Molecular and morphological evidence together support the idea that the anterior end of the amphioxus nerve cord contains regions homologous with vertebrate forebrain and hindbrain 1,2
, though it is generally the ventral portion of these regions that are best represented 3
. The presence of a midbrain homolog is more controversial. Its most probable position, as defined by gene expression patterns, would be from somewhere forward of the caudal limit of otx
expression, which in vertebrates extends through the midbrain, to a point close to the beginning of the zone of Hox gene expression. In young amphioxus larvae, this corresponds with a region extending from the infundibular cells, which lie at a level roughly midway along somite 1, through the posterior part of the cerebral vesicle to a level somewhere near or just beyond the middle of somite 2 (Fig. A). This domain begins with an anterior zone of ventral neuropile and commissural fibers followed, near the junction between somites 1 and 2, by a complex of ventrally-positioned somatic motoneurons and interneurons with caudal projections that initiate and control larval swimming behavior 4
. The first members of the visceral motoneuron series lie further back, near the caudal end of somite 2. From there, visceral motoneurons recur at regular intervals along the anterior nerve cord and innervate the body via an extended series of peripheral nerves.
Figure 1 Dorsal views of the larval nerve cords of (A) amphioxus and (B) Ciona, a representative ascidian, showing expression domains for the main CNS patterning genes. This is somewhat simplified, as the exact extent of the expression domains can vary with developmental (more ...)
Any attempt to identify an amphioxus homolog of the vertebrate midbrain is hampered by the fact that we currently lack unambiguous criteria for recognizing its presence. The quintessential identifying feature in vertebrates is the dorsal optic tectum, but this is absent in amphioxus. In fact, except for a pineal homolog, amphioxus appears to lack all of the dorsal structures of the vertebrate brain. Further, because the amphioxus nerve cord is of uniform dimension along its length, there are no morphological constrictions to separate sub-domains in the anterior cord from one another in the way vertebrate isthmus separates midbrain from hindbrain. The isthmus is notable as the site of an important organizing center, the midbrain-hindbrain boundary (MHB), characterized in vertebrates by the expression of a highly conserved set of gene, including fgf8
, and wnt1 5,6
. The comparable site in amphioxus lies somewhere close to the caudal limit of somite 1, which is where the anterior zone of otx
expression abuts that of gbx 7
. However, though engrailed
is expressed in small clusters of cells in the embryonic nerve cord 8
, these lie further forward in the cerebral vesicle, at a level near the midpoint of somite 1. In addition, wnt1
is not expressed at all in the anterior cord, nor does the expression of the amphioxus pax2
, match the vertebrate pattern. Instead, the latter is expressed caudally through much of the nerve cord 9
, and though there is a small anterior zone of later expression, it is too far forward for an MHB marker.
There is thus no clear molecular evidence for a focal center in amphioxus with the expression profile of an MHB. This is perhaps not surprising, considering that the structures organized by the MHB are primarily dorsal ones not present in amphioxus. The MHB is, however, also required for normal organization of some ventral brain regions in vertebrates 10
. Since the ventral midbrain of vertebrates very likely combines vertebrate-specific neuronal cell types along with cell groupings with more primitive organizational features, it would be useful to know whether all of these are MHB-dependent or only the former. It may well be that only vertebrate innovations, whether dorsal or ventral, are under the specific control of the MHB. This leaves open the question of whether the amphioxus pattern differs from that of vertebrates because it reflects an earlier stage in the evolution of a vertebrate-type MHB 7
, or whether amphioxus has secondarily diverged from a pattern that was once closer to the vertebrate one. In addition, the vertebrate midbrain marker dmbx
is not expressed in the amphioxus nerve cord 11
. The available molecular data thus argues against the presence, in amphioxus, of precise homologs of either the vertebrate midbrain or MHB.
Among tunicates, the other group of protochordates, it is the ascidians that are best studied, and for these there is again good molecular evidence for homologs of both forebrain and hindbrain in the larval CNS 11,12
, corresponding roughly with the sensory vesicle and caudal ganglion respectively. In addition, cells in the narrow “neck” region that separates these structures express at least some characteristic MHB genes (Fig. B), notably pax2/5/8
, and fgf8 13,14
. Data on pax2/5/8
led initially to the proposal that ascidians had an exact counterpart of the vertebrate MHB and that amphioxus, being a later offshoot of the chordate lineage, must have lost this feature secondarily 12
. However, the precise spatial arrangement of the expression zones for several key genes in ascidians differs from that in vertebrates, e.g. fgf
in ascidian larvae are expressed in cells immediately caudal to those expressing pax2/5/8
, whereas in vertebrates, dmbx
lies forward of the other three genes, whose expression overlaps. Expression patterns of the same genes in larvaceans, another group of tunicates, is somewhat different yet again 13
, which further complicates the problem of determining the nature of the ancestral pattern.
