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A recent study used 61 extant animal genomes to reconstruct the chromosomes of the hypothetical amniote ancestor. Comparison of this karyotype to the 17 chordate linkage groups previously inferred in the ancestral chordate indicated that two whole genome duplications probably occurred in the lineage preceding the ancestral vertebrate.

For postmetamorphic specimens of amphioxus (Cephalochordata), serial block‐face scanning electron microscopy (SBSEM) is used to describe the long‐ignored Rohde‐like cells (RLCs) at the extreme posterior end of the dorsal nerve cord. These cells, numbering about three dozen in all, are divisible into a group with larger diameters running near the dorsal side of the cord and a more ventral group with smaller diameters closely associated with the central canal of the neurocoel. It is possible that the smaller ventral cells might be generated at the ependymal zone of the dorsal nerve cord and later migrate to a dorsal position, although a functional reason for this remains a mystery. All the RLCs have conspicuous regions of microvilli covering as much as 40% of their surface; limited data (by others) on the more anterior bona fide Rohde cells also indicate an extensive microvillar surface. Thus, both the RLCs and the better‐known Rohde cells appear to be rhabdomeric photoreceptors, although a specific function for this feature is currently unknown. Even more perplexingly, although the Rohde cells are quintessential neurons extending giant processes, each RLC comprises a perikaryon that does not bear any neurites. [ABSTRACT FROM AUTHOR]

Cephalochordates, the sister group of vertebrates + tunicates, are evolving particularly slowly. Therefore, genome comparisons between two congeners of Branchiostoma revealed so many conserved noncoding elements (CNEs), that it was not clear how many are functional regulatory elements. To more effectively identify CNEs with potential regulatory functions, we compared noncoding sequences of genomes of the most phylogenetically distant cephalochordate genera, Asymmetron and Branchiostoma, which diverged approximately 120-160 million years ago. We found 113,070 noncoding elements conserved between the two species, amounting to 3.3% of the genome. The genomic distribution, target gene ontology, and enriched motifs of these CNEs all suggest that many of them are probably cis-regulatory elements. More than 90% of previously verified amphioxus regulatory elements were re-captured in this study. A search of the cephalochordate CNEs around 50 developmental genes in several vertebrate genomes revealed eight CNEs conserved between cephalochordates and vertebrates, indicating sequence conservation over >500 million years of divergence. The function of five CNEs was tested in reporter assays in zebrafish, and one was also tested in amphioxus. All five CNEs proved to be tissue-specific enhancers. Taken together, these findings indicate that even though Branchiostoma and Asymmetron are distantly related, as they are evolving slowly, comparisons between them are likely optimal for identifying most of their tissue-specific cis-regulatory elements laying the foundation for functional characterizations and a better understanding of the evolution of developmental regulation in cephalochordates.

In the past 40 years, comparisons of developmental gene expression and mechanisms of development (evodevo) joined comparative morphology as tools for reconstructing long-extinct ancestral forms. Unfortunately, both approaches typically give congruent answers only with closely related organisms. Chordate nervous systems are good examples. Classical studies alone left open whether the vertebrate brain was a new structure or evolved from the anterior end of an ancestral nerve cord like that of modern amphioxus. Evodevo plus electron microscopy showed that the amphioxus brain has a diencephalic forebrain, small midbrain, hindbrain and spinal cord with parts of the genetic mechanisms for the midbrain/hindbrain boundary, zona limitans intrathalamica and neural crest. Evodevo also showed how extra genes resulting from whole-genome duplications in vertebrates facilitated evolution of new structures like neural crest. Understanding how the chordate central nervous system (CNS) evolved from that of the ancestral deuterostome has been truly challenging. The majority view is that this ancestor had a CNS with a brain that gave rise to the chordate CNS and, with loss of a discrete brain, to one of the two hemichordate nerve cords. The minority view is that this ancestor had no nerve cord; those in chordates and hemichordates evolved independently. New techniques such as phylostratigraphy may help resolve this conundrum.

