Cross references: Amphioxus PDFs Chordates Hemichordates
Protochordates Cephalochordate Amphioxus Amphioxus Evolution Amphioxus Nervous System Abstract Basic features of the anterior nerve cord in amphioxus larvae are summarized to highlight its essential similarity with the vertebrate brain. Except for a pineal homolog, the amphioxus brain consists of a much simplified version of the ventral brainstem, including a region probably homologous with the hypothalamus, and a locomotory control center roughly comparable to the vertebrate tegmentum and reticulospinal system. Amphioxus has direct pathways for activating its locomotory circuits in response to mechanical stimuli via epithelial sensory cells, but this response is evidently modulated by inputs from diverse sensory-type cells located in the putative hypothalamic homolog, and from the lamellar body, the pineal homolog. This implies that a basic function of the amphioxus brain is to switch between locomotory activities, of which there are several, and the principal non-locomotory one, namely feeding. A similar involvement in switching between behavioral modes may thus have been a core brain function in ancestral chordates. Currently, however, incomplete knowledge of the physiology and behavior of amphioxus limits how effectively it can be used as an evolutionary model. Eye evolution is briefly discussed to illustrate how a better understanding of living forms can inform the evolutionary debate. An account of recent data on dorsoventral inversion is also included, as this bears directly on the question of where the chordate brain originated in relation to other structures. It now appears likely that key components of the ancestral brain were originally located around the mouth. A secondary repositioning of the latter would therefore have been required before a unitary brain could be assembled and internalized. This association between the mouth and the evolving brain reinforces the idea of a fundamental early connection between core brain structures and the control of feeding activity. 1. Introduction When speculating about the events of early chordate evolution, one needs to beware of taking any living protochordate too literally as a model for the proximate invertebrate ancestor of vertebrates. A close analysis of living organisms is certainly a key source of information about the course of past evolution, but all are modified from the ancestral form to a greater or lesser degree. The problem, of course, is to identify which organisms, and which characteristics, provide the most reliable guide to the ancestral condition. There are two groups of organisms to consider here, cephalochordates (amphioxus) and urochordates (tunicates). Of these, tunicates have evidently experienced much more rapid genomic change, and the peculiarities of their life history and morphology are considered to be secondary specializations [6,9]. At the molecular level, even core features of the developmental control program have been modified in tunicates, probably due to the evolution of lineage-dependent control mechanisms [6]. Thus, while fascinating subjects for studies of the changes that can occur with evolution, tunicates are of somewhat limited value as models of the ancestral form. In contrast, cephalochordates appear to be less modified in both genetic and morphological terms, and hence are potentially more directly informative. When it comes to the evolution of the central nervous system (CNS), there is a further problem with tunicates. This relates to the extreme reduction of the nervous system in some forms (larvaceans), and the alterations to it that have accompanied the evolution of a sedentary habit in others (ascidians). The life history of ascidians imposes a nearly complete separation between locomotion and feeding. During the larval phase the animal swims but does not feed, while the reverse is true for the adult. The larval CNS is designed to perform a limited repertoire of sensory and locomotory functions, but has no visceral control functions. The adult has only the latter, the larval system having degenerated at metamorphosis. There is thus no need for neural circuitry to switch between locomotion and non-locomotory functions. Amphioxus, in contrast, swims and feeds throughout its post-embryonic life just as vertebrates do, and a key function of the anterior brain-like region of its CNS[see below] appears to be to modulate these activities and switch between them. There is thus some justification for expecting the brains of amphioxus and vertebrates to share basic organizational features, even to the level of circuitry, so long as there has been at least a degree of continuity through time in the core tasks they are required to perform. Here I provide a brief summary of what is known of the circuitry and organization of the brain-like region of the anterior nerve cord of young amphioxus larvae, which, from molecular data, is regionally subdivided in a comparable fashion to the vertebrate brain. There follows a discussion of two related subjects. First, using the visual system as an example, I highlight the need for more research on all aspects of amphioxus biology as a necessary step towards understanding ancestral chordates as fully functioning organisms. Second, I illustrate how in-depth comparative analysis can provide an unexpected resolution of even the most puzzling evolutionary problems. The example here comes from recent gene expression studies in hemichordates, which are clarifying the role that dorsoventral inversion has played in chordate evolution. A consequence of inversion is that the mouth must shift its position before core components of the brain can assemble into a unitary structure, and this may, in itself, tell us something useful about the nature of those components. 