The long-controversial structure of the lancelet notochord
Nineteenth century embryologists correctly understood that the embryonic lancelet notochord begins as a predominantly cellular structure [26]. However, with rare exceptions [6, 7], they mistakenly thought that the post-embryonic stages had notochords consisting chiefly of extracellular material, described as amorphous, plate-like, or fibrous. The correct view, that the core of the organ consists of nucleated lamellar cells, was not fully accepted until revealed by TEM in the twentieth century [8, 18, 27, 28]. Contemporaneously, it was also discovered that the fibrous component in the lamellar cell cytoplasm was a contractile apparatus rich in paramyosin (also called tropomyosin A) [29, 30]. In addition, TEM showed that dorsal extensions of lamellar cells in the trunk region receive neural input at synapses on the surface of the overlying CNS [8]. These features are unique to lancelets [31] and have caused some to question the notochord as a chordate homologue [32], although this is counterbalanced by other recent findings that support the homology [33,34,35,36].
Intergeneric differences in lancelet notochords
The genera of cephalochordates comprise Branchiostoma and Asymmetron as well as the poorly known Epigonichthys. The details of notochord structure differ somewhat between Branchiostoma and Asymmetron. First, the Müller cells of Branchiostoma, as compared to Asymmetron [6, 8, 18] are: stellate, not oval; run in dorsal and ventral rows several abreast, not single-file; run in two additional rows, one in each dorsolateral position; are surrounded by voluminous fluid-filled spaces; and include some filament bundles and a conspicuous Golgi complex. It is unclear if these differences are real or simply result from comparing the trunk of one species with the tail of another species. In addition, the lamellar cells in adults of Branchiostoma, as compared to Asymmetron, contain less conspicuous vacuoles and are often separated by intercellular spaces, although the latter might be artifacts [18]. Moreover, the synaptic associations already mentioned between the lamellar cells and the CNS of Branchiostoma [8] were not found in the tail of Asymmetron, possibly because such synapses are limited to the trunk region of lancelets to influence notochord biomechanics there. Finally, it would be interesting to determine if the fine-structural details of the posterior terminus of the notochord in the genus-specific caudal process of Asymmetron are comparable to those in the more muscular tail of Branchiostoma.
Cell population dynamics in the lancelet notochord
The present results suggest that the notochord of adult cephalochordates comprises populations of likely stem cells, progenitor cells, and terminally differentiated cells. Each of these is distinguishable from the others by distinctive morphology. Moreover, unequivocal intermediate cells are found at the boundaries between cell populations. This clarity of structure contrasts with some vertebrate stem cells and progenitor cells that can be distinguished only by their different gene expression and immunochemistry [37]. Because of the substantial collagenous sheath, mentioned above as surrounding the entire notochord, the notochordal cell populations appear well isolated from other tissues. This relative isolation from the surrounding tissues is established by the late embryonic stage [38]. Therefore, it would not be surprising to find that the notochordal stem cells (if one assumes that their identity will ultimately be confirmed) are tissue-specific in cephalochordates.
At the posterior end of the lancelet notochord, the presumed stem cells probably shift their positions only gradually and only over short distances as they self-renew and/or differentiate. It is also likely that the lamellar cells, under conditions of normal growth, do not move around in the core of the notochord. In contrast, more uncertainty surrounds the movements of the Müller cells. Some might differentiate into lamellar cells while still in the core of the notochord (as suggested by the arrangement of the nuclei in Fig. 2a), but most evidently reach the surface of the organ to join the dorsal or ventral row of their counterparts. It is not known whether Müller cells are added to the surface rows only posteriorly or more haphazardly. It even remains possible that these cells are motile and able to change places with their neighbors. The superficial Müller cells of Branchiostoma can proliferate, as suggested for adults in the older literature and more recently indicated by nuclear incorporation of bromodeoxyuridine in larvae [39]. However, an important question, yet unanswered, is whether they are constrained to divide not more than a finite number of times before differentiating into a lamellar cell—and thus fit the definition of progenitor cells as opposed to stem cells [40]. This question needs to be addressed by studying the details of notochordal cell proliferation. Although counting mitotic figures by electron microscopy underestimates the abundance of dividing cells in somatic tissues of adult cephalochordates [41], proliferation markers like BrdU [39] and phosphohistone H3 [42] can provide more reliable results.
