The notochord is a dorsally located rod of tensile mesodermal tissue, which lies beneath the neural tube, and is the defining characteristic of the chordate embryo. Over the last couple of decades, different genetic and embryological studies have informed us about the development of the notochord, and have shown that in addition to its structural function, it plays a potentially critical role in the patterning of ectodermal, mesodermal and endodermal tissues.
In vertebrates, the notochord is derived from the dorsal organiser, first identified by Spemann and Mangold in amphibians, following the transplantation the organizer into prospective lateral or ventral regions of a host embryo, where it was found that it induced the formation of a second embryonic axis, while only contributing to notochord and prechordal mesendoderm (Harland and Gerhart, 1997). In amphibians, the organiser region is the dorsal lip of the blastopore, and homologous structures in other species also exist (Beddington, 1994).
Figure 1 | Embryonic neurulation is a fundamental stage in development of the future central nervous system and skeleton.
The initial major transition in notochord development is from dorsal organiser to chordamesoderm (the direct antecedent of the notochord), which becomes morphologically and molecularly distinct from other mesoderm in the early gastrula stages – through inductive interactions involving the transforming growth factor β (TGFβ)-superfamily and fibroblast growth factor (FGF)-family signalling molecules (Ang and Rossant, 1994). Subsequent conformational changes (mediolateral intercalation and convergence of cells towards the dorsal midline) force the chordamesoderm into an elongated stack of cells. As development proceeds, cells of the chordamesoderm acquire a thick extracellular sheath (composed of predominantly extracellular matrix, collagens and proteoglycans) and a vacuole – the osmotic pressure within which, acts against the sheath, giving the characteristic rod-like appearance of the notochord as well as elongating and straightening the notochord (Adams et al., 1990).
A number of other factors are involved in this complex process, specifically, the Brachury gene T that codes for the T-box transcription factor-commonly referred to as “T”, as well as Foxa2/HNF-3β (a key transcription factor required for the development of the node, notochord, and also the floor plate of the neural tube). The T gene is expressed in all mesodermal cells and is expressed within the notochord until formation of the developing embryonic body is completed, though; other mesodermal cells only express it transiently demonstrating its directed activity in notochordal development (Herrmann and Kispert 1994).
“Brachyury is required for the differentiation of the axial midline mesoderm into notochord as well as for the formation of posterior mesodermal tissues”
Studies in the mouse, Xenopus, and zebrafish have demonstrated that Brachyury is required for the differentiation of the axial midline mesoderm into notochord as well as for the formation of posterior mesodermal tissues (Schulte-Merker et al., 1997). In ascidians, expression of Brachyury is maintained exclusively in the notochord, in contrast to the situation in vertebrate embryos, where Brachyury is expressed in both notochord and ventral–posterior mesoderm. Takahashi et al. (1999) extended this observation, demonstrating that misexpression of Brachyury in the endoderm of ascidian embryos is sufficient to induce fully differentiated ectopic notochord; contrasting the observed induction of ventral–posterior mesoderm when Brachyury was misexpressed in Xenopus animal caps (Cunliffe and Smith 1994). Only by coexpressing noggin (a Bone Morphogenic Protein Inhibitor) with Brachyury could notochord be induced in this tissue (Cunliffe and Smith 1994). Thus, coexpression removes the ventralizing constraint of BMP signalling on mesodermal fate induced by Brachyury, yielding notochord and somitic muscle.
The transition from chordamesoderm to mature notochord requires a host of factors, some of which, have been identified in zebrafish genetic screen studies (Odenthal et al., 1996). These have been helpful in investigating the relationship between dorsal organiser activity and specification of notochord fate; and have demonstrated that activities of the two are independent. Bozozok mutant embryos, for example, lack a morphologically distinct shield, and both bozozok and floating head mutant embryos fail to form a notochord (Fekany et al., 1999), nevertheless, in both types of mutants, the embryonic axes form with clear dorsal-ventral and anterior-posterior identities (Fekany et al., 1999).
Once developed, the notochord assumes its crucial patterning and structural roles. In the case of patterning, independent studies have provided evidence that notochord signalling is important for development of the heart, the vasculature, and for establishing the lateralization of organs.
Arguably, an important role of the notochord is patterning the neural tube. Studies where the notochord was transplanted or removed during embryo development showed that the notochord possessed the capability to signal the formation of the floor plate, the ventral-most fate of the spinal cord (Yamada et al., 1991). Such secreted signals were the Hedgehog (Hh) proteins, and in particular, Sonic Hedgehog, which induced a range of graded ventral spinal cord fates, while simultaneously suppressing the expression of dorsal genes (Yamada et al., 1991).
