Evolutionary Origin of the Notochord

The notochord is a mesoderm-derived, rod-shaped organ found in embryos of all chordates that defines the craniocaudal axis of the embryo. Chordates comprise cephalochordates, urochordates, and vertebrates. They probably originated more than 560 million years ago from a common ancestor shared with nonchordate deuterostomes (echinoderms and hemichordates), and the occurrence of a novel fishlike (tadpole-type) larva was a key evolutionary event leading to the evolution of chordates. With the notochord providing necessary stiffness, these larvae swim using bilateral caudal muscles to produce a lateral undulatory motion. During vertebrate embryonic development, the notochord is replaced with vertebrae. A member of the T-box transcription factor family, Brachyury, plays a pivotal role in notochord development. Brachyury functions as a primary regulator in the cellular migration of metazoan gastrulation, essential to the development of embryos with two or three germ layers. During chordate evolution, this gene has apparently acquired a secondary expression domain in the mesoderm from which the notochord and bilateral muscles develop. In urochordates and vertebrates, Brachyury expression in muscle is somehow suppressed, leaving only notochord-specific expression of the gene. The WNT/β-catenin pathway, the transforming growth factor-β (TGF-β bone morphogenic protein [BMP]/Nodal) signaling pathway, and fibroblast growth factor (FGF) signaling pathways are involved in upstream transcriptional activation of Brachyury, and various genes downstream are associated with notochord formation and function. Further studies of the complex regulation of Brachyury expression and function are essential to understand the notochord from both basic biological and medical points of view.

3.1 Chordate Evolution

Since Charles Darwin proposed the evolution of animals by means of natural selection¹ the origin and evolution of chordates have been investigated and debated for more than 150 years.²^,³^,⁴^,⁵^,⁶^,⁷ Chordates consist of three distinct animal groups: cephalochordates, urochordates (tunicates), and vertebrates. Chordates are members of a larger group, the deuterostomes, together with echinoderms and hemichordates. Major questions of chordate evolution concern (a) phylogenetic relationships of the five groups and (b) how chordates originated from a common ancestor shared with nonchordate deuterostomes.

3.2 Phylogenetic Relationship

Studies of molecular phylogeny, comparative genomics, and evolutionary developmental biology have significantly advanced our understanding of chordate evolution ( ▶ Fig. 3.1 ⁷). Human beings and other vertebrates have definitive features that are absent in invertebrates, including a neural crest a placode, a hard, mineralized endoskeleton, an adaptive immune system, a specific genomic composition, and others.⁸^,⁹ The phylum Vertebrata belongs to the superphylum Chordata, together with two other phyla, the Cephalochordata (lancelets) and the Urochordata (Tunicata, ascidians) ( ▶ Fig. 3.1). Chordates are characterized by possession of the notochord, a dorsal, hollow neural tube, somites, and a postanal tail. The Chordata is one of two superphyla of the intrakingdom Deuterostomia ( ▶ Fig. 3.1). The other is the Ambulacraria, which includes two phyla, the Echinodermata (sea stars and sea urchins) and the Hemichordata (acorn worms). Deuterostomes are defined by embryonic development in which the blastopore, the first opening in the embryo, becomes the anus, whereas the mouth is formed secondarily on the other side of the embryo. Deuterostomes also have a mesoderm-oriented coelom and pharyngeal gills (although extant echinoderms have lost this character). Analyses using a relaxed molecular clock estimate that deuterostome ancestors lived in the middle Ediacaran period (~ 570 million years ago [MYA]), and chordates evolved in late Ediacaran (~ 560 MYA).

Fig. 3.1 Schematic representation of deuterostome groups and the evolution of chordates. Representative developmental events associated with the evolution of chordates are included. The notochord originated during the evolution of chordates. (Reproduced with permission from Satoh et al 2014.⁷)

3.3 Evolutionary Scenarios

Various hypotheses have been proposed to explain the origin and evolution of chordates. The four major ones are the paedomorphosis hypothesis, the auricularia hypothesis, the inversion hypothesis, and the aboral-dorsalization hypothesis.⁵^,⁷^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶ The first of these hypotheses debated whether ancestral chordates were sessile or free-living. Recent molecular phylogeny has demonstrated that free-living cephalochordates diverged first among chordates, indicating that the chordate ancestor was a free-living, vermiform creature.¹⁷^,¹⁸