2. The larval brain of amphioxus: organization and circuitry Past studies of the adult CNS in amphioxus have provided a catalog of neuronal cell types, but the circuitry responsible for observed behaviors has so far been examined in detail only in young larvae. The latter are sufficiently small that their CNS can be examined and reconstructed at the EM-level, a task that occupied my own lab for much of the past 15 years [ref. [17] and citations therein]. Of special interest is the anterior-most ca. 90mu of the nerve cord (Fig. 1), comprising the cerebral vesicle and the region just caudal to it to about the end of myotome 1. This corresponds with the zone expressing genes that are markers for vertebrate forebrain and midbrain, notably Otx [5]. Fig. 1.  Fig. 1. Organization of the anterior nerve cord in a 12-day amphioxus larva, seen here in lateral view, anterior to the left, and extending to just beyond the junction between myotomes 1 and 2. The figure shows roughly 120 microns of tissue containing, in total, ca. 150 neurons at this stage. The infundibular cells (inf, dark shading) mark the transition between the largely sensory anterior part of the cerebral vesicle and the posterior part, occupied dorsally by the lamellar body (light shading, including cells within it) and ventrally by the post-infundibular neuropile. Sensory input, from both peripheral and centrally located sensory neurons enters the cord via both the rostral and anterodorsal nerves and from caudal sources (not shown). Somewhat schematic; see refs. [7,17] for further details. A ventral cluster of secretory infundibular cells serves as a useful landmark at the transition between the anterior and posterior parts of the cerebral vesicle. Immediately behind the infundibular cells is a region of ventral neuropile, and behind this is the primary motor center (PMC). There are qualitative differences between the neurons in the anterior-most part of the nerve cord to the level of the post-infundibular neuropile, and those further back. The former, whether in the frontal eye (pigment cells, photoreceptors and some neurons) or the preinfundibular and infundibular region (sensory-type cells) have neurites that mainly project to the neuropile, where they form irregular varicosities. The varicosities are typically large, lack synapses, and contain mixed populations of the vesicles, including a large proportion of dense-cored vesicles. Cells of the dorsal lamellar body, a putative pineal homolog, also contribute to the neruropile, and their terminals also lack synapses. The absence of synapses implies that the anterior neurons act largely through paracrine release and, while the transmitters have not been identified, the morphology indicates they likely include a variety of neuropeptides. As one moves caudally into the primary motor center, the most conspicuous neurons are sets of large interneurons and somatic motoneurons with descending projections. Among the former are three pairs of especially large cells that, from their pattern of projections and synapses, function as a pacemaker. The presynaptic terminals in this region are of conventional appearance, and clear vesicles predominate. The locomotory circuits thus probably depend on fast transmission involving acetylcholine and various amino acid transmitters, a conclusion supported by preliminary pharmacological studies. Dendrites from the pacemaker cells and some of the other PMC neurons project forward to the post-infundibular neuropile. There they receive a highly redundant input, via well-defined synapses, from sensory axons entering via the rostral and anterodorsal nerves and from fibers originating in the tail. The input in young larvae appears to be predominantly if not entirely mechanosensory, and larvae respond very strongly to touch by initiating several types of escape response. They are also capable of more prolonged periods of slow swimming, which may, in nature, drive the diurnal vertical migrations observed in some species. There is, in summary, ample morphological evidence for the circuits needed to drive most of the locomotory behaviors observed in larvae, though many details have yet to be worked out. The principal non-locomotory activity larvae engage in is feeding, which takes place while they are suspended vertically and more-or-less motionless at the water surface [16]. Swimming, whether for escape or migration, is thus incompatible with feeding. One can argue that, to optimize chances of survival, there is an advantage to modulating locomotory behavior depending on the available food supply and nutritional state of the animal. In other words, when it is starved or surrounded by abundant food, the best strategy might be to suppress locomotion in order to feed with minimal interruption. While the synaptic connections identified by EM do not indicate how this is done, dendrites from the locomotory control cells do ramify within the post-infundibular neuropile, which itself receives inputs from a variety of sensory-type cells located dorsally and further forward that could, in principle, act to modulate the locomotory response. Some have cilia modified in ways that suggest they monitor physical displacement, i.e. to act as a balance organ. In most cases, however, function is not evident from the morphology. Instead, because the cells reside in a region that, from molecular data, is probably homologous with the hypothalamus, one is left to speculate on their probable function based on what hypothalamic neurons are known to do. The hypothalamus in vertebrates monitors various physical and chemical stimuli, including temperature, ionic balance, and concentrations of a wide range of specific metabolites, and is involved in setting levels of alertness, activity and appetite. As a working hypothesis, it is reasonable to suppose that the closest amphioxus counterpart of this brain region might do much the same by playing a key role in modulating levels of activity in response to changes in both external environment and internal conditions, including the nutritional state of the animal. The importance of paracrine transmission to circuits originating from the putative amphioxus homolog of the hypothalamus also accords with a concept developed by Nieuwenhuys [14,15] concerning the primitive paracrine (or limbic) core of the vertebrate brain. The latter consists of a series of core brainstem systems organized in a loose, reticular fashion and dependent on paracrine release of a range of neuropeptides. The main hypothalamic centers involved in homeostasis and metabolic control are included among these, and the system as a whole is largely ventral in location. Since it is predominantly counterparts of ventral brainstem structures that are represented in amphioxus (Fig. 2), it will be very interesting to see what further research reveals these cells to be doing. Fig. 2.  