Efforts to understand nervous system structure and function have received new impetus from the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. Comparative analyses can contribute to this effort by leading to the discovery of general principles of neural circuit design, information processing, and gene-structure-function relationships that are not apparent from studies on single species. We here propose to extend the comparative approach to nervous system 'maps' comprising molecular, anatomical, and physiological data. This research will identify which neural features are likely to generalize across species, and which are unlikely to be broadly conserved. It will also suggest causal relationships between genes, development, adult anatomy, physiology, and, ultimately, behavior. These causal hypotheses can then be tested experimentally. Finally, insights from comparative research can inspire and guide technological development. To promote this research agenda, we recommend that teams of investigators coalesce around specific research questions and select a set of 'reference species' to anchor their comparative analyses. These reference species should be chosen not just for practical advantages, but also with regard for their phylogenetic position, behavioral repertoire, well-annotated genome, or other strategic reasons. We envision that the nervous systems of these reference species will be mapped in more detail than those of other species. The collected data may range from the molecular to the behavioral, depending on the research question. To integrate across levels of analysis and across species, standards for data collection, annotation, archiving, and distribution must be developed and respected. To that end, it will help to form networks or consortia of researchers and centers for science, technology, and education that focus on organized data collection, distribution, and training. These activities could be supported, at least in part, through existing mechanisms at NSF, NIH, and other agencies. It will also be important to develop new integrated software and database systems for cross-species data analyses. Multidisciplinary efforts to develop such analytical tools should be supported financially. Finally, training opportunities should be created to stimulate multidisciplinary, integrative research into brain structure, function, and evolution.

Abstract The question of whether the ancestral bilaterian had a central nervous system (CNS) or a diffuse ectodermal nervous system has been hotly debated. Considerable evidence supports the theory that a CNS evolved just once. However, an alternative view proposes that the chordate CNS evolved from the ectodermal nerve net of a hemichordate-like ancestral deuterostome, implying independent evolution of the CNS in chordates and protostomes. To specify morphological divisions along the anterior/posterior axis, this ancestor used gene networks homologous to those patterning three organizing centers in the vertebrate brain: the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer, and subsequent evolution of the vertebrate brain involved elaboration of these ancestral signaling centers; however, all or part of these signaling centers were lost from the CNS of invertebrate chordates. The present review analyzes the evidence for and against these theories. The bulk of the evidence indicates that a CNS evolved just once – in the ancestral bilaterian. Importantly, in both protostomes and deuterostomes, the CNS represents a portion of a generally neurogenic ectoderm that is internalized and receives and integrates inputs from sensory cells in the remainder of the ectoderm. The expression patterns of genes involved in medio/lateral (dorso/ventral) patterning of the CNS are similar in protostomes and chordates; however, these genes are not similarly expressed in the ectoderm outside the CNS. Thus, their expression is a better criterion for CNS homologs than the expression of anterior/posterior patterning genes, many of which (for example, Hox genes) are similarly expressed both in the CNS and in the remainder of the ectoderm in many bilaterians. The evidence leaves hemichordates in an ambiguous position – either CNS centralization was lost to some extent at the base of the hemichordates, or even earlier, at the base of the hemichordates + echinoderms, or one of the two hemichordate nerve cords is homologous to the CNS of protostomes and chordates. In any event, the presence of part of the genetic machinery for the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer in invertebrate chordates together with similar morphology indicates that these organizers were present, at least in part, at the base of the chordates and were probably elaborated upon in the vertebrate lineage.

Over the past 200 years, almost every invertebrate phylum has been proposed as a starting point for evolving vertebrates. Most of these scenarios are outdated, but several are still seriously considered. The short-range transition from ancestral invertebrate chordates (similar to amphioxus and tunicates) to vertebrates is well accepted. However, longer-range transitions leading up to the invertebrate chordates themselves are more controversial. Opinion is divided between the annelid and the enteropneust scenarios, predicting, respectively, a complex or a simple ancestor for bilaterian animals. Deciding between these ideas will be facilitated by further comparative studies of multicellular animals, including enigmatic taxa such as xenacoelomorphs., Biologists have considered nearly every major taxon of animals as the key starting point for the evolution of vertebrates. We survey these ideas, many of which are no longer tenable [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.05.016 Byline: Takayuki Onai (a), Jr-Kai Yu (b), Ira L. Blitz (c), Ken W.Y. Cho (c), Linda Z. Holland (a) Keywords: Branchiostoma; Amphioxus; BMP signaling; Nodal/Vg1 signaling; Axial patterning Abstract: The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution. Author Affiliation: (a) Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093-0202, USA (b) Institute of Cellular and Organismic Biology, Academia Sinica,128 Academia Road, Sec., Nankang, Taipei 11529, Taiwan (c) Department of Developmental and Cell Biology, and Developmental Biology Center, University of California Irvine, Irvine CA 92697-2300, USA Article History: Received 14 March 2010; Revised 10 May 2010; Accepted 11 May 2010

