Evolutionary modification of mouth position in deuterostomes

In chordates, the oral ectoderm is positioned at the anterior neural boundary and is characterized by pituitary homeobox (Pitx) and overlapping Dlx and Six3 expressions. Recent studies have shown that the ectoderm molecular map is also conserved in hemichordates and echinoderms. However, the mouth develops in a more posterior position in these animals, in a domain characterized by Nkx2.1 and Goosecoid expression, in a manner similar to that observed in protostomes. Furthermore, BMP signaling antagonizes mouth development in echinoderms and hemichordates, but seems to promote oral ectoderm specification in chordates. Conversely, Nodal signaling appears to be required for oral ectoderm specification in sea urchins but not in chordates. The Nodal/BMP antagonism at work during ectoderm patterning thus seems to constitute a conserved feature in deuterostomes, and mouth relocation may have been accompanied by a change in the influence of BMP/Nodal signals on oral ectoderm specification. We suggest that the mouth primordium was located at the anterior neural boundary, in early chordate evolution. In extant chordate embryos, subsequent mouth positioning differ between urochordates and vertebrates, presumably as a consequence of surrounding tissues remodelling. We illustrate these morphogenetic movements by means of morphological data obtained by the confocal imaging of ascidian tailbud embryos, and provide a table for determining the tailbud stages of this model organism.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.     

Pitx homeobox genes in Ciona and amphioxus show left-right asymmetry is a conserved chordate character and define the ascidian adenohypophysis

All vertebrates have directional asymmetries in the organization of their internal organs. In jawed vertebrates, development of asymmetry is controlled by a conserved molecular pathway that includes Pitx2, which is expressed by lateral plate mesoderm cells on the left side of the embryo. Pitx2 is a member of the Pitx homeobox gene family, the expression of which also marks stomodeal ectoderm and the adenohypophysis. Here we report the characterization of Pitx genes from Branchiostoma floridae (an amphioxus) and Ciona intestinalis (a urochordate), representatives of two basal chordate lineages and successively deeper outgroups to the vertebrates. Expression of B. floridae Pitx is similar to that reported from B. belcheri, a different amphioxus species. Expression of the Ciona Pitx ortholog in the embryonic primordial pharynx and adult neural complex leads us to propose the Ciona primordial pharynx and ciliated funnel are homologous to the adenohypophyseal placode and adenohypophysis, respectively. Additionally, in both species we identify asymmetrical left-sided expression of Pitx genes during embryonic development. This shows that asymmetrical Pitx gene expression, and by inference directional asymmetry, evolved before the radiation of living chordates and should be considered a chordate character.     

Early development of coelomic structures in an echinoderm larva and a similarity with coelomic structures in a chordate embryo

Early coelomic development in the abbreviated development of the sea urchin Holopneustes purpurescens is described and then used in a comparison with coelomic development in chordate embryos to support homology between a single arm of the five-armed radial body plan of an echinoderm and the single bilateral axis of a chordate. The homology depends on a positional similarity between the origin of the hydrocoele in echinoderm development and the origin of the notochord in chordate development, and a positional similarity between the respective origins of the coelomic mesoderm and chordate mesoderm in echinoderm and chordate development. The hydrocoele is homologous with the notochord and the secondary podia are homologous with the somites. The homology between a single echinoderm arm and the chordate axis becomes clear when the aboral to oral growth from the archenteron in the echinoderm larva is turned anteriorly, more in line with the anterior–posterior axis of the early zygote. A dorsoventral axis inversion in chordates is not required in the proposed homology.     

