Amphioxus neuroglia: Molecular characterization and evidence for early compartmentalization of the developing nerve cord.

Glial cells play important roles in the development and homeostasis of metazoan nervous systems. However, while their involvement in the development and function in the central nervous system (CNS) of vertebrates is increasingly well understood, much less is known about invertebrate glia and the evolutionary history of glial cells more generally. An investigation into amphioxus glia is therefore timely, as this organism is the best living proxy for the last common ancestor of all chordates, and hence provides a window into the role of glial cell development and function at the transition of invertebrates and vertebrates. We report here our findings on amphioxus glia as characterized by molecular probes correlated with anatomical data at the transmission electron microscopy (TEM) level. The results show that amphioxus glial lineages express genes typical of vertebrate astroglia and radial glia, and that they segregate early in development, forming what appears to be a spatially separate cell proliferation zone positioned laterally, between the dorsal and ventral zones of neural cell proliferation. Our study provides strong evidence for the presence of vertebrate-type glial cells in amphioxus, while highlighting the role played by segregated progenitor cell pools in CNS development. There are implications also for our understanding of glial cells in a broader evolutionary context, and insights into patterns of precursor cell deployment in the chordate nerve cord.

Patterning, From Conifers to Consciousness: Turing's Theory and Order From Fluctuations.

This is a brief account of Turing's ideas on biological pattern and the events that led to their wider acceptance by biologists as a valid way to investigate developmental pattern, and of the value of theory more generally in biology. Periodic patterns have played a key role in this process, especially 2D arrays of oriented stripes, which proved a disappointment in theoretical terms in the case of Drosophila segmentation, but a boost to theory as applied to skin patterns in fish and model chemical reactions. The concept of "order from fluctuations" is a key component of Turing's theory, wherein pattern arises by selective amplification of spatial components concealed in the random disorder of molecular and/or cellular processes. For biological examples, a crucial point from an analytical standpoint is knowing the nature of the fluctuations, where the amplifier resides, and the timescale over which selective amplification occurs. The answer clarifies the difference between "inelegant" examples such as Drosophila segmentation, which is perhaps better understood as a programmatic assembly process, and "elegant" ones expressible in equations like Turing's: that the fluctuations and selection process occur predominantly in evolutionary time for the former, but in real time for the latter, and likewise for error suppression, which for Drosophila is historical, in being lodged firmly in past evolutionary events. The prospects for a further extension of Turing's ideas to the complexities of brain development and consciousness is discussed, where a case can be made that it could well be in neuroscience that his ideas find their most important application.

Basic features of the ancestral chordate brain: a protochordate perspective.

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.

The origin of dopaminergic systems in chordate brains: insights from amphioxus.

The basic anatomy of the central nervous system (CNS) is well conserved within the vertebrates and differs in significant ways from that of non-vertebrate chordates. Of the latter, amphioxus is of special interest, being the best available stand-in for the basal chordate condition. Immunohistochemical and gene expression studies on the developing CNS of amphioxus embryos and larvae are now sufficiently advanced that we can begin to assign specific neurotransmitter phenotypes to neurons identified by transmission electron microscopy (TEM), and then compare the distribution of cell types to that in vertebrate brains. Here, by monitoring tyrosine hydroxylase (TH) transcripts and protein, along with serial TEM, we identify a population of catecholamine-containing neurons in the anterior nerve cord of amphioxus larvae and describe their pattern of synaptic inputs and outputs. Inputs parallel those to the large paired neurons that control the larval escape response, suggesting that the TH+ system functions as an accessory excitatory and perhaps modulatory pathway in larval locomotion, with the added feature of recruiting an assortment of additional interneurons to the circuitry. The TH+ cells probably contain either L-DOPA or dopamine, and correspond closely with a cell population known from previous work on adult amphioxus to be dopaminergic. This population lies in a CNS domain now thought to comprise a combined vertebrate diencephalon plus mesencephalon, the implication being that dopaminergic nuclei in both of these brain regions could derive from a single dien-mesencephalic population in the last common ancestor of amphioxus and vertebrates.

Ventral neurons in the anterior nerve cord of amphioxus larvae. II. Further data on the pacemaker circuit.