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.04.457 Byline: Michael Schubert (a)(b), Nicholas D. Holland (c), Vincent Laudet (a), Linda Z. Holland (c) Keywords: Branchiostoma; Chordate evolution; Hindbrain regionalization; Hox collinearity; Morpholino; Motor neuron; Neurogenesis; Retinoic acid-antagonist Abstract: Retinoic acid (RA) mediates both anterior/posterior patterning and neuronal specification in the vertebrate central nervous system (CNS). However, the molecular mechanisms downstream of RA are not well understood. To investigate these mechanisms, we used the invertebrate chordate amphioxus, in which the CNS, although containing only about 20,000 neurons in adults, like the vertebrate CNS, has a forebrain, midbrain, hindbrain, and spinal cord and is regionalized by RA-signaling. Here we show, first, that domains of genes with expression normally limited to diencephalon and midbrain are generally not affected by altered RA-signaling, second, that contrary to previous reports, not only Hox1, 3, and 4, but also Hox2 and Hox6 are collinearly expressed in the amphioxus CNS, and third, that collinear expression of all these Hox genes is controlled by RA-signaling. Finally, we show that Hox1 is involved in mediating both the role of RA-signaling in regionalization of the hindbrain and in specification of hindbrain motor neurons. Thus, morpholino knock-down of the single amphioxus Hox1 mimics the effects of treatments with an RA-antagonist. This analysis establishes RA-dependent regulation of collinear Hox expression as a feature common to the chordate CNS and indicates that the RA-Hox hierarchy functions both in proper anterior/posterior patterning of the developing CNS and in specification of neuronal identity. Author Affiliation: (a) Laboratoire de Biologie Moleculaire de la Cellule, CNRS UMR5161/INRA UMR1237/ENS Lyon, IFR128 BioSciences Lyon-Gerland, Ecole Normale Superieure de Lyon, 46 allee d'Italie, 69364 Lyon Cedex 07, France (b) Institut des Sciences de l'Evolution-Montpellier, CNRS-UMR5554, Universite de Montpellier II, CC 065, 34095 Montpellier Cedex 05, France (c) Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, 8750 Biological Grade, La Jolla, CA, 92093-0202, USA Article History: Received 1 February 2006; Revised 9 April 2006; Accepted 19 April 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.03.003 Byline: L. Filipe C. Castro (a), Stacy L.K. Rasmussen (b), Peter W.H. Holland (a), Nicholas D. Holland (b), Linda Z. Holland (b) Keywords: Brain patterning; Midbrain/hindbrain organizer; Branchiostoma; Otx; Gbx; Chordate evolution Abstract: In the vertebrate central nervous system (CNS), mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain/hindbrain boundary (MHB), but does not induce MHB organizer genes such as En, Pax2/5/8 and Wnt1. In the CNS of the cephalochordate amphioxus, Otx is also expressed anteriorly, but En, Pax2/5/8 and Wnt1 are not expressed near the caudal limit of Otx, raising questions about the existence of an MHB organizer in amphioxus. To investigate the evolutionary origins of the MHB, we cloned the single amphioxus Gbx gene. Fluorescence in situ hybridization showed that, as in vertebrates, amphioxus Gbx and the Hox cluster are on the same chromosome. From analysis of linked genes, we argue that during evolution a single ancestral Gbx gene duplicated fourfold in vertebrates, with subsequent loss of two duplicates. Amphioxus Gbx is expressed in all germ layers in the posterior 75% of the embryo, and in the CNS, the Gbx and Otx domains abut at the boundary between the cerebral vesicle (forebrain/midbrain) and the hindbrain. Thus, the genetic machinery to position the MHB was present in the protochordate ancestors of the vertebrates, but is insufficient for induction of organizer genes. Comparison with hemichordates suggests that anterior Otx and posterior Gbx domains were probably overlapping in the ancestral deuterostome and came to abut at the MHB early in the chordate lineage before MHB organizer properties evolved. Author Affiliation: (a) Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK (b) Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093-0202, USA Article History: Received 8 February 2006; Revised 28 February 2006; Accepted 1 March 2006

