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Onai T
,
Aramaki T
,
Inomata H
,
Hirai T
,
Kuratani S
.
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INTRODUCTION: Somites, blocks of mesodermtissue located on either side of the neural tube in the developing vertebrate embryo, are derived from mesenchymal cells in the presomitic mesoderm (PSM) and are a defining characteristic of vertebrates. In vertebrates, the somite segmental boundary is determined by Notch signalling and the antagonistic relationship of the downstream targets of Notch, Lfng, and Delta1 in the anterior PSM. The presence of somites in the basal chordate amphioxus (Branchiostoma floridae) indicates that the last common ancestor of chordates also had somites. However, it remains unclear how the genetic mechanisms underlying somitogenesis in vertebrates evolved from those in ancestral chordates.
RESULTS: We demonstrate that during the gastrula stages of amphioxus embryos, BfFringe expression in the endoderm of the archenteron is detected ventrally to the ventral limit of BfDelta expression in the presumptive rostral somites along the dorsal/ventral (D/V) body axis. Suppression of Notch signalling by DAPT (a γ-secretase inhibitor that indirectly inhibits Notch) treatment from the late blastula stage reduced late gastrula stage expression of BfFringe in the endodermal archenteron and somite markers BfDelta and BfHairy-b in the mesodermal archenteron. Later in development, somites in the DAPT-treated embryo did not separate completely from the dorsal roof of the archenteron. In addition, clear segmental boundaries between somites were not detected in DAPT-treated amphioxus embryos at the larva stage. Similarly, in vertebrates, DAPT treatment from the late blastula stage in Xenopus (Xenopus laevis) embryos resulted in disruption of somite XlDelta-2 expression at the late gastrula stage. At the tail bud stage, the segmental expression of XlMyoD in myotomes was diminished.
CONCLUSIONS: We propose that Notch signalling and the Fringe/Delta cassette for dorso-ventral boundary formation in the archenteron that separates somites from the gut in an amphioxus-like ancestral chordate were co-opted for anteroposterior segmental boundary formation in the vertebrate anterior PSM during evolution.
Fig. 1 Development of amphioxus rostral somites. a At the early
neurula stage, the dorsal roof of the anterior archenteron begins to
expand dorsolateral to the ectoderm. b At the mid-neurula stage,
the rostral somites swell and form a U-shape that remains part of
the dorsal roof of archenteron. c At the late neurula stage, the somites
pinch off from the archenteron roof. nc notochord
Fig. 2 Notch signalling controls the pinching off process of the
rostral somites. a–b BfFringe expressed in the anterior endoderm
(ventral part of the archenteron) and BfDelta was expressed in the
presumptive rostral somites at the late gastrula stage. Blastopore
views with the dorsal side up. The white dotted circle indicates the
archenteron. s somite, ar archenteron. Scale bar, 50 μm. c–f Effect of
100 μM DAPT treatment on BfFringe (n = 7, 100 %) or BfDelta (n = 8,
100 %). Anterior views with the dorsal side up. g In the DMSO-treated
control larval embryo, the segmental boundary between the somite
and the dorsal gut roof was clear (n = 1). h In larval embryos treated
with DAPT, the boundary was unclear and ectopic expression of
Muscle-actin was observed (n = 1). Transverse sections with the
dorsal side up. White arrowheads indicate somite and gut fusion
locations. The white dotted circle indicates the border between
the gut and somites
Fig. 3 DAPT treatment affects somitogenesis in amphioxus embryos. Effect of treatment with 100 μM DAPT at the late blastula stage on
mesodermal gene expression (a, b Delta; c, d, Hairy-b; e, f, Gsc; g, h, Brachyury; n = 10 each, 100 %) at the gastrula stage. Dorsal views with the
anterior side up. s, somite. Scale bar, 50 μm (a)
Fig. 4 Effect of DAPT treatment on BfMuscle-actin expression. a, c, e DMSO-treated control embryo (n = 5, 100 %). Anterior to the left. Dorsal view.
nt, notochord; s, somite; m, myotome. CellMask (Red) labelled the plasma membrane. BfMuscle-actin (Green) was stained by fluorescence in situ
hybridization. The white dotted circle indicates a myotome. The white line indicates the segmental border between myotomes. b, d, f DAPTtreated
embryo (n = 5, 100 %). Anterior to the left. Dorsal view. The white arrowhead indicates a possible segmental border between myotomes.
Scale bars, 20 μm
Fig. 5 Filament formation of actin is suppressed by DAPT treatment. a DMSO-treated control embryos display filamentous actin in the rostral
somites (n = 12, 100 %). b F-actin staining in the somites. Treatment with 100 μM DAPT from the late gastrula stage onwards (n = 10, 100 %). The
images were taken using an LSM 710 confocal microscope (Zeiss). c Dorsal view of DMSO-treated control embryos. d Dorsal view of DAPT-treated
embryos. s somite, nt notochord, n neural tube, g, gut. Nuclei were labelled in blue and the plasma membrane was labelled in red
Fig. 6 Inhibition of Notch signalling results in loss of segment formation in Xenopus embryos. a–f DAPT treatment did not affect the expression
pattern of Gsc (a n = 42, 93 %; b n = 38, 87 %) and Brachyury (c n = 20, 95 %; d n = 14, 94 %), whereas it disrupted the paired-stripe expression
pattern of Delta-2 (e n = 33, 100 %; f n = 47, 51 %). a–b Anterior views with dorsal side up. c–f Dorsal views with the anterior side up. g–h DAPT
treatment resulted in loss of segmental expression of Mrf1 in amphioxus (g n = 12, 100 %; h n = 11, 100 %. Dorsal views with anterior to the left),
MyoD in Xenopus (i n = 23, 100 %; j n = 15, 93 %), and Tbx1 (k n = 23, 100 %; l n = 13, 54 %). i, j The lower left panel displays the magnification of
the somite expression of MyoD
Fig. 7 A new evolutionary scenario of the chordate somite
formation. a In amphioxus, mesoderm/endoderm boundary formation
is organized by Fringe/Delta cassette under Notch signalling. b The
Notch signalling-dependent mechanism of segmental boundary
formation in (a) was co-opted to the future segments in the
anterior PSM of amniotes embryos. The mesoderm is presented
in green. The endoderm is in light blue. The dotted line indicates a
segmental border
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