Previous serial EM studies of the anterior nerve cord of amphioxus larvae implicate the third pair of large paired neurons (LPN3s) as key components of the pacemaker responsible for oscillatory premotor output in somites 1 and 2. Here the synaptic relationship between the LPN3s and a fourth such pair (LPN4s), located in somite 3, is examined from a second series of sections. Because of limited overlap between the two series, fiber identity can only be inferred in most instances. To act as pacemakers, the LPN3s must inhibit each other; the current data show similar patterns of synaptic contact, presumably also inhibitory, with the LPN4s. The oscillatory signal appears therefore to be relayed from one LPN pair to the next. Despite their evident importance in the anterior cord, the role these cells play more caudally in signal propagation is not clear.

The dorsal compartment locomotory control system in amphioxus larvae.

Amphioxus myotomes consist of separate sets of superficial and deep muscle fibers, each with its own innervation, that are thought to be responsible for slow swimming and escape behavior, respectively. Tracings from serial EM sections of the anterior nerve cord in the larva show that the motoneurons and premotor interneurons controlling the superficial fibers (the dorsal compartment, or DC pathway) are linked by specialized junctions of a previously undescribed type, referred to here as juxta-reticular (JR) junctions for the characteristic presence of a cisterna of endoplasmic reticulum on each side. JR junctions link the DC motoneurons with each other, with the largest of the anterior paired neurons (LPN3s) and with one class of ipsilateral projection neurons (IPNs), but occur nowhere else. Because of the paucity of synaptic input to the DC system, larval behavior can only be explained if the JR junctions act as functional links between cells. An analysis of the pattern of cell contacts also suggests that the LPN3s are probably pacemakers for both slow and fast locomotion, but act through junctions in the former case and conventional synapses in the latter. The only major synaptic input to the DC system identified in somites 1 and 2 was from four neurons located in the cerebral vesicle, referred to here as Type 2 preinfundibular projection neurons (PPN2s). They have unusually large varicosities, arranged in series, that make periodic contacts with the DC motoneurons. More caudally, the DC motoneurons receive additional input via similar large varicosities from the receptor cells of the first dorsal ocellus, located in somite 5. The overall circuitry of the locomotory control system suggests that the PPN2s may be instrumental in sustaining slow swimming, whereas mechanical stimulation, especially of the rostrum, preferentially activates the fast mode.

Serial EM analysis of a copepod larval nervous system: Naupliar eye, optic circuitry, and prospects for full CNS reconstruction.

The medial eye and optic center of the first nauplius of Dactylopusia (=Dactylopodia) tisboides, a harpacticoid copepod, were reconstructed from serial EM micrographs. Axons from the eye project to a set of matching cartridges defined by glial cells processes, and input is then processed in sequence through two synaptic fields. A single class of local relay neurons provides the main pathway between these, subject to modulatory input from a class of densely stained neurons with distinctive dense terminals. The importance of other outside sources of synaptic input to the second synaptic field indicates that the latter is a major site for integrating the optic input with signals originating elsewhere in the CNS. This accords with physiological data on the shadow response in barnacles, whose visual system is also derived from a naupliar eye. With a body length of ca. 80microns, copepod larvae like that of Dactylopusia are arguably among the smallest functional metazoans with a complex nervous system. Hence they are promising subjects for full reconstruction of neural circuitry at the EM level that could, in principle, reveal where key decision-making functions are localized.

Head organization and the head/trunk relationship in protochordates: problems and prospects.

The fossil record has been an invaluable aid for reconstructing the major events of vertebrate evolution. There is no comparable record for protochordates, however, which severely limits our knowledge of their ancestral morphology, habits, and mode of life. The alternative is inference based on an interpretation of living protochordates but this is fraught with problems, not least being our own biases of what we think an ancestral chordate ought to look like. Relevant to the present symposium is the problem of head/trunk relationships and whether or not the myotomes of the trunk originally extended into the head in vertebrates. I will review what is currently known of patterns of innervation in tunicates and amphioxus in relation to Romer's somaticovisceral concept of the vertebrate body to show how little progress has been made in resolving this problem. There are, in contrast, surprisingly good prospects for solving some other puzzles concerning chordate origins. Dorsoventral inversion provides a good example. A consensus is now emerging, based largely on molecular data from hemichordates that casts new light on the asymmetry of the head in amphioxus. Specifically, the morphogenetic growth process that reestablishes symmetry in late-stage larvae can now be seen, at least in part, as a recapitulation of past evolutionary events, and this has important implications for the origin and basic organization of the brain.

