On the origin of vertebrate somites

Segmented structures composed of repetitive units, called somites, that arise transiently
during embryogenesis are a key feature of the vertebrate body plan. The somites lie
laterally to the notochord, and a spinal nerve forms a segmental unit assigned to
somitic derivatives in the trunk 1], 2]. During development, somites differentiate into myotomes and skeletal elements that
form the basic supporting structure in adults. From an evolutionary perspective, somites
are conserved between vertebrates and amphioxus but are secondarily lost in tunicates
3]–5]. Amphioxus (Branchiostoma floridae), also known as cephalochordate or lancelet, has a notochord, neural tube, and pharynx,
but lacks a neural crest and placodes. In amphioxus, somites extend into the anterior
end, unlike in vertebrates, and amphioxus is thus considered not to possess a homolog
of the vertebrate unsegmented head mesoderm; however, this contention remains controversial
(Additional file 1: Figure S1) 6], 7]. A recent genome analysis indicated that amphioxus retains most of the developmental
genes, such as Hox clusters, present in vertebrates and is thus the best proxy to
address the origin of the vertebrate body plan 8].

Although somites are common in chordates, there are some differences in developmental
sequences between amphioxus and vertebrates. For example, in amphioxus, the rostral
somites develop from the dorsal mesoderm during early embryogenesis. The mesoderm
is a single layer located in the dorsal roof of the archenteron (endodermal/mesodermal
structure) at the early neurula stage (Fig. 1a). By the mid-neurula stage, a prospective somite swells dorsolaterally and the bottom
narrows (Fig. 1b). At the late neurula stage, all the rostral somites pinch off from the archenteron
roof simultaneously and line up laterally to the notochord (Fig. 1c). Beginning from the late neurula stage, the caudal somites bud off one by one directly
from the lateral epithelial cells in the tail bud by schizocoely (a coelom is formed
by separating mesenchymal cells) 9]. Whereas, in vertebrates, somites are formed in pairs in the anterior end of the
PSM through gradual epithelialization 10]. These developmental differences raise the question of the ancestral origin of somitogenesis.

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

Somites do not occur in any group other than chordates. However, a previous study
indicated that the evolutionary origin of somites could be found in cnidarians, diploblastic
animals that are thought to be ancestral to the bilaterians 11]. According to Sedgwick’s enterocoel theory, chordate somites are derived from the
alimentary pouches of coelenterates (ctenophores and cnidarians) 11]. Nonetheless, it is still debated whether coelomic cavities are ancestral to all
bilaterians (e.g., the basal bilaterian Acoela does not have coelomic cavities), as
there is no persuasive evidence to support this hypothesis 12]–14]. The enterocoel theory promotes the view that the rostral somites are more ancestral
than the caudal somites in amphioxus and the vertebrate somites. However, without
grounding in molecular genetic comparisons, the somite evolutionary scenario remains
enigmatic.

Recent molecular studies have revealed the developmental mechanisms underlying somitogenesis.
In vertebrates, somites are formed by several developmental sequences. Starting from
gastrulation, the PSM is internalized around the blastopore and becomes part of the
tail bud at the posterior end of the body. A future pair of somites is gradually epithelialized
in the anterior PSM, and a boundary between each somite is determined. One pair of
somites is formed every 120 min in mice and every 25 min in zebrafish, indicating
the interspecies differences in somitogenetic rhythms among vertebrates 15]. Molecular oscillators control this rhythmic somitogenesis. Of these, Notch signalling
is a key factor in the anterior PSM, or “determination front”, and is essential for
the boundary formation of future segments 10]. At the determination front, the lunatic fringe (Lfng) glycosyltransferase, a downstream modulator of Notch signalling, has been reported
to be essential for generating a boundary by modifying the Notch receptor in mouse
embryos 15]. Additionally, in zebrafish, Lfng is expressed in the tail bud where, unlike in mouse and chick embryos, expression
does not oscillate, suggesting a diverse role for somitogenesis, which may have been
secondarily lost in the anole lizard, as Lfng is not expressed in the PSM in lizard embryos 16], 17]. Upstream of Lfng, mesoderm posterior (Mesp2), a member of the basic helix-loop-helix transcriptional factor family, regulates
Notch signalling input 18].

In amphioxus, the dorsal mesoderm includes somites and the notochord the initial somite
boundary formation is recognized in the dorsal mesoderm at the mid-gastrula stage.
A pair of stripes of BfDelta (the homolog of DLL1) expression is detected in the first somites 19]. Additionally, several homologs of vertebrate segmentation genes, such as Hairy and Uncx4.1, are expressed in somites from the gastrula to the larval stages 9]. However, no direct evidence of periodic expression of cyclic genes in the tail bud
has been reported. Although the amphioxus genome harbours a Mesp homolog, it is not likely to be expressed during embryogenesis 9]. BfFringe is expressed in the neural tube and the endodermal gut, but not in the somites, during
the neurula stages 20]. Evidence suggests that the periodic expression of cyclic genes and the establishment
of the determination front by the Mesp and Lfng genes are vertebrate-specific genetic networks.

In this study, we investigated the molecular mechanisms underlying somitogenesis in
amphioxus embryos with a focus on Notch signalling. Since current evidence suggests
that vertebrate somites evolved from somites of an amphioxus-like ancestral animal,
we performed gene expression and functional analyses using amphioxus and Xenopus embryos.