The next three offered embryological and/or evolutionary developmental biology explanations of how the chordate body plan or bauplan, especially its adult form, originated from the common ancestor(s) of deuterostomes. The auricularia hypothesis, originally proposed by Garstang,¹⁹ emphasized the significance of changes in larval form, namely, that pterobranch-like, sessile animals with dipleurula (auricularia-like) larvae led to primitive ascidians (as the latest common ancestor of chordates) through morphological changes both in larvae and adults. In larvae, the ancestor’s circumoral, ciliated bands and their associated underlying nerve tracts moved dorsally to meet and fuse at the dorsal midline, forming a dorsal nerve cord in the chordate body.²⁰ On the other hand, the inversion hypothesis emphasized inversion of the dorsal–ventral (D-V) axis of the chordate body, compared with protostomes.¹¹^,¹²^,¹³ In arthropods and annelids, the central nervous system (CNS) runs ventral to the digestive tract, whereas in vertebrates, the CNS runs dorsal to the digestive system. Evolutionary developmental (evo-devo) studies revealed that an interaction of bone morphogenic proteins (BMPs) and their antagonists (chordin and/or anti-dorsalizing morphogenetic protein [Admp]) provide the molecular basis for this inversion phenomenon.²¹^,²² Further studies revealed that the inversion occurred between nonchordate deuterostomes and chordates, at the time chordates originated.²³ However, none of the three hypotheses necessarily explains the occurrence of the notochord during chordate evolution, despite its being the most prominent chordate feature (chordates are named for this structure).

The aboral-dorsalization hypothesis is based upon recent deuterostome phylogeny and emphasizes the occurrence of tadpole-like larvae as the critical developmental event that led to chordate evolution.⁷^,¹⁴^,¹⁵ All key chordate characteristics are associated with the formation of tadpole-like larvae, which are able to swim faster, and to catch prey more efficiently than larvae with cilia (pluteus or tornaria larvae of nonchordate deuterostomes). Since nonchordates (e.g., acorn worms) lack the notochord, dorsal hollow neural tube, and somites found in chordate embryos, it is reasonable to ask how these structural novelties formed in chordate embryos rather than to seek possible homologies with structures of acorn worm embryos and larvae. Viewed from the vegetal pole, the early embryo of nonchordate deuterostomes is radially symmetrical, suggesting that dorsal-midline structures could be formed anywhere. However, in chordate embryos, these structures are only formed on the dorsal side, which corresponds to the aboral side of nonchordate deuterostome embryos. That is, the aboral-dorsalization hypothesis speculates that the oral side is spatially limited due to formation of the mouth so that the dorsal-midline organs were allowed to form on the aboral side of ancestral chordate embryos.

3.4 Notochord

As mentioned above, the notochord supports the tail by providing stiffness for efficient muscular function.¹⁴^,¹⁵ In this sense, the stomochord of adult acorn worms (hemichordate)²^,²⁴ and the axochord of adult annelids²⁵ are not organs evolutionarily linked to the notochord.

Interestingly, the developmental mode and structural components of the notochord differ between cephalochordates and Olfactores (urochordates + vertebrates). Cephalochordate notochord provides material to infer the origin of this organ, and Olfactores notochord the evolution of this organ. In cephalochordates, after gastrulation and during neural tube formation, the notochord is formed by “pouching-off” from the dorsal region of the archenteron ( ▶ Fig. 3.2 a–f). The cephalochordate notochord displays muscle properties, and its constituent cells possess myofibrils.²⁶ Expressed sequence tag (EST) analysis indicated that approximately 11% of genes expressed in the notochord of Branchiostoma belcheri adults encode muscle components, including actin, tropomyosin, troponin I, and creatine kinase.²⁷^,²⁸ On the other hand, in Olfactores, the notochord is formed by convergent extension of precursor cells that are bilaterally positioned in the early embryo ( ▶ Fig. 3.2 g–j)²⁹; these cells do not possess any muscle properties. Vacuolation within the cells provides both stiffness and increased cell volume; this is the case in both ascidians and amphibians.³⁰ Convergence, intercalation, and extension of notochord cells are among the most significant morphogenetic events in formation of the dorsal-midline organs of chordate embryos.³¹ In addition, in vertebrates, the embryonic region that gives rise to the notochord serves as a signaling center, or “organizer,” and induces epidermal cells overlying it to differentiate into the nervous system.³²

3.5 Brachyury: A Key Transcription Factor for Notochord Formation

3.5.1 Brachyury

In 1990, Herrmann and colleagues succeeded in cloning mouse Brachyury via an elaborate chromosomal walk.³⁷ Brachyury (short tail) or T (tail) is named after its mutation in which homozygous embryos die in utero after 10 days of gestation due to deficient mesodermal development, whereas heterozygous embryos are born with short tails.³⁸^,³⁹ The gene is expressed in early-stage mesoderm and is subsequently restricted to the notochord. This was a turning point of studies on molecular mechanisms involved in notochord formation of chordate embryos.