The cholinergic gene locus in amphioxus: molecular characterization and developmental expression patterns.

The cholinergic gene locus (CGL), consisting of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT) gene, encodes two specific cholinergic neuronal markers used extensively to study cholinergic transmission. In the present work, we isolated the amphioxus homologs of VAChT and ChAT and examined their expression during development. Analysis of the 5' untranslated region of VAChT and ChAT suggests that the splicing of the VAChT/ChAT mRNA has been evolutionarily conserved in amphioxus and mammals. By double whole-mount in situ hybridization, we demonstrate that VAChT and ChAT are coexpressed in the same cells. They are first expressed in four pairs of differentiating cells in the neural plate. Their later expression is primarily in the anterior nerve cord in several types of motoneurons, some of the interneurons and in the receptor cells of the larval ocellus.

Prospective protochordate homologs of vertebrate midbrain and MHB, with some thoughts on MHB origins.

The MHB (midbrain-hindbrain boundary) is a key organizing center in the vertebrate brain characterized by highly conserved patterns of gene expression. The evidence for an MHB homolog in protochordates is equivocal, the "neck" region immediately caudal to the sensory vesicle in ascidian larvae being the best accepted candidate. It is argued here that similarities in expression patterns between the MHB and the ascidian neck region are more likely due to the latter being the principal source of neurons in the adult brain, and hence where all the genes involved in patterning the latter will necessarily be expressed. The contrast with amphioxus is exemplified by pax2/5/8, expressed in the neck region in ascidian larvae, but more caudally, along much of the nerve cord in amphioxus. The zone of expression in each case corresponds with that part of the nerve cord ultimately responsible for innervating the adult body, which suggests the spatially restricted MHB-like expression pattern in ascidians is secondarily reduced from a condition more like that in amphioxus. Patterns resembling those of the vertebrate MHB are nevertheless found elsewhere among metazoans. This suggests that, irrespective of its modern function, the MHB marks the site of an organizing center of considerable antiquity. Any explanation for how such a center became incorporated into the chordate brain must take account of the dorsoventral inversion chordates have experienced relative to other metazoans. Especially relevant here is a concept developed by Claus Nielsen, in which the brain is derived from a neural center located behind the ancestral mouth. While this is somewhat counterintuitive, it accords well with emerging molecular data.

Vetulicolians--are they deuterostomes? chordates?

A recent paper by Shu et al.(1) reinterprets the fossil Vetulicola and related forms, all from the Lower Cambrian, as basal deuterostomes, assigning them their own phylum, Vetulicolia. Their conclusion is based on the presence of structures resembling gill slits and a trunk-like region that shows evidence of segmentation. This report summarizes the fossil evidence for their interpretation and evaluates a possible alternative, that vetulicolians may instead be tunicate-like chordates. Implications for our understanding of the nature of the primitive deuterostome (and chordate) body plan are discussed. .

Sensory systems in amphioxus: a window on the ancestral chordate condition.

Amphioxus has an assortment of cells and organs for sensing light and mechanical stimuli. Vertebrate counterparts of these structures are not always apparent, and a strong case can be made for homology in only a few instances. For example, amphioxus has anatomically simple but plausible homologs of both the pineal and paired eyes of vertebrates. Placodal and neural crest derivatives are, however, more problematic: the evidence for an olfactory system in amphioxus is only circumstantial and, despite the variety of secondary sensory cell types that occur on the body surface in amphioxus, none are obvious homologs of vertebrate taste buds, neuromasts or acoustic hair cells. A useful perspective can nevertheless be gained by examining differences in amphioxus and vertebrate development, specifically how each specifies and positions sensory precursors, controls their proliferation, and deploys them through the body. The much larger size of vertebrate embryos and the need to cope developmentally with increased scale and cell numbers may account for some key vertebrate innovations, including placodes and neural crest. The presence or absence of specific structural adaptations, like the latter, is therefore less useful for judging homology between amphioxus and vertebrates than shared features of specific cell types. It is also clear that the duration of embryogenesis in vertebrates has been significantly extended in comparison with ancestral chordates so as to incorporate events that would originally have occurred during the post-embryonic growth period, including events of neurogenesis. Consequently, no scenario for the origin of vertebrates can be considered complete unless it deals explicitly with the whole of the life history and changes to it.