Building divergent body plans with similar genetic pathways

Deuterostome animals exhibit widely divergent body plans. Echinoderms have either radial or bilateral symmetry, hemichordates include bilateral enteropneust worms and colonial pterobranchs, and chordates possess a defined dorsal-ventral axis imposed on their anterior-posterior axis. Tunicates are chordates only as larvae, following metamorphosis the adults acquire a body plan unique for the deuterostomes. This paper examines larval and adult body plans in the deuterostomes and discusses two distinct ways of evolving divergent body plans. First, echinoderms and hemichordates have similar feeding larvae, but build a new adult body within or around their larvae. In hemichordates and many direct-developing echinoderms, the adult is built onto the larva, with the larval axes becoming the adult axes and the larval mouth becoming the adult mouth. In contrast, indirect-developing echinoderms undergo radical metamorphosis where adult axes are not the same as larval axes. A second way of evolving a divergent body plan is to become colonial, as seen in hemichordates and tunicates. Early embryonic development and gastrulation are similar in all deuterostomes, but, in chordates, the anterior-posterior axis is established at right angles to the animal-vegetal axis, in contrast to hemichordates and indirect-developing echinoderms. Hox gene sequences and anterior-posterior expression patterns illuminate deuterostome phylogenetic relationships and the evolution of unique adult body plans within monophyletic groups. Many genes that are considered vertebrate 'mesodermal' genes, such as nodal and brachyury T, are likely to ancestrally have been involved in the formation of the mouth and anus, and later were evolutionarily co-opted into mesoderm during vertebrate development.     

The deuterostome ancestor

Hemichordates, the phylum of bilateral animals closest to chordates, can illuminate the evolutionary origins of various chordate traits to determine whether these were already present in a shared ancestor (the deuterostome ancestor) or were evolved within the chordate line. We find that an anteroposterior map of gene expression domains, representing 42 genes of neural patterning, is closely similar in hemichordates and chordates, though it is restricted to the neural ectoderm in chordates whereas in hemichordates, which have a diffuse nervous system, it encircles the whole body. This map allows an accurate alignment of the anterioposterior axes of members of the two groups. We propose that this map dates back at least to the deuterostome ancestor. The map of dorsoventral expression domains, organized along a Bmp-Chordin developmental axis, is also similar in the two groups in terms of many gene expression domains and for the placement of the gill slits, heart, and post-anal tail. The two groups, however, differ in two major respects along this axis. The nervous system and epidermis are not segregated into distinct territories in hemichordates, as they are in chordates, and furthermore, the mouth is on the Chordin side in hemichordates but the Bmp side in chordates. The dorsoventral dimension has undergone extensive modification in the chordate line, including centralization of the nervous system, segregation of epidermis, derivation of the notochord, perhaps from the gut midline, and relocation of the mouth. Based on the shared domain maps, speculations can be made for the remodeling of the body axis in the chordate line.     

Anteroposterior patterning in hemichordates and the origins of the chordate nervous system

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, we examined the hemichordate Saccoglossus kowalevskii and studied the expression of orthologs of genes that are involved in patterning the chordate central nervous system. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. We propose that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage.     

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.     

Cloning and expression of a Pitx homeobox gene from the lamprey,a jawless vertebrate

The Pitx homeobox gene family has important roles in vertebrate pituitary, eye, branchial arch, hindlimb and brain development, as well as a key function in regulating left-right asymmetry. Here we report the isolation of a Pitx gene, PitxA, from two lamprey species, Lampetra planeri and Petromyzon marinus. Molecular phylogenetics show PitxA is most closely related to the Pitx1 and Pitx2 genes of jawed vertebrates, however resolution in the trees is insufficient to determine if PitxA is orthologous to a specific jawed vertebrate gene. In situ hybridisation studies show lamprey PitxA is expressed in the developing nasohypohyseal system and stomodeal ectoderm from early development through to early ammocoette larvae. PitxA expression was also detected in several areas of the developing brain, in the developing optic system, in pharyngeal endoderm and endostyle and in the lateral somite. These results show some key aspects of Pitx gene expression in gnathostomes are primitive for all living vertebrates.     

Retinoids and nonvertebrate chordate development

Retinoic acid (RA) is required for the differentiation and morphogenesis of chordate-specific features, such as the antero-posterior regionalization of the dorsal hollow nerve cord and neural crest cells. RA receptors (RARs) have been reported exclusively in chordates, suggesting that the acquisition of the RAR gene was important for chordate evolution. A scenario is presented here for the establishment of an RAR-mediated developmental regulatory system during the course of chordate evolution. In the common chordate ancestor, RAR came to control the spatial expression pattern of Hox genes in the ectoderm and endoderm along the antero-posterior axis. In these germ layers, RA was required for the differentiation of epidermal sensory neurons and the morphogenesis of pharyngeal gill slits, respectively. As the diffuse epidermal nerve net in the chordate ancestor became centralized to form the dorsal nerve cord, the epidermal Hox expression pattern was carried into the central nervous system. Because the Hox code here came to specify neuronal identity along the antero-posterior axis, RA became inextricably linked to the antero-posterior patterning of the chordate central nervous system.     

