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Neuroectodermal signalling centres induce and pattern many novel vertebrate brain structures but are absent, or divergent, in invertebrate chordates. This has led to the idea that signalling-centre genetic programs were first assembled in stem vertebrates and potentially drove morphological innovations of the brain. However, this scenario presumes that extant cephalochordates accurately represent ancestral chordate characters, which has not been tested using close chordate outgroups. Here we report that genetic programs homologous to three vertebrate signalling centres-the anterior neural ridge, zona limitans intrathalamica and isthmic organizer-are present in the hemichordate Saccoglossus kowalevskii. Fgf8/17/18 (a single gene homologous to vertebrate Fgf8, Fgf17 and Fgf18), sfrp1/5, hh and wnt1 are expressed in vertebrate-like arrangements in hemichordate ectoderm, and homologous genetic mechanisms regulate ectodermal patterning in both animals. We propose that these genetic programs were components of an unexpectedly complex, ancient genetic regulatory scaffold for deuterostome body patterning that degenerated in amphioxus and ascidians, but was retained to pattern divergent structures in hemichordates and vertebrates.

The “agitator”, a device designed for culturing lecithotrophic echinoderm larvae, is described. The unit consists of an open system which is easily constructed and is made up of a base which holds the culturing baskets as well as a lid or manifold carrying a tube which transports fresh seawater into each culture, gently stirring it and eliminating any decaying embryos. The device is scalable and water flow can be adjusted for laminar flow or slow dripping. The method has been used to successfully rear large numbers of lecithotrophic larvae of the starfish Solaster stimpsoni. The agitator could be used to culture any fragile planktonic organisms that require an extended rearing time.

Deuterostomes comprise vertebrates, the related invertebrate chordates (tunicates and cephalochordates) and three other invertebrate taxa: hemichordates, echinoderms and Xenoturbella. The relationships between invertebrate and vertebrate deuterostomes are clearly important for understanding our own distant origins. Recent phylogenetic studies of chordate classes and a sea urchin have indicated that urochordates might be the closest invertebrate sister group of vertebrates, rather than cephalochordates, as traditionally believed. More remarkable is the suggestion that cephalochordates are closer to echinoderms than to vertebrates and urochordates, meaning that chordates are paraphyletic. To study the relationships among all deuterostome groups, we have assembled an alignment of more than 35,000 homologous amino acids, including new data from a hemichordate, starfish and Xenoturbella. We have also sequenced the mitochondrial genome of Xenoturbella. We support the clades Olfactores (urochordates and vertebrates) and Ambulacraria (hemichordates and echinoderms). Analyses using our new data, however, do not support a cephalochordate and echinoderm grouping and we conclude that chordates are monophyletic. Finally, nuclear and mitochondrial data place Xenoturbella as the sister group of the two ambulacrarian phyla. As such, Xenoturbella is shown to be an independent phylum, Xenoturbellida, bringing the number of living deuterostome phyla to four.

The evolutionary origins of the chordate central nervous system remain uncertain. Conclusions drawn from classical morphological comparisons and from a broad range of metazoan phyla conflict with the new molecular genetic information from developmental model systems characterized by central nervous systems. This has led to debate as to the nature of the ancestral deuterostome nervous system. Hemichordates as basal deuterostomes occupy a phylogenetically critical position for addressing hypotheses on the evolution of the chordate nervous system. Characterizing the molecular basis of the development of their diffuse nervous system offers insights into the role of conserved body patterning genes in the evolution of specific neural anatomies. We present a description of hox gene expression during the development of the direct-developing hemichordate Saccoglossus kowalevskii. The nested ectodermal expression of these genes in a hemichordate suggest that they play a general patterning role in the anterior/posterior regionalization of the ectoderm of bilaterians rather than being uniquely associated with the development and evolution of central nervous systems.

While some aspects of the phylogeny of the five living echinoderm classes are clear, the position of the ophiuroids (brittlestars) relative to asteroids (starfish), echinoids (sea urchins) and holothurians (sea cucumbers) is controversial. Ophiuroids have a pluteus-type larva in common with echinoids giving some support to an ophiuroid/echinoid/holothurian clade named Cryptosyringida. Most molecular phylogenetic studies, however, support an ophiuroid/asteroid clade (Asterozoa) implying either convergent evolution of the pluteus or reversals to an auricularia-type larva in asteroids and holothurians. A recent study of 10 genes from four of the five echinoderm classes used ‘phylogenetic signal dissection’ to separate alignment positions into subsets of (i) suboptimal, heterogeneously evolving sites (invariant plus rapidly changing) and (ii) the remaining optimal, homogeneously evolving sites. Along with most previous molecular phylogenetic studies, their set of heterogeneous sites, expected to be more prone to systematic error, support Asterozoa. The homogeneous sites, in contrast, support an ophiuroid/echinoid grouping, consistent with the cryptosyringid clade, leading them to posit homology of the ophiopluteus and echinopluteus. Our new dataset comprises 219 genes from all echinoderm classes; analyses using probabilistic Bayesian phylogenetic methods strongly support Asterozoa. The most reliable, slowly evolving quartile of genes also gives highest support for Asterozoa; this support diminishes in second and third quartiles and the fastest changing quartile places the ophiuroids close to the root. Using phylogenetic signal dissection, we find heterogenous sites support an unlikely grouping of Ophiuroidea + Holothuria while homogeneous sites again strongly support Asterozoa. Our large and taxonomically complete dataset finds no support for the cryptosyringid hypothesis; in showing strong support for the Asterozoa, our preferred topology leaves the question of homology of pluteus larvae open.

