November 15, 2001;
Hypaxial muscle migration during primary myogenesis in Xenopus laevis.
In contrast to many vertebrates, the ventral
body wall muscles and limb
muscles of Xenopus develop at different times. The ventral
body wall forms in the tadpole
, while limb
(appendicular) muscles form during metamorphosis to the adult frog. In organisms that have been examined thus far, a conserved mechanism has been shown to control migratory muscle
precursor specification, migration, and differentiation. Here, we show that the process of ventral
body wall formation in Xenopus laevis is similar to hypaxial muscle
development in chickens and mice. Cells specified for the migratory lineage display an upregulation of pax3
in the ventro-lateral
region of the somite
. These pax3
-positive cells migrate ventrally, away from the somite
, and undergo terminal differentiation with the expression of myf-5, followed by myoD
. Several other genes are selectively expressed in the migrating muscle
precursor population, including neural cell adhesion molecule (NCAM), Xenopus kit
related kinase (Xkrk1), and Xenopus SRY
box 5 (sox5). We have also found that muscle
precursor migration is highly coordinated with the migration of neural crest-derived melanophores. However, by extirpating neural crest at an early stage and allowing embryos to develop, we determined that muscle
precursor migration is not dependent on physical or genetic interaction with melanophores.
[+] show captions
FIG. 1. Stage series showing the development of the ventral body wall. Immunohistochemistry was performed by using the 12/101 antibody on four different stages of embryos beginning with stages 31 (A), 37/38 (B), 41 (C), and 46 (D). The ﬁrst appearance of differentiated
ventral body wall muscle is at stage 37/38 (B), indicated by arrows. By stage 41, the ventral body wall has expanded ventrally and retains segmental identity, as indicated by the lack of fusion between adjacent myotubules (C, arrow heads). At stage 46, the ventral expansion of the body wall has ceased (D, arrows). The two sides of the ventral body wall fail to meet at the ventral midline (D). (E) A transverse section through a stage-46 embryo in the trunk region. Arrows indicate ventral and dorsal extent of the ventral body wall musculature. (F) A high-magniﬁcation view of a serial section to that shown in (E). The arrow marks the ventral limit of differentiated body wall, while the arrow head points to nonskeletal muscle tissue connecting the body wall to the somite it arose from. Embryos in (A) are oriented with
anterior to the left and dorsal to the top of the page. Embryo in (D) is seen from a ventral viewpoint with anterior to the left, while in (E)
and (F), dorsal is to the top of the page. In all subsequent ﬁgures embryos are seen with anterior to the left and dorsal to the top of the page except where otherwise noted.
FIG. 2. Expression patterns of the myogenic transcription factors pax3, myf-5, and myoD. Upregulation of pax3 in the dorso-lateral lip of
the anterior trunk somites is apparent at stage 26 (A, arrows). By stage 28/29 pax3-positive cells can be seen leaving the somites and
migrating ventrally (B, arrows). At stage 37/38, pax3 cells can be seen at the front of the migration (D) as they continue to stream out of
the somites (D, arrow head). Stage-41 embryos have faint pax3 expression at the front of the migrating cells (E, arrows). Early myf-5
expression is restricted to the dorso-medial and ventro-lateral lips of the somites, marking the expanding epaxial myotome (F, G). At stage
31 (H, arrows), myf-5-expressing cells can clearly be seen outside of the somitic region migrating ventrally, and continuing to do so through
stages 37/38 (I) and 41 (J, arrows). Expression of myoD is present only in the somitic region (K) until stage 37/38 (N), when it appears
in discrete patches clearly separate from the somites (arrows). By stage 41, there are long stripes of myoD expression corresponding to the
differentiated ventral body wall muscle (O). Expression is greatest in the ventral-most region of the body wall, where skeletal muscle has
most recently differentiated.
FIG. 3. Embryos stained for either pax3 (A) or myoD (E) followed by 12/101 staining. Stage-37 embryos stained for pax3 expression
(A, B) reveal the pax3-expressing cells are just ventral to differentiated muscle (arrow heads), with little overlap, as indicated from a
transverse section (B) of embryo in (A) (arrow). The lack of overlap is more dramatic in stage-41 embryos (C, arrow). (D) A transverse section
of embryo in (C). The arrow shows pax3 expression which does not overlap with 12/101 staining (arrow heads). Stage-37 embryos stained
formyoD (E, F) indicate signiﬁcant overlap ofmyoD expression and differentiatedmuscle (arrow heads in E, arrow in F). At stage 41, overlap
of myoD expression continues over a broad range of differentiated muscle (G), which can also be seen in (H) (arrows). In (B), (D), (F), and (H),
dorsal is toward the top of the page.
FIG. 4. Expression patterns of NCAM, Xkrk1, and sox5. Heavy NCAM expression can be seen in the neural tissues of the head and neural tube throughout the stage series (A). NCAM-expressing cells can be seen moving out of the somites at stage 28/29 (B, arrow) and continuing to move ventrally at later stages (C, arrows). At stage 37/38, expression can be seen from the leading edge of the migrating
cells all the way back to the somites (D). At stage 41, NCAM-expressing cells are present at the leading edge of migration (E, arrows). The
expression of Xkrk1 can be seen in the developing lateral line system, particularly at stage 37/38 (I, arrow heads). Expression is also found
in the migrating myoblasts that have left the somite, beginning at stage 28/29 (G, arrow), and continuing to move ventrally through stage 37/38 (I, arrows), where expression in the developing ventral body wall is particularly strong. By stage 41, there is no longer any expression
of Xkrk1 in the ventral region of the embryo (J). The expression of sox5 can be seen in neural tissue and somitic mesoderm throughout the stage series (K). Its expression in the ventral body wall is similar to myoD expression, ﬁrst appearing in small clusters of cells at stage 37/38 (N, arrows) and moving ventrally at stage 41 (O, arrows).
FIG. 5. Trunk somites I-VIII contribute to the ventral body wall. Embryos were stained for pax3 followed by 12/101 antibody staining. A
stage-31 embryo (A) shows that the three remaining head somites (arrow heads) do not contribute pax3-positive cells to the ventral body
wall. A stage-36 embryo (B) shows that trunk somites VII and VIII (arrows) are the last somites to contribute to the ventral body wall.
FIG. 6. DiI labeling of migrating myoblasts (A). Arrows mark three points of injection along the anteriorosterior axis targeting the
ventro-lateral region of the somites (A). As development of this embryo proceeds, cells labeled by the anterior-most injection migrate ﬁrst
and farthest (B, C, arrows). The cells labeled by the posterior-most injection in the tail region show very little migration.
FIG. 7. Neural crest-derived melanophores comigrate with muscle precursors. Expression of pax3 at stages 31 (A), 37/38 (B), and 41 (C) in
unbleached embryos. (B, C) Comigration of pax3 staining cells with pigmented melanophores can be seen. Close-ups of pax3 (D), myoD (E),
and NCAM (F) expression in stage-39 embryos showing close spatial and temporal movements of melanophores and myoblast precursors.
Melanophores can be seen overlying the stained cells. 12/101 staining in stages 37/38 (G) and 41 (H) also matches the ventral extent of the
melanophores. Expression of slug in a stage-37 embryo marks melanophores in the ventral trunk region and in the head (I).