Ion flow regulates left–right asymmetry in sea urchin development

The degree of conservation among phyla of early mechanisms that pattern the left–right (LR) axis is poorly understood. Larvae of sea urchins exhibit consistently oriented LR asymmetry. The main part of the adult rudiment is formed from the left coelomic sac of larvae, the left hydrocoel. Although this left preference is conserved among all echinoderm larvae, its mechanism is largely not understood. Using two marker genes, HpNot and HpFoxFQ-like, which are asymmetrically expressed during larval development of the sea urchin Hemicentrotus pulcherrimus, we examined in this study the possibility that the recently discovered ion flux mechanism controls asymmetry in sea urchins as it does in several vertebrate species. Several ion-transporter inhibitors were screened for the ability to alter the expression of the asymmetric marker genes. Blockers of the H⁺/K⁺-ATPase (omeprazole, lansoprazole and SCH28080), as well as a calcium ionophore (A23187), significantly altered the normal sidedness of asymmetric gene expression. Exposure to omeprazole disrupted the consistent asymmetry of adult rudiment formation in larvae. Immuno-detection revealed that H⁺/K⁺-ATPase-like antigens in sea urchin embryos were present through blastula stage and exhibited a striking asymmetry being present in a single blastomere in 32-cell embryos. These results suggest that, as in vertebrates, endogenous spatially-regulated early transport of H⁺ and/or K⁺, and also of Ca²⁺, functions in the establishment of LR asymmetry in sea urchin development.Electronic Supplementary Material Supplementary material is available for this article at     

Opposing Nodal and BMP Signals Regulate Left–Right Asymmetry in the Sea Urchin Larva

Nodal and BMP signals are important for establishing left-right (LR) asymmetry in vertebrates. In sea urchins, Nodal signaling prevents the formation of the rudiment on the right side. However, the opposing pathway to Nodal signaling during LR axis establishment is not clear. Here, we revealed that BMP signaling is activated in the left coelomic pouch, specifically in the veg2 lineage, but not in the small micromeres. By perturbing BMP activities, we demonstrated that BMP signaling is required for activating the expression of the left-sided genes and the formation of the left-sided structures. On the other hand, Nodal signals on the right side inhibit BMP signaling and control LR asymmetric separation and apoptosis of the small micromeres. Our findings show that BMP signaling is the positive signal for left-sided development in sea urchins, suggesting that the opposing roles of Nodal and BMP signals in establishing LR asymmetry are conserved in deuterostomes.     

Ventralization of an indirect developing hemichordate by NiCl₂ suggests a conserved mechanism of dorso-ventral (D/V) patterning in Ambulacraria (hemichordates and echinoderms)

One of the earliest steps in embryonic development is the establishment of the future body axes. Morphological and molecular data place the Ambulacraria (echinoderms and hemichordates) within the Deuterostomia and as the sister taxon to chordates. Extensive work over the last decades in echinoid (sea urchins) echinoderms has led to the characterization of gene regulatory networks underlying germ layer specification and axis formation during embryogenesis. However, with the exception of recent studies from a direct developing hemichordate (Saccoglossus kowalevskii), very little is known about the molecular mechanism underlying early hemichordate development. Unlike echinoids, indirect developing hemichordates retain the larval body axes and major larval tissues after metamorphosis into the adult worm. In order to gain insight into dorso-ventral (D/V) patterning, we used nickel chloride (NiCl?), a potent ventralizing agent on echinoderm embryos, on the indirect developing enteropneust hemichordate, Ptychodera flava. Our present study shows that NiCl? disrupts the D/V axis and induces formation of a circumferential mouth when treated before the onset of gastrulation. Molecular analysis, using newly isolated tissue-specific markers, shows that the ventral ectoderm is expanded at expense of dorsal ectoderm in treated embryos, but has little effect on germ layer or anterior-posterior markers. The resulting ventralized phenotype, the effective dose, and the NiCl? sensitive response period of Ptychodera flava, is very similar to the effects of nickel on embryonic development described in larval echinoderms. These strong similarities allow one to speculate that a NiCl? sensitive pathway involved in dorso-ventral patterning may be shared between echinoderms, hemichordates and a putative ambulacrarian ancestor. Furthermore, nickel treatments ventralize the direct developing hemichordate, S. kowalevskii indicating that a common pathway patterns both larval and adult body plans of the ambulacrarian ancestor and provides insight in to the origin of the chordate body plan.     

