bias in a rostro-caudal direction in the PSM. Since both
Mesp2-null and Mesp1/Mesp2 double-null cells have the ability to form caudal
cells, it is likely that if wild-type cells could occupy the rostral part of
future somite regions and have the ability to sort in the PSM, a normal
rostro-caudal patterning would be generated. We did not observe this, however,
and conclude that the presence of mutant cells lacking Mesp factors must have
disrupted normal cellular interactions via Notch signaling. Thus these
non-cell-autonomous effects of our mutant cells are strongly supportive of our
previous contention that rostro-caudal pattering is generated by cellular
interactions via Notch signaling.
 |
ACKNOWLEDGMENTS
|
---|
We thank Mariko Ikumi, Seiko Shinzawa, Eriko Ikeno and Shinobu Watanabe for
general technical assistance. This work was supported by Grants-in-Aid for
Science Research on Priority Areas (B) and the Organized Research Combination
System of the Ministry of Education, Culture, Sports, Science and Technology,
Japan.
 |
REFERENCES
|
---|
Bettenhausen, B., Hrabe de Angelis, M., Simon, D.,
Guénet, J.-L. and Gossler, A. (1995). Transient and
restricted expression during mouse embryogenesis of Dll1, a murine gene
closely related to Drosophila Delta.
Development 121,2407
-2418.[Abstract/Free Full Text]
Borycki, A. G. and Emerson, C. P., Jr (2000).
Multiple tissue interactions and signal transduction pathways control somite
myogenesis. Curr. Top. Dev. Biol.
48,165
-224.[Medline]
Brown, D., Wagner, D., Li, X., Richardson, D. A. and Olson, E.
N. (1999). Dual role of the basic helix-loop-helix
transcription factor scleraxis in mesoderm formation and chondrogenesis during
mouse embryogenesis. Development
126,4317
-4329.[Abstract/Free Full Text]
Burgess, R., Cserjesi, P., Ligon, K. L. and Olson, E. N.
(1995). Paraxis: a basic helix-loop-helix protein expressed in
paraxial mesoderm and developing somites. Dev. Biol.
168,296
-306.[CrossRef][Medline]
Burgess, R., Rawls, A., Brown, D., Bradley, A. and Olson, E.
N. (1996). Requirement of the paraxis gene for
somite formation and musculoskeletal patterning.
Nature 384,570
-573.[CrossRef][Medline]
Ciruna, B. G., Schwartz, L., Harpal, K., Yamaguchi, T. P. and
Rossant, J. (1997). Chimeric analysis of fibroblast
growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in
morphogenetic movement through the primitive streak.
Development 124,2829
-2841.[Abstract/Free Full Text]
Duband, J. L., Dufour, S., Hatta, K., Takeichi, M., Edelman, G.
M. and Thiery, J. P. (1987). Adhesion molecules during
somitogenesis in the avian embryo. J. Cell Biol.
104,1361
-1374.[Abstract]
Fan, C. M. and Tessier Lavigne, M. (1994).
Patterning of mammalian somites by surface ectoderm and notochord: Evidence
for sclerotome induction by a hedgehog homolog. Cell
79,1175
-1186.[Medline]
Gossler, A. and Hrabe de Angelis, M. (1997).
Somitogenesis. Curr. Top. Dev. Biol.
38,225
-287.
Johnson, J., Rhee, J., Parsons, S. M., Brown, D., Olson, E. N.
and Rawls, A. (2001). The anterior/posterior polarity of
somites is disrupted in Paraxis-deficient mice. Dev.
Biol. 229,176
-187.[CrossRef][Medline]
Kitajima, S., Takagi, A., Inoue, T. and Saga, Y.
(2000). MesP1 and MesP2 are essential for the development of
cardiac mesoderm. Development
127,3215
-3226.[Abstract/Free Full Text]
Koizumi, K., Nakajima, M., Yuasa, S., Saga, Y., Sakai, T.,
Kuriyama, T., Shirasawa, T. and Koseki, H. (2001). The role
of presenilin 1 during somite segmentation.