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

The in situ staining patterns of Fig 4C and 4D were incorrectly assigned to the nkx2.2 gene of Saccoglossus kowalevskii. Subsequent sequencing and analysis has shown that these images were actually expression patterns for the foxA gene. Fig 4 has been updated with images of the in situ staining patterns of the nkx2.2 gene of Saccoglossus kowalevskii and the figure legend has been amended to reflect this. Fig 4 Expression in S. kowalevskii of Orthologs of Chordate Genes Important in Dorsoventral Patterning of the Neural Tube. The corrected legend for Fig 4 appears below. Additionally, the authors would like to amend the text description of nk2-2 expression on p.1610 (right hand column), which incorrectly concerned foxa; and restate the conclusion about nk2-2 expression in the Discussion on p.1615 (left column). The authors confirm this conclusion is unchanged, but the old foxa data were irrelevant to the cross-phylum comparison of nk2-2 expression among S. kowalevskii, chordates, and Drosophila. The corrected text appears below. Page 1610: In S. kowalevskii, we find that the expression profiles of the hh, nk2.2, and msx orthologs do not at all resemble the expression domains of vertebrates or Drosophila, as shown in Fig 4. We could isolate only one kind of hedgehog ortholog from S. kowalevskii, but this may exhaust the hemichordate repertoire since basal deuterostomes such as amphioxus and sea urchins have but one gene [86,87], whereas vertebrates have up to four paralogs of hh [86]. The expression of hh in S. kowalevskii begins at day 2, in a small patch at the apical tip of the prosome ectoderm (Fig 4A), and it continues in the same domain throughout all stages examined (Fig 4B). Low level expression also occurs in the anterior gut (unpublished data). At no time is hh expressed in a dorsal or ventral midline domain, for example, close to the netrin ventral domain. The nk2-2 gene (also called nk2.2 and nkx2.2) of S. kowalevskii was cloned by low stringency hybridization. During gastrulation, it is expressed in the archenteron endoderm (Fig 4C) rather than ectoderm, as has also been found in amphioxus, though not other chordates [88]. Expression in S. kowalevskii is excluded from the anterior mesendoderm that becomes the prosome mesoderm, and the level of expression is reduced in the posterior endoderm. During days 2 and 3 of development, nk2-2 expression is further down-regulated in in the posterior and midline endoderm, leaving by day 3.5 (Fig 4D) a left and right patch of expression in the pharyngeal endoderm, ventral to the site of gill pore formation. At no stage was nk2-2 expressed in an ectodermal dorsal or ventral midline domain. Thus, like hh of S. kowalevksii, the nk2-2 gene, which in chordates depends on Hh signaling for expression, is not expressed in a chordate-like ectodermal midline domain. Page 1615: Nonetheless hemichordates use the Bmp-Chordin axis for a second phase, the “morphogenetic phase” for patterning the three germ layers. The patterning of neuronal cell types within the diffuse nervous system is included in the patterning, for example, the giant neurons near the dorsal midline (the Bmp pole); a nerve tract rich in motor neuron axons at the ventral midline, a specialized patch of sensory neurons near the mouth, and a line of poxN expressing cells, perhaps specialized neurons, on the posterior dorsal midline. However, chordates appear to achieve much more patterning of neuronal cell types than do hemichordates. Indeed, the entire Bmp-Hh double gradient used by chordates to select and place ten gene expression domains in the dorsoventral axis of the neural tube [96] is absent from hemichordates. Hh is localized to the apical ectoderm and anterior gut in hemichordates; it has no dorsoventral expression. Whereas the neural tube domains of chordates include those of pax6, dbx, en, irx, nk2.2, and msx, none of these genes shows a dorsoventral ectodermal midline domain in S. kowalevskii (see Results here and in [45]). Yet all of these are expressed in the hemichordate embryo in anteroposterior domains, as they are in chordates in domains additional to the dorsoventral domains. Particularly noteworthy are the nk2.2 and msx domains since these are thought to have similar expression in Drosophila neurectoderm and the chordate neural ectoderm of the neural tube [15], with roles in neural patterning. Yet neither gene has a dorsal or ventral ectodermal domain in S. kowalevskii. These differences suggest that much of the regulatory architecture involved in dorsoventral patterning of chordate nervous system evolved subsequent to the divergence of hemichordates and chordates. If true, the dorsoventral axis would have been a locus of much more evolution in chordates than was the anteroposterior axis since, as we showed previously [45], the gene expression domains of this axis are extensively similar in both groups, hence in the deuterostome ancestor.