The Early Phylogeny of Chordates and Echinoderms and the Origin of Chordate Left–Right Asymmetry and Bilateral Symmetry

Abstract Left–right asymmetry in Dexiothetica (= echinoderms + chordates) results mainly from dexiothetism—an episode in their ancestry when an animal resembling the Recent pterobranch Cephalodiscus lay right-side-downwards on the sea floor. Castericystis sprinklei belongs to the dexiothete stem group. The history of the echinoderm stem group is reconstructed. Chordate bilateral symmetry evolved by six successive steps. Tail–head overlap occurred independently in craniates and acraniates. The neural crest would have existed in the latest common ancestor of extant chordates, or even earlier. Gross asymmetries occur in extant chordates in organs derived from the calcichordate head, but not in those derived from the calcichordate tail. The anterior boundary of hox gene expression in vertebrates corresponds to the anterior end of the calcichordate tail. Left–right organ pairing (an important step in the origin of chordate bilateral symmetry) may have involved the interaction of a symmetrizing morphogen, produced from the anterior end of the tail, with a lateral morphogen (Wilhelmi's morphogen), produced in ontogeny at first from the left and later from the right. This mechanism may still act in the metamorphosis of amphioxus and in mirror-imaging in vertebrate twins. Wilhelmi's morphogen may be related to one or more members of the dorsal cascade of Drosophila.     

Left-right lineage analysis of the embryonic Xenopus heart reveals a novel framework linking congenital cardiac defects and laterality disease

The significant morbidity and mortality associated with laterality disease almost always are attributed to complex congenital heart defects (CHDs), reflecting the extreme susceptibility of the developing heart to disturbances in the left-right (LR) body plan. To determine how LR positional information becomes ;translated' into anatomical asymmetry, left versus right side cardiomyocyte cell lineages were traced in normal and laterality defective embryos of the frog, Xenopus laevis. In normal embryos, myocytes in some regions of the heart were derived consistently from a unilateral lineage, whereas other regions were derived consistently from both left and right side lineages. However, in heterotaxic embryos experimentally induced by ectopic activation or attenuation of ALK4 signaling, hearts contained variable LR cell composition, not only compared with controls but also compared with hearts from other heterotaxic embryos. In most cases, LR cell lineage defects were associated with abnormal cardiac morphology and were preceded by abnormal Pitx2c expression in the lateral plate mesoderm. In situs inversus embryos there was a mirror image reversal in Pitx2c expression and LR lineage composition. Surprisingly, most of the embryos that failed to develop heterotaxy or situs inversus in response to misregulated ALK4 signaling nevertheless had altered Pitx2c expression, abnormal cardiomyocyte LR lineage composition and abnormal heart structure, demonstrating that cardiac laterality defects can occur even in instances of otherwise normal body situs. These results indicate that: (1) different regions of the heart contain distinct LR myocyte compositions; (2) LR cardiomyocyte lineages and Pitx2c expression are altered in laterality defective embryos; and (3) abnormal LR cardiac lineage composition frequently is associated with cardiac malformations. We propose that proper LR cell composition is necessary for normal morphogenesis, and that misallocated LR cell lineages may be causatively linked with CHDs that are present in heterotaxic individuals, as well as some 'isolated' CHDs that are found in individuals lacking overt features of laterality disease.     

Hemichordates and the origin of chordates

Hemichordates, the phylum of bilateral animals most closely related to chordates, could reveal the evolutionary origins of chordate traits such as the nerve cord, notochord, gill slits and tail. The anteroposterior maps of gene expression domains for 38 genes of chordate neural patterning are highly similar for hemichordates and chordates, even though hemichordates have a diffuse nerve-net. About 40% of the domains are not present in protostome maps. We propose that this map, the gill slits and the tail date to the deuterostome ancestor. The map of dorsoventral expression domains, centered on a Bmp-Chordin axis, differs between the two groups; hemichordates resemble protostomes more than they do chordates. The dorsoventral axis might have undergone extensive modification in the chordate line, including centralization of the nervous system, segregation of epidermis, derivation of the notochord, and an inversion of organization.