Following its cloning, mouse Brachyury was shown to act as a tissue-specific transcription factor by specifically binding to a palindrome of 20 bp, the T-site, including 5′-AGGTGTGAAATT-3′.⁴⁰^,⁴¹ The protein consists of a large N-terminal DNA-binding domain (amino acids 1–180, T-domain) and two pairs of transcription activation and repression domains in the C-terminal protein half ( ▶ Fig. 3.3 a).

Fig. 3.2 Development of the notochord in amphioxus and ascidians. (a–f) Schematic drawings of development of the notochord in amphioxus embryos (based on Conklin 1932,³³ Hatschek 1893, ³⁴ and Hirakow and Kajita 1994³⁵). (a, b) Midneurula, (c, d) mid- to late neurula, and (e, f) late neurula. (a, c, e) Midsagittal section, (b, d, f) cross-section. During the time of neural tube formation, the notochord develops from the adjacent chordamesodermal plate that constitutes the roof of the archenteron. The notochord is formed by an upward pouching off of midline cells along the chordamesodermal plate. (g–j) Ascidian notochord development from the 64-cell stage embryo (Reproduced with permission from Satoh et al 2014.³⁶) (g) The 64-cell stage embryo, (h) 110-cell stage embryo, and (i, j) late tailbud embryo. Infolding and convergent extension transform the notochord precursor into a column of 40 stacked cells. (k–n) Schematic drawings showing the transition from primary gene expression in the blastopore (k, l) to secondary expression in the notochord (m, n) (see text for details) (Reproduced with permission from Satoh et al 2012.¹⁵)

Crystallographic structural analysis showed that the protein binds DNA as a dimer, interacting with major and minor grooves of DNA, with a specific DNA contact that had not been previously observed.⁴² Thus, Brachyury (T) was the founding member of a novel class of T-box transcription factors, including Tbx2, Tbx6, and Tbr (reviewed by Papaioannou,⁴³ Showell et al, ⁴⁴ Papaioannou ⁴⁵). Of these, Brachyury likely represents the ancestral form of the family, with ancient and/or primary functions.

Reflecting its significant role in embryogenesis, Brachyury orthologs have been isolated from various metazoans, including frogs,⁵⁵ zebrafish⁵⁴ ( ▶ Fig. 3.3 k), chickens,⁴⁰ urochordates⁵²^,⁵⁶ ( ▶ Fig. 3.3 h), cephalochordates⁵³ ( ▶ Fig. 3.3 i, j), hemichordates⁵¹ ( ▶ Fig. 3.3 g), echinoderms,⁵⁷^,⁵⁸ annelids,⁴⁹ fruit flies⁵⁰ ( ▶ Fig. 3.3 f), cnidarians⁴⁷^,⁵⁹ ( ▶ Fig. 3.3 c), ctenophores⁴⁸ ( ▶ Fig. 3.3 d), placozoans⁴⁶ ( ▶ Fig. 3.3 b), and others (see Satoh et al¹⁵). Expression profiles and possible functions of Brachyury in relation to notochord formation will be discussed below. Comparative genomics suggests that Brachyury appeared at the time of multicellular animal evolution.⁶⁰

Fig. 3.3 Brachyury and its expression (Reproduced with permission from Satoh et al 2012¹⁵). (a) Domain structure of mouse brachyury protein. TA, transactivation domain; R, repression domain. Numbers indicates amino acid residues in the sequence. The location of the nuclear localization signal (NLS) is indicated by a bracket.⁴⁰^,⁴¹ (b–k) The expression of Brachyury gene in metazoans. (b) Placozoan: Brachyury is expressed in a few isolated cells, mainly near the edge of the adult animal.⁴⁶ (c) Nematostella: blastopore (arrow) region of the gastrula (Reproduced with permission from Scholz and Technau 2003⁴⁷). (d) Ctenophore: in the blastopore (bp) and stomodeal (sd) cells. ph, pharynx (Reproduced with permission from Yamada et al 2010⁴⁸). (e) Polychaete: stomodeum (sto) and proctodaeum (pro) of 22-hour embryo.⁴⁹ (f) Drosophila: in the hindgut and anal pads.⁵⁰ (g) Hemichordate: in the blastopore region and sd invagination region. ar, archenteron; pc, protocoel (Reproduced with permission from Tagawa et al 1998⁵¹). (h) Ciona: in primordial notochord cells of the 110-cell stage embryo.⁵² (i, j) Amphioxus: in cells of the blastopore (b) of the gastrula (i), and notochord (n) and tailbud of 18-hour embryos (j) (Reproduced with permission from Holland et al 1995⁵³). (k) no tail expression in the bp and notochord of zebrafish embryo (Reproduced with permission from Schulte-Merker et al 1994⁵⁴).