Mesodermal gene expression in the acoel isodiametra pulchra indicates a low number of mesodermal cell types and the endomesodermal origin of the gonads

Acoelomorphs are bilaterally symmetric small marine worms that lack a coelom and possess a digestive system with a single opening. Two alternative phylogenetic positions of this group within the animal tree are currently debated. In one view, Acoelomorpha is the sister group to all remaining Bilateria and as such, is a morphologically simple stepping stone in bilaterian evolution. In the other, the group is a lineage within the Deuterostomia, and therefore, has derived a simple morphology from a more complex ancestor. Acoels and the closely related Nemertodermatida and Xenoturbellida, which together form the Acoelomorpha, possess a very limited number of cell types. To further investigate the diversity and origin of mesodermal cell types we describe the expression pattern of 12 orthologs of bilaterian mesodermal markers including Six1/2, Twist, FoxC, GATA4/5/6, in the acoel Isodiametra pulchra. All the genes are expressed in stem cells (neoblasts), gonads, and at least subsets of the acoel musculature. Most are expressed in endomesodermal compartments of I. pulchra developing embryos similar to what has been described in cnidarians. Our molecular evidence indicates a very limited number of mesodermal cell types and suggests an endomesodermal origin of the gonads and the stem cell system. We discuss our results in light of the two prevailing phylogenetic positions of Acoelomorpha

The nervous system of Xenacoelomorpha: a genomic perspective

Xenacoelomorpha is, most probably, a monophyletic group that includes three clades: Acoela, Nemertodermatida and Xenoturbellida. The group still has contentious phylogenetic affinities; though most authors place it as the sister group of the remaining bilaterians, some would include it as a fourth phylum within the Deuterostomia. Over the past few years, our group, along with others, has undertaken a systematic study of the microscopic anatomy of these worms; our main aim is to understand the structure and development of the nervous system. This research plan has been aided by the use of molecular/developmental tools, the most important of which has been the sequencing of the complete genomes and transcriptomes of different members of the three clades. The data obtained has been used to analyse the evolutionary history of gene families and to study their expression patterns during development, in both space and time. A major focus of our research is the origin of 'cephalized' (centralized) nervous systems. How complex brains are assembled from simpler neuronal arrays has been a matter of intense debate for at least 100 years. We are now tackling this issue using Xenacoelomorpha models. These represent an ideal system for this work because the members of the three clades have nervous systems with different degrees of cephalization; from the relatively simple sub-epithelial net of Xenoturbella to the compact brain of acoels. How this process of 'progressive' cephalization is reflected in the genomes or transcriptomes of these three groups of animals is the subject of this paper

The evolution of bilaterian body-plan: perspectives from the developmental genetics of the Acoela (Aeoelomorpha)