Studies in zebrafish show a role for the notochord in regulation of early cardiac development (Goldstein and Fishman, 1998); where, by laser ablation of the anterior extremity of the notochord, an increased expression of the homeobox gene Nkx2-5 (a marker for the presumptive heart field) was observed – suggesting that the notochord may normally act to suppress cardiogenic fate. A study attuned with this suggestion showed that the addition of a neural plate/notochord combination to chick anteromedial mesendoderm explants reduced BMP2-induced cardiac differentiation – though critically, this applied to BMP2 and not Nkx2.5 expression (Schultheiss et al., 1997). It might then be the case that the target genes of suppression by notochord could include Nkx2.5, which is capable of driving some components of cardiac differentiation, but others may also be involved as well.
Notochord signals have also been associated with dorsal aorta formation, with studies showing that zebrafish mutants ntl and flh, both of which lack a notochord, fail to form the dorsal aorta (Fouquet et al., 1997); and that with transplantation of wild-type notochord cells into flh mutants, notochord development could be restored to some extent and an aortic primordium formed (Fouquet et al., 1997).
In addition to the reinforcement and maintenance of earlier developmental events, notochord-derived signals may also contribute to the establishment of left-right asymmetry. This notion was supported by an experiment where the notochord was ablated in Xenopus gastrulae, resulting in the randomisation of asymmetry (Lohr et al., 1997).
Recently, a study demonstrated that the notochord and floor plate are required for the correct positioning of the metanephric kidney, and that their disruption (and of Shh from these structures) can affect mediolateral organization of developing midline and lateral structures – leading to a more medial position for the metanephric mesenchyme, setting the stage for kidney fusions to occur (Tripathi et al., 2010). Although this study raises the possibility that mediolateral patterning defects during embryogenesis can be a potential cause for horseshoe kidneys in human patients (Tripathi et al., 2010), it is yet to be fully substantiated.
Finally, with respect to patterning, the notochord is important for the normal development of early endoderm and the pancreas (Kim et al., 1997). In a study, the notochord was identified as the source of the signals required for pancreatic differentiation, as its removal prevented the expression of genes required for pancreatic development and function (Kim et al., 1997). The results also suggested that competence of the dorsal endoderm to respond to notochord signals and initiate pancreatic gene expression were localized – so overall, showing that, in addition to patterning neural and somite differentiation, the notochord is capable of patterning endoderm (Kim et al., 1997).
Although the patterning roles of the notochord are essential for normal vertebrate development, the notochord also has an essential structural role; especially since it is the main axial skeletal element of the early chordate embryo, without which, embryos fail to elongate (Odenthal et al., 1996). Zebrafish mutation screen studies have been useful in that respect, as they have shown that a number of factors are involved (Odenthal et al., 1996) – some of which are important for the formation of the chordamesoderm, highlighting important signalling roles of the notochord, and others that are profoundly important for structural aspects of notochord function.
Noteworthy, notochord development may be indirectly influenced by abnormal vertebrae formation. Thus, mouse mutants lacking collagen type II, the homeobox transcription factor Bapx1, and the paired box transcription factors Pax1 and Pax9 (expressed in the sclerotome), display abnormal development of the vertebral bodies and notochord (Risbud et al., 2010). In these mutants, notochord formation is normal but the notochord is not removed from the vertebral bodies – compromising the formation of the nucleus pulposus.
Two other genes that are expressed in sclerotome-derived cells and the notochord are Sox5 and Sox6, encoding the highly homologous transcription factors L-Sox5 and Sox6, respectively; and these are required for peri-notochordal sheath formation, cell survival, and development of the nucleus pulposus (Smits and Lefebvre, 2003) – thus, it is not a revelation that Sox5-/-/Sox6-/- embryos exhibit extreme defects in notochord development and formation of the axial skeleton (Smits and Lefebvre, 2003).
Conclusion
The complex processes of notochord development appropriately enable it to undertake complex responsibilities that will, in turn, have widespread implications for the developing embryo – making it an imperative structure for the normal development of chordates. Research in this area has progressed such that there seems to be more focus and attention on the patterning roles of the notochord, however it is also important to appreciate its structural roles. Studies employing mutational analyses will hopefully provide us with a more comprehensive insight into the mechanical basis of the notochord. Further to its germ-layer-patterning responsibilities, the notochord has nominated itself as a convenient candidate to model organogenesis, particularly for integrating extracellular matrix formation with cellular differentiation.
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