Fig. 2. The current consensus, based on combined anatomical and molecular data, for how the anterior nerve cord in amphioxus larvae maps to the vertebrate brain. Most of the former finds its closest counterparts and probable homologs in the ventral region (variously shaded) of the latter, with the exception of the lamellar body, a pineal homolog. The Otx-positive zone in amphioxus, marking the putative forebrain plus hindbrain homologs, extends through the cerebral vesicle (c.v.) and primary motor center to a level somewhere in the front half of myotome 2. Caudal to this point, the nerve cord becomes more hindbrain-like in character, but the exact point of the transition is uncertain (indicated by ?) (from ref. [17]). Genomic analysis will provide a more complete account of the neuropeptides and peptide receptors used by amphioxus than is currently available, and eventually these will be localized within the CNS. In the author’s view, there is a reasonable prospect that many of the basic homeostatic control systems that operate in vertebrates will prove to be ancient and fundamental to CNS organization, traceable to the protochordate level of organization at least, if not earlier. 3. Amphioxus photoreception: multiple receptors for diverse needs We are used to thinking of the vertebrate visual system as consisting first and foremost of paired, image-forming eyes, with at most a few accessory structures to perform related functions, notably the pineal. Explaining how this situation arose in evolution requires some knowledge of the ancestral structures from which the eyes evolved, and of how they were used. Amphioxus has four known photoreceptor systems, two of which are ciliary and two rhabdomeric [8]. The former have vertebrate homologs while the latter evidently do not. The two ciliary receptors are the frontal eye, which is almost certainly the homolog and precursor of the paired eyes, and the pineal-like lamellar body. The two differ structurally in amphioxus in ways that reflect their different functions. Photoreceptor cells in the lamellar body have massive arrays of lamellae to maximize light absorption. The lamellar body is well developed in larvae but less so in the adult, reflecting its probable role monitoring light levels during vertical migration in the water column, which is primarily a larval activity. At depth, there will be a reduction in light intensity, so maximizing light absorption makes sense. In contrast, the photoreceptors in the frontal eye in amphioxus are simple unspecialized cells. There is, in fact, nothing morphological to suggest they are photoreceptors except their close association with the pigment cup, beside which they are ranged in rows that form essentially a one-dimensional array. Behavioral observations [16] indicate the frontal eye acts as a shadow detector to orient the animal while suspended at the water surface, i.e. in conditions of bright light. How then might an image-forming eye evolve starting from something like the amphioxus frontal eye? Since the image formation for a grazing animal is chiefly concerned with identifying approaching predators, any existing shadow detector could be co-opted and improved for this purpose. Visual predators in the Cambrian would have required light for hunting prey, so a shadow detector used during daylight hours is the obvious choice for an eye, which lets the lamellar body out on two counts: it is non-directional and is probably too light-sensitive to operate at the surface in daylight. With regard to the sensitivity-enhancing specializations of vertebrate rods and cones, it is clear these must have come later, since it is only as the receptors are packed in two-dimensional arrays that efficiency of photon capture again becomes an issue. While we are still a long way from a complete story of eye evolution, my intent here is to illustrate how an understanding of amphioxus behavior clarifies some aspects of the earliest events of vertebrate eye evolution, including the issue of why increasing predation produced a burst of adaptive change affecting the frontal eye while leaving the lamellar body essentially unchanged. 4. Dorsoventral inversion and the implied linkage between brain and mouth For much of the past century, comparative zoologists designated the ventral surface of the body as the one bearing the mouth, and assumed this to be the correct orientation for comparing animal body plans. Starting in the mid-1990s, new data on the genes specifying the dorsoventral body axis reawakened an interest in a hypothesis with roots in the 19th century, that chordates are dorsoventrally inverted relative to non-chordates, notably arthropods [1,2]. If true, then the position of the nerve cord (ventral in arthropods, dorsal in vertebrates) is a more reliable indicator of body orientation than the mouth (ventral in both). This raises a further problem, however, for if the body is inverted in chordates so that the mouth becomes dorsal, the mouth has then to migrate or be otherwise shifted to what was the dorsal surface and is now the ventral one. Recent work on hemichordates goes a long way towards resolving this issue. Hemichordates are basal deuterostomes with a bilaterian body plan, so they are exactly what is needed to bridge the gap between protostomes and chordates. The available molecular data now indicate strongly that hemichordates share the dorsoventral orientation of protostomes [12], so it