It would be especially interesting to follow the fate of the Müller cells during regeneration after tail removal. Those remaining in the stump of the notochord would presumably be the source of new lamellar cells to lengthen the regenerating organ. This possibility is strengthened by the finding of Joseph [43] that a localized injury in the lancelet notochord is followed by proliferation and internalization of nearby Müller cells to repair the damaged core (which he mistakenly thought consisted primarily of extracellular matrix). A study of cephalochordate tail regeneration [42] found that a mass of small cells accumulated just posterior to the differentiated lamellar cells in the regenerating notochord. It has yet to be clearly demonstrated that these small cells were proliferating Müller cells, but it would not be surprising if they were. It also seems likely that, towards the completion of notochord regeneration, some of these actively proliferating cells would reverse the course of differentiation to re-establish the lost stem cells, as is known to happen in some regenerating vertebrate tissues [44, 45].
The present study focused on the posterior end of the notochord. However, in cephalochordates, the organ extends all the way up to the anterior tip the body, where the histological organization is virtually a mirror image of the posterior end. In a study of cell proliferation in larval cephalochordates [39], a few dividing cells were found at either extremity of the notochord. Moreover, in adult cephalochordates, the anterior tip of the notochord, if removed, can regenerate [42]. Thus there is little doubt that the notochord can grow anteriorly as well as posteriorly. It could well be that the population dynamics of the notochord cells are very similar at either end of the organ.
Evolution of notochordal cell population dynamics in the chordates
The present study suggests stem cells might account for notochord growth in an adult lancelet, the best available proxy for the ancestral chordate. It is, therefore, interesting to consider how this feature may have evolved in the two other main groups of chordates—the tunicates and the vertebrates.
Notochordal stem cells in tunicates?
In ascidian tunicates, determinate cleavage [46] produces a larval notochord without the participation of stem cells, and the organ persists for only a day or two before being destroyed at larval metamorphosis. The small, transitory notochord in ascidians accords with their general tendency to simplify and even lose ancestral features [47].
In another tunicate group, the appendicularians, the situation is more interesting because the notochord cells originally form in the embryo by an invariant cell lineage, but then continue proliferating along the length of the organ in the early adult [48]. However, because the appendicularian life span is typically only about 1 week, notochord growth might not involve a sub-population of stem cells. Instead, it remains possible that the increase in cell number results from divisions of progenitor cells with a limited potential for proliferation or from direct entry of differentiated cells into the cell cycle, as happens when the mammalian liver grows by proliferation of differentiated hepatocytes [49].
Notochordal stem cells in non-mammalian vertebrates?
This section is focused on possible stem cells in adult notochords and omits the initial establishment of the organ during early embryology—when it can sometimes be problematical where and how to apply the concept of stem cells [50]. Even so, some of the work on late larval stages of teleosts and amphibians is relevant here and will be covered.
In adults of agnathans [51, 52] and several basal clades of fishes [53, 54], the notochord remains unconstricted by encroaching skeletal elements. Histologically, the notochord in these species comprises a thin peripheral layer of non-vacuolated cortical cells (a name that we will use here instead of the several synonymous terms in the literature) surrounding vacuolated cells of the inner core. The cortical cells have been called “chordoblasts” to indicate that they are thought to differentiate into core cells [51,52,53,54]. However, these studies include no speculation that a population of stem cells might be present in addition. It is conceivable that future work will show that the cortical cells are a progenitor population derived from stem cells that have escaped detection so far—either at the posterior end or perhaps arranged in some other pattern in the notochord.
Vertebrates other than those mentioned above often partially replace the notochord with cartilage and/or bone by the adult stage, dividing the organ into a series of relicts. In adult elasmobranchs [55] and teleosts [56, 57], each notochordal remnant is a conspicuous pad of tissue housed in a cavity formed intervertebrally by the apposition of the concave ends of neighboring vertebrae. In adult elasmobranchs [55] and teleosts [57], each notochordal pad consists of a cortical layer of small cells proposed to be chordoblasts that differentiate into the deeper vacuolated cells. Neighboring intervertebral pads often remain interconnected—but only by a tenuous strand of tissue passing through a small canal in the calcified and/or boney vertebrae. An added complexity, the notochordal pads of teleosts can include voluminous intercellular spaces [57]; such spaces within epithelia and mesothelia are relatively uncommon, but a good example is the tunnel of Corti in the inner ear epithelium of mammals [58].