[spa] Los acelos son unos gusanos, principalmente marinos, de simetría bilateral y aplastados según el eje dorso ventral, que pertenecen al grupo de los acelomorfos (acelos +nemertodermatidos+xenoturbellidos), cuya posición filogenética es tema de debate entre los biólogos evolucionistas. Los acelomorfos carecen de cavidades corporales, su sistema digestivo es ciego y su sistema nervioso consiste de una concentración neuronal anterior y cuerda nerviosas no claramente desplazadas hacía el lado dorsal o ventral. La simplicidad morfológica de los acelos, entremedia entre la de cnidarios y bilaterales superiores, les hace buenos candidatos para el estudio de la transición de animales radiales-diploblastos a bilaterales-triploblastos. En esta tesis se presentan datos sobre el desarrollo e la especificación molecular del mesodermo, que ha sido una de las innovaciones claves para la radiación de los bilaterales. S. roscoffensis que como todos los acelos tiene exclusivamente musculatura de tipo liso, expresa un gen ortólogo a la troponina, un proteína clave para la regulación de los músculos estriados, y que no existe en cnidarios. La explicación más parsimoniosa es que las bases moleculares de evolución de músculos estriados se han implantado en los acelos, aunque estos no hayan alcanzado la condición completa (explicación favorecida si los acelomorfos son confirmados como grupo hermano de los demás bilaterales). Por otra parte se puede considerar esta condición como debida a una reducción secundaria (explicación favorecida en el caso que los acelos se confirmen ser deuteróstomos). Los ortólogos de genes endodermales de cnidarios y con clara expresión mesodermal en bilaterales se expresan en la músculatura del acelo l. pulchra. Estos datos concuerdan perfectamente con la evolución del mesodermo a partir del endodermo de animales diploblásticos. Aun así, es difícil proponer un modelo específico de evolución de miocitos hasta que la posición filogenética de los acelomorfos no esté resuelta.[eng] The mesoderm is the third germ layer, which is formed at gastrulation between the endoderm and the ectoderm in triploblastsc animals (Bilateria). The mesoderm differentiates into muscles, connective tissues and coelomic cavities. These structures have been key evolutionary innovations that prompted the enormous radiation of the bilaterians that at present make up for the 90% of animal species. As such, understanding the evolution of the mesoderm and its derivatives it is pivotal to understand the evolution of animals. In this thesis I have characterized the molecular patterning of the mesoderm and its derivatives (mainly muscles) in two different acoel species: Symsagittifera roscoffensis and Isodiametra pulchra. The acoels belong to the phylum Acoelomorpha (togheter with nemertodermatids and Xenoturbella). The phylogenetic placement of the Acoelomorpha is highly debated between a position basal to the bilaterians or nested inside the deuterostomes. The Acoelomorpha are morphologically simple animals and a trait sometimes considered a direct link to the cnidarians, the Bilateria sister group. With them they the acoelomorphs share a blind gut and a non centralized nervous system. Within the acoelomorphs, the acoels present the most derived body plan, however it is still rather simple if compared to other bilaterians. The nervous system for example is condensed anteriorly but not clear dorso ventral centralization exists as in most of the remaining bilaterians (the nerve cords are distributed circumferentially around the body). The mesoderm only develops from endodermal precursors, and this might be ancestral, since it is thought that the mesoderm evolved from the endoderm of a diploblastic, cnidarian-like ancestor. The muscles are the only mesodermal derivative in most basal acoelomorphs taxa, although in more advanced ones a parenchymal tissue, stem cells, and gonads also occupy the mesodermal space. The embryonic origins of the latter though, are at present still unknown. Thus acoelomorphs present most of traits considered to be eumetazoan ancestral traits (i.e. most of traits are also part of the cnidarians ground pattern), but still that the possibility that their body plan evolved in consequence of a secondary reduction must be considered as they could be more related to other deuterostomes than cnidarians. I have first investigated the molecular architecture of the muscles in the acoel Symsagittifera roscoffensis and found that although they have a smooth ultrastructural aspect they are molecularly more similar to the bilaterian striated muscles given that tey express key genes that control the contraction in the striated cells. This could be considered a first step into the evolution of the striated musculature without fully reaching it. Indeed, cnidarians have smooth epithelio muscular cells likely regulated by the same bilaterian smooth muscle proteins. However, the possibility of a secondary loss of the striation pattern cannot be discarded given that this already happened in some other bilaterians. Second, I have analyzed the expression of bilaterian mesodermal genes during embryogenesis and postembryonic development of Isodiametra pulchra and found that all but one (a FoxA ortholog) are expressed at the anterior pole, the site where the first myocytes start to differentiate. In juveniles and adults these genes are all expressed in muscles or at least a subset of them. Moreover the same genes are expressed in the gonads of I. pulchra and therefore it suggests that they could orginate in the endo-mesoderm of the worm. The cnidarians orthologues of these genes are expressed in the endoderm, which is moreover the site of the gametes differentation. The similarity between cnidarians endoderm and acoels mesoderm are astonishing, however before drawing conclusions we need a solid phylogenetic fram

Mesodermal gene expression in the acoel isodiametra pulchra indicates a low number of mesodermal cell types and the endomesodermal origin of the gonads

Acoelomorphs are bilaterally symmetric small marine worms that lack a coelom and possess a digestive system with a single opening. Two alternative phylogenetic positions of this group within the animal tree are currently debated. In one view, Acoelomorpha is the sister group to all remaining Bilateria and as such, is a morphologically simple stepping stone in bilaterian evolution. In the other, the group is a lineage within the Deuterostomia, and therefore, has derived a simple morphology from a more complex ancestor. Acoels and the closely related Nemertodermatida and Xenoturbellida, which together form the Acoelomorpha, possess a very limited number of cell types. To further investigate the diversity and origin of mesodermal cell types we describe the expression pattern of 12 orthologs of bilaterian mesodermal markers including Six1/2, Twist, FoxC, GATA4/5/6, in the acoel Isodiametra pulchra. All the genes are expressed in stem cells (neoblasts), gonads, and at least subsets of the acoel musculature. Most are expressed in endomesodermal compartments of I. pulchra developing embryos similar to what has been described in cnidarians. Our molecular evidence indicates a very limited number of mesodermal cell types and suggests an endomesodermal origin of the gonads and the stem cell system. We discuss our results in light of the two prevailing phylogenetic positions of Acoelomorpha

Expression of orthologs of bilaterian mesodermal genes that are broadly expressed in <i>I. Pulchra.</i>