Cephalochordates (amphioxus), the closest living invertebrate relatives of the vertebrates, are key to understanding the evolution of developmental mechanisms during the invertebrata-to-vertebrate transition. However, a major impediment to amphioxus as a model organism for developmental biology has been the inability to introduce transgenes or other macromolecules into the embryos. Here, we report the development of a reproducible method for microinjection of amphioxus eggs. Specifically, we show that expression of a LacZ reporter construct including 6.3 kb of AmphiFoxD upstream regulatory DNA recapitulates expression of the endogenous gene in the nerve cord, somites, and notochord. We have also identified the 1.6 kb at the 5' end of this region as essential for expression in the first two of these domains and the 4.7 kb at the 3' end as sufficient for expression in the notochord. This study, which is the first report of a method for introduction of large molecules such as DNA into amphioxus embryos, opens the way for studies of gene regulation and function in amphioxus and for comparative studies with vertebrates to understand the relationship between the extensive gene duplications that occurred within the vertebrate lineage and the evolution of vertebrate innovations such as neural crest. Keywords: Branchiostoma floridae; Cis-regulatory sequence; Reporter gene construct; Fox domain transcription factor; Somite; Notochord; Forebrain; Neural crest; Evolution; Repetitive DNA

We isolated a full-length cDNA clone of amphioxus AmphiNk2-tin, an NK2 gene similar in sequence to vertebrate NK2 cardiac genes, suggesting a potentially similar function to Drosophila tinman and to vertebrate NK2 cardiac genes during heart development. During the neurula stage of amphioxus, AmphiNk2-tin is expressed first within the foregut endoderm, then transiently in muscle precursor cells in the somites, and finally in some mesoderm cells of the visceral peritoneum arranged in an approximately midventral row running beneath the midgut and hindgut. The peritoneal cells that express AmphiNk2-tin are evidently precursors of the myocardium of the heart, which subsequently becomes morphologically detectable ventral to the gut. The amphioxus heart is a rostrocaudally extended tube consisting entirely of myocardial cells (at both the larval and adult stages); there are no chambers, valves, endocardium, epicardium, or other differentiated features of vertebrate hearts. Phylogenetic analysis of the AmphiNk2-tin sequence documents its close relationship to vertebrate NK2 class cardiac genes, and ancillary evidence suggests a relationship with the Drosophila NK2 gene tinman. Apparently, an amphioxus-like heart, and the developmental program directing its development, was the foundation upon which the vertebrate heart evolved by progressive modular innovations at the genetic and morphological levels of organization. Keywords: Cephalochordate; Lancelet; Cardiogenesis; NK2 class homeobox

In Xenopus, the canonical Wnt-signaling pathway acting through [beta]-catenin functions both in establishing the dorso-ventral axis and in patterning the anterior--posterior axis. This pathway also acts in patterning the animal--vegetal axis in sea urchins. However, because sea urchin development is typically indirect, and adult sea urchins have pentamerous symmetry and lack a longitudinal nerve cord, it has not been clear how the roles of the canonical Wnt-signaling pathway in axial patterning in sea urchins and vertebrates are evolutionarily related. The developmental expression patterns of Notch, brachyury, caudal, and eight Wnt genes have now been determined for the invertebrate chordate amphioxus, which, like sea urchins, has an early embryo that gastrulates by invagination, but like vertebrates, has a later embryo with a dorsal hollow nerve cord that elongates posteriorly from a tail bud. Comparisons of amphioxus with other deuterostomes suggest that patterning of the ancestral deuterostome embryo along its anterior-posterior axis during the late blastula and subsequent stages involved a posterior signaling center including Wnts, Notch, and transcription factors such as brachyury and caudal. In tunicate embryos, in which cell numbers are reduced and cell fates largely determined during cleavage stages, only vestiges of this signaling center are still apparent; these include localization of Wnt-5 mRNA to the posterior cytoplasm shortly after fertilization and localization of [beta]-catenin to vegetal nuclei during cleavage stages. Neither in tunicates nor in amphioxus is there any evidence that the canonical Wnt-signaling pathway functions in establishment of the dorso-ventral axis. Thus, roles for Wnt-signaling in dorso-ventral patterning of embryos may be a vertebrate innovation that arose in connection with the evolution of yolky eggs and gastrulation by extensive involution. Key Words: amphioxus; sea urchin; tunicate; echinoderm; Xenopus; caudal; hemichordate; tail bud; catenin; Wnt; wingless; Notch; brachyury.