In larval teleosts, the still-unconstricted notochord consists of three cell populations: cortical cells covering the surface, vacuolated cells at the core, and discoid cells at the posterior end of the organ [59,60,61]. At first glance, the arrangement of these three cell populations suggests a similarity with the adult lancelet notochord. However, in normally developing teleost larvae, the discoid cells do not behave like stem cells but evidently differentiate into cortical and core cells without dividing and thus disappear shortly before the vertebrae begin forming. Also in larval teleost notochords, experimentally damaged core cells are replaced by ingression of some cortical cells, although the internalized cells only form a dense plug without differentiating into typical vacuolated cells [62].
To date, no stem cells have been demonstrated in notochordal tissue of adult elasmobranchs and teleosts. However, in many such fishes, overall body growth continues throughout adult life, and their component tissues grow correspondingly. It is known that the slow, continuous enlargement of some organs in adult fishes and other vertebrates is supported by stem cells [63, 64]. These findings strengthen the likelihood that stem cells are similarly involved in the growth of the intervertebral pads of teleosts.
In adult amphibians and reptiles (in the broad sense to include birds), the fate of the notochord varies considerably from one species to the next. It is only rarely unconstricted, in which case a layer of cortical cells is presumed to differentiate into vacuolated cells of the core [65, 66]. The unconstricted notochords in larval frog tails have a similar structure and a similar conversion of cortical to vacuolated cells [67]. Conversely, the notochord of some amphibians and reptiles can be totally destroyed at the adult stage [68, 69].
In contrast to the above extremes, notochordal tissue of many amphibians and reptiles survives into adulthood, but becomes constricted [70,71,72]. For example, some species maintain the notochordal tissue in intervertebral regions (where it still contributes to limited flexibility of the vertebral column). In these species, the notochordal tissue becomes discontinuous due to the insertion of a cartilaginous plug in the mid-region of each vertebra.
In adults of other species of amphibians and reptiles, the notochordal relicts are arranged in just the reverse pattern. The notochordal tissue disappears intervertebrally, but remains in the mid-region of each vertebra with no role in spinal column flexibility. In such species, adjacent vertebrae often articulate at a joint comprising a skeletal convex surface fitting into a shallow skeletal socket with an intervening zone of cartilage—either solid (a synchondrosis) or incorporating an inconspicuous synovial cavity [68]. In one lizard with mid-vertebral relicts of notochord, cell proliferation was demonstrated, not in the vacuolated notochord cells, but only in cartilage cells in neighboring tissues [73]. The implication seemed to be that any growth of the notochordal relicts would require importation of cells from nearby cartilaginous regions, as is discussed below for mammals.
Notochordal stem cells in mammals?
The previous section on non-mammalian vertebrates emphasizes that their adult notochord tissues are structurally diverse and that little attention has been paid to their possible stem cell component. Quite the opposite can be said of adult mammalian notochords: their vestigial structure is relatively uniform from one species to the next, and they have been intensively studied by stem cell biologists.
The subdivision of the embryonic mammalian notochord into relicts by the interposition of cartilaginous and bony vertebrae was described accurately over a century ago [74]. Each remnant in adult mammals is the nucleus pulposus, which represents the inner core of an intervertebral disc. The nucleus pulposus is not invested by a layer of chordoblast cells, but instead is bounded rostrally and caudally by flat plates of hyaline cartilage and peripherally by a thick layer of fibrous cartilage (the annulus fibrosus). Histologically, the nucleus pulposus of young mammals consists of a relatively voluminous extracellular material with vacuolated cells sparsely embedded in it. In some mammalian species, this disc histology persists throughout life [75]. However, in other species, vacuolated cells predominate in youth, but, with age, disappear as more and more chondrocyte-like cells become mixed with them. In older humans, even the latter begin to disappear.
The degenerative change in the nucleus pulposus regions of the human spinal column is currently driving the search for stem cell-based rejuvenation of the tissue. The approaches may involve introducing extra-vertebral cells into the tissue [76] or stimulating the proliferation of stem cells and progenitor cells already present [77]. This second approach is the one of interest here. The presence of proliferating cells in intervertebral discs of adult mammals is controversial. On the one hand, tracing of labeled proliferating cells indicates that augmentation of cell number in the nucleus pulposus depends on import of chondrocytes from more peripheral tissues [78,79,80]. On the other hand, cell type-specific targeting in Cre-Lox mice indicates that endogenous cell proliferation occurs within the adult nucleus pulposus itself [81, 82].