is chordates alone that seem to be inverted relative to both non-chordate deuterostomes and protostomes. Further, and quite remarkably, the genes involved in axial patterning are expressed in a series of circumferential bands in hemichordates, providing a precise map of position along the longitudinal body axis for comparison with other taxa [4]. Fig. 3  Fig. 3. Consequences of dorsoventral inversion for the site of origin and composition of the chordate brain. (A) Embryo of an enteropneust hemichordate, a close relative of basal chordates that has an epithelial nervous system. Circumferential bands of gene expression mark axial position with considerable precision in ways the closely mirror vertebrates. The expression zone corresponding with the vertebrate mid-hindbrain boundary (MHB) lies behind the mouth (m) as shown by the shaded box; shows also anterior pole marked an apical plate (ap) which, though poorly defined in direct-developing species, is evident in those with larvae. In the inverted ancestral chordate (B) the ancestral (a-) dorsal surface is now on the underside of the body and the mouth is on top. If axial markers are to be believed, however, the boxed region will still be post-oral. The epithelial zone from which the anterior nervous tissue most probably arises in chordates is shown as a U-shaped domain (shaded) embracing the mouth and positioned such that the MHB and midbrain structures would lie within the boxed post-oral domain. More anterior regions, including some or all of the forebrain, would likely come from zones lateral to or even forward of the mouth. However, in order for these structures to coalesce into a unitary brain, the mouth must be moved out of the way. This evidently happened by having the mouth shift laterally down the left side of the head to reach the new ventral surface (see text), as in fact it does today during amphioxus development. Notably, the genes expressed at the level of the mid-hindbrain boundary in vertebrates are post-oral in hemichordates (Fig. 3), so that part of the brain is evidently a post-oral structure in vertebrates as well [10]. The source of more anterior brain structures is not yet clear, but it is reasonable to suppose that at least some could derive from sites further forward, along the sides of the mouth, as shown in Fig. 3, and in front of it. It may be significant that similarly positioned adoral neural centers are key components of the larval nervous system in echinoderms and hemichordates. There is a problem for any evolutionary scheme that derives the mouth from an epithelial domain that also forms a significant part of the CNS. Since a functional mouth requires an external opening, the components of the CNS that surround it cannot coalesce to form a unitary, internalized structure until the mouth is moved out of the way. This implies an ancestral condition in which the brain was not at first an internalized structure. There are then two main options for moving the mouth. One, suggested by Nielsen [13], is to have it move forward over the anterior end of the animal. This requires that preoral structures like the apical organ be displaced to a ventral position, below the mouth, which forever breaks their connection with the brain. An alternative is for the mouth to move laterally over the side of the head. Preoral and apical structures could then retain some connection with the evolving brain. Interestingly, among the structures in question is the preoral ciliary organ of hemichordates, a putative homolog of the adenohypophysis [3]. The latter retains associations with both the mouth and brain throughout the vertebrates, which could reflect a more ancient condition when its precursors where in contact with both, at a time before the mouth had shifted its position. Evidence that the mouth may have shifted laterally, down the side of the head, comes from two sources. First, the hemichordate data show that suppression of the dorsalizing signal allows the mouth to expand laterally along both sides of the head until, in some cases, the proboscis is completely separated from the body [12]. This implies that there is a restricted circumferential zone of tissue within which the dorsoventral extent of the mouth is specified. Its position could in principle, be respecified by altering relevant patterns of gene expression so as to shift the orientation of the dorsoventral axis. This would move the mouth down one side of the head, like a tram on a track, and away from the dorsal midline, without altering its position along the longitudinal axis. A second, less direct form of evidence comes from an analysis of pharyngeal development in amphioxus larvae [11]. The initial asymmetry of the larval head is extreme, and to restore symmetry requires a process of repatterning and differential growth that is exceedingly complex so long as the mouth is assumed to be part of the ventral patterning system. If it is not, i.e. if it originates dorsally, re-patterning is much less of a problem. If this interpretation is correct, then the initial appearance of the mouth on the left side of the larval head of amphioxus, and its subsequent movement to the ventral midline, is in part a recapitulation of a past evolutionary event. A further consequence of inversion, relevant to the circuitry analysis presented above, is that, by requiring a close connection between the mouth and evolving CNS, it is consistent with and, indeed, strongly reinforces the idea of a deep connection between core centers in the brain in chordates and the control of feeding-related activities. Acknowledgements Supported by NSERC Canada. I thank Nick Holland and Chris Lowe for insights and discussion, and the organizers of this symposium. References [1] D. Arendt, K. Nubler-Jung, Inversion of dorsoventral axis? Nature 371 (1994) 26. [2] D. Arendt, K. Nubler-Jung, Comparison of early nerve cord development in insects and vertebrates, Development 126 (1999) 2309–2325. [3] S. Candiani, N.D. Holland, D. 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