Development 128,1391
-1402.[Abstract/Free Full Text]
Mansouri, A., Yokota, Y., Wehr, R., Copeland, N. G., Jenkins, N.
A. and Gruss, P. (1997). Paired-related murine homeobox gene
expressed in the developing sclerotome, kidney, and nervous system.
Dev. Dyn. 210,53
-65.[CrossRef][Medline]
Nakaya, Y., Kuroda, S., Katagiri, Y. T., Kaibuchi, K. and
Takahashi, Y. (2004). Mesenchymal-epithelial transition
during somitic segmentation is regulated by differential roles of Cdc42 and
Rac1. Dev. Cell 7,425
-438.[CrossRef][Medline]
Neidhardt, L. M., Kispert, A. and Herrmann, B. G.
(1997). A mouse gene of the paired-related homeobox class
expressed in the caudal somite compartment and in the developing vertebral
column, kidney and nervous system. Dev. Genes Evol.
207,330
-339.[CrossRef]
Nieto, M. A., Gilardi-Hebenstreit, P., Charnay, P. and
Wilkinson, D. G. (1992). A receptor protein tyrosine kinase
implicated in the segmental patterning of the hindbrain and mesoderm.
Development 116,1137
-1150.[Abstract/Free Full Text]
Nomura-Kitabayashi, A., Takahashi, Y., Kitajima, S., Inoue, T.,
Takeda, H. and Saga, Y. (2002). Hypomorphic Mesp
allele distinguishes establishment of rostro-caudal polarity and segment
border formation in somitogenesis. Development
129,2473
-2481.[Medline]
Pourquié, O. (2001). Vertebrate
somitogenesis. Annu. Rev. Cell. Dev. Biol.
17,311
-350.[CrossRef][Medline]
Saga, Y. (1998). Genetic rescue of segmentation
defect in MesP2-deficient mice by MesP1 gene replacement. Mech.
Dev. 75,53
-66.[CrossRef][Medline]
Saga, Y. and Takeda, H. (2001). The making of
the somite: molecular events in vertebrate segmentation. Nat. Rev.
Genet. 2,835
-845.[CrossRef][Medline]
Saga, Y., Hata, N., Kobayashi, S., Magnuson, T., Seldin, M. and
Taketo, M. M. (1996). MesP1: A novel basic helix-loop-helix
protein expressed in the nascent mesodermal cells during mouse gastrulation.
Development 122,2769
-2778.[Abstract/Free Full Text]
Saga, Y., Hata, N., Koseki, H. and Taketo, M. M.
(1997). Mesp2: a novel mouse gene expressed in the
presegmented mesoderm and essential for segmentation initiation.
Genes Dev. 11,1827
-1839.[Abstract]
Saga, Y., Miyagawa-Tomita, S., Takagi, A., Kitajima S.,
Miyazaki, J. and Inoue, T (1999). MesP1 is expressed in the
heart precursor cells and required for the formation of a single heart tube.
Development 126,3437
-3447.[Abstract/Free Full Text]
Sato, Y., Yasuda, K. and Takahashi, Y. (2002).
Morphological boundary forms by a novel inductive event mediated by Lunatic
fringe and Notch during somitic segmentation.
Development 129,3633
-3644.[Abstract/Free Full Text]
Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T.,
Koseki, H. and Saga, Y. (2000). Mesp2 initiates somite
segmentation through the Notch signalling pathway. Nat.
Genet. 25,390
-396.[CrossRef][Medline]
Takahashi, Y., Inoue, T., Gossler, A. and Saga, Y.
(2003). Feedback loops comprising Dll1, Dll3 and Mesp2, and
differential involvement of Psen1 are essential for rostrocaudal patterning of
somites. Development
130,4259
-4268.[Abstract/Free Full Text]
Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A.,
Kerr, W. G. and Soriano, P. (1997). Disruption of overlapping
transcripts in the ROSA beta geo 26 gene trap strain leads to widespread
expression of beta-galactosidase in mouse embryos and hematopoietic cells.
Proc. Natl. Acad. Sci. USA
94,3789
-3794.[Abstract/Free Full Text]