The molecular data is thus somewhat inconclusive regarding protochordate homologs of either the vertebrate midbrain or MHB. If anatomical and functional considerations are taken into consideration, however, a somewhat stronger case can be made that amphioxus may have an approximate counterpart of the midbrain. The key point is that some of the cell types and neural circuits located in the caudal part of the zone of otx
expression are similar to those found in the ventral midbrain in vertebrates. In amphioxus, as pointed out above, only ventral markers are available for comparison. Of these, the infundibular cells probably mark the anterior limit of any prospective midbrain-like territory, as their homology with the ventral infundibular region of the vertebrate diencephalon seems to be fairly well accepted. Immediately behind this point, in amphioxus, there is a zone of ventral neuropile in some ways comparable with the ventral tegmental commissure, which forms part of the early axonal scaffolding in embryonic vertebrate brains. This same region in amphioxus also contains the anterior-most motoneurons in the nerve cord along with populations of large interneurons with descending projections, features found in the tegmentum and the reticulospinal plexus of lower vertebrates beginning at midbrain level. The ventral midbrain in vertebrates is also where dopaminergic neurons with projections to the forebrain first develop, and these are a key component of the motivational circuitry linking basal brainstem centers with the forebrain. Dopaminergic neurons are found in the cerebral vesicle in amphioxus and nowhere else in the nerve cord. Two main populations develop, one in a dorsal position in the anterior cerebral vesicle, the other more ventrally near the junction between somites 1 and 2 14,15
. Further research is needed on these cells to determine their precise pattern of projections and function, but the more caudal of the two populations is well positioned to be a homolog of the midbrain dopaminergic neurons in vertebrates.
If the anatomical and functional criteria outlined above are meaningful indicators that amphioxus does indeed have a midbrain homolog, the value of the molecular markers used to date to test this would have to be reconsidered. The same could be said of ascidian larvae, but for a different reason, for here there is an alternative explanation for the expression patterns observed. The CNS of adult ascidians consists of a simple brain-like dorsal ganglion, from which nerves radiate to the body musculature and visceral organs. The ganglion is present in only a rudimentary form in the larva, however, and the source of its cells has never been clear. In the few species where this has been investigated in the past, the ganglion develops in contact with the neck region of the larval nerve cord near the site where it contacts the neurohypophyseal duct. The latter is partly a stomodeal derivative, which raises the question of whether a significant part of the ganglion might be derived from stomodeal ectoderm. An analysis of the salp ganglion, compared with the data available on compound ascidians 16,17
, supports the idea that most if not all of the dorsal ganglion is of neural origin and that, in ascidians, it develops as part of the neck region. Recent data on Ciona 18
tends to support this interpretation, as its ganglion develops in contact with the base of an outgrowth that arises from the caudal part of the sensory vesicle very near the neck. More importantly, experimental work now in progress on Ciona
is confirming the neck region as the source of major classes of neurons within the adult ganglion, including motoneurons innervating the visceral organs [J.F. Brunet, personal communication].
Evidently then, the cells of the neck region in ascidian larvae serve as a pool of precursors from which most if not all post-metamorphic CNS neurons are derived. Assuming the genes expressed in the vertebrate MHB include major players in the overall process of neuronal specification and differentiation, one can argue that their expression in the neck region is to be expected, irrespective of any homology between this site and the MHB. One would predict the genes would be expressed in combinatorial patterns and in a few cells at most, which is precisely what is observed.
Current thinking on the nature of ancestral chordates favors the view that the separation of adult and larval tissues in ascidians is a secondary specialization, and that the ancestral body plan was a more fully integrated one, as in amphioxus 19,20
. This may explain why the expression patterns of some key developmental control genes differ so dramatically between ascidians and amphioxus. Consider pax2/5/8
, which has an extended zone of expression in amphioxus, but a very restricted one in ascidians (cf. Figs. A, B). Paralleling these, there are differences in innervation patterns of, for example, the visceral organs. These are strictly adult structures in ascidians, and the cells responsible for their innervation arise from the neck region, whereas the Hox-expressing part of the nerve cord, which innervates the tail, is entirely lost at metamorphosis. In amphioxus, in contrast, visceral motoneurons, along with the rest of the locomotory control system, arise from an extended region of the nerve cord extending well into the Hox zone. Whether the zone supplying visceral innervation corresponds precisely with that expressing pax2/5/8
has yet to be determined. One can nevertheless predict that any gene essential to the development of the visceral innervation will necessarily be expressed over a much greater length of the nerve cord in amphioxus than in ascidians. For at least some of the genes associated with the MHB, therefore, the reason their homologs have a very restricted expression zone in ascidian larvae likely has less to do with the presence of a vertebrate-type MHB, and more with the functional necessity of generating a full complement of adult neurons from a single site within the nerve cord. What one then wants to know, to determine whether the ascidian pattern and the vertebrate one are more than coincidentally related, is what functional role the genes play in each instance. With regard to pax2/5/8
specifically, a further clue might come from knowing whether its expression in hemichordate embryos is restricted, as in ascidians, or extended, as in amphioxus. If the latter, this would be further evidence for the view that the ascidian pattern is indeed the derived one.