<p>In the left panel whole-mount <i>in situ</i> hybridization of juvenile (left column) and adult (central and right columns) specimens are shown. Expression in the specimens in the right column is detected with fluorescent signal (purple). The arrow points to the female genital organ, arrowhead to the male genital organ. The asterisk indicates the cross parenchymal muscles. In the right panel the neoblasts localization of the transcript is shown. Left columns all show double stained worms with EdU and with the fluorescent antisense probe. The center and right columns show RNA transcripts and neoblasts staining alone. All aspects show a single confocal plane. The empty arrowheads point to colocalized EdU-RNA signal. Anterior to the left, scale bar 50 µm in all aspects<b>. A–F</b>. <i>IpmuscleLIM</i> expression. hm: head myocytes; nb/go: neoblasts and gonads pa:parenchymal cells; ds: digestive syncytium. <b>D–L</b>. <i>IpPitx</i> expression. The mouth is surrounded by specialized ring muscles (m) that express <i>IpPitx</i> (<b>H</b> and <b>I</b>). <b>M–R </b><i>IpFoxA1</i> expression. <i>IpFoxA1</i> is expressed in the endodermal digestive syncytium (ds) as well as in the mesodermal musculature (<i>e.g.</i>, the mouth ring muscles, <b>N</b>). The black empty arrowheads in <b>M</b> and <b>N,</b> show paired muscles associated to the copulatory organs. <b>S–X</b>. <i>IpFoxC</i> expression.</p

Different scenarios about mesoderm evolution depending on the phylogenetic position of Acoelomorpha.

<p>Two possible phylogenetic positions of Acoelomorpha either as sister to the remaining Bilateria (scenarios 1 and 2) or as sister group to Ambulacraria (scenarios 3 and 4). Musculature in red. Four possible scenarios are numbered. Scenario 1: A cnidarian-like ancestor with epithelial-muscle cells that form circular and longitudinal musculature form the orthogonal musculature of acoels. The musculature would be the first cell type of mesoderm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055499#pone.0055499-Smith2" target="_blank">[63]</a>. Scenario 2: A similar cnidarian-like ancestor is forming myoepithelial coelomic cavities as outpouchings from the gastric cavity (according to enterocoely hypothesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055499#pone.0055499-Ax1" target="_blank">[113]</a>). In the lineage to the Acoelomorpha the orthogonal muscle grid of acoels is formed from the coeloms. After the formation of the muscle grid coeloms got reduced. This scenario includes several losses and gains and is thus not parsimonious and can be rejected. Scenario 3: In case the last common ancestor of Deuterostomia had coeloms, the coeloms got reduced in the lineage to the Acoelomorpha without any traces <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055499#pone.0055499-Remane2" target="_blank">[12]</a>. Scenario 4: Coelomic cavities of Ambulacraria are not homologous with those in other animal lineages <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055499#pone.0055499-Philippe1" target="_blank">[22]</a> and are formed independently from the endoderm of a acoelomorph-like ancestor (<i>e.g.</i>, by enterocoely).</p

Mesodermal Gene Expression in the Acoel <em>Isodiametra pulchra</em> Indicates a Low Number of Mesodermal Cell Types and the Endomesodermal Origin of the Gonads

<div><p>Acoelomorphs are bilaterally symmetric small marine worms that lack a coelom and possess a digestive system with a single opening. Two alternative phylogenetic positions of this group within the animal tree are currently debated. In one view, Acoelomorpha is the sister group to all remaining Bilateria and as such, is a morphologically simple stepping stone in bilaterian evolution. In the other, the group is a lineage within the Deuterostomia, and therefore, has derived a simple morphology from a more complex ancestor. Acoels and the closely related Nemertodermatida and Xenoturbellida, which together form the Acoelomorpha, possess a very limited number of cell types. To further investigate the diversity and origin of mesodermal cell types we describe the expression pattern of 12 orthologs of bilaterian mesodermal markers including <em>Six1/2</em>, <em>Twist, FoxC</em>, <em>GATA4/5/6</em>, in the acoel <em>Isodiametra pulchra</em>. All the genes are expressed in stem cells (neoblasts), gonads, and at least subsets of the acoel musculature. Most are expressed in endomesodermal compartments of <em>I. pulchra</em> developing embryos similar to what has been described in cnidarians. Our molecular evidence indicates a very limited number of mesodermal cell types and suggests an endomesodermal origin of the gonads and the stem cell system. We discuss our results in light of the two prevailing phylogenetic positions of Acoelomorpha.</p> </div

Anatomy of adult <i>Isodiametra pulchra.</i>

<p>Gross anatomy and confocal images of the musculature. <b>A.</b> Living adult under Differential Interference Contrast, showing the statocyst (st), digestive syncytium (ds) and gonads (go). Positions of following inconspicuous structures are outlined: mouth (m), bursal nozzle (bn), postero-ventral female organ (for) and male genital organ (mo). <b>B–D.</b> Musculature of anterior, central and posterior regions of body visualized by fluorescent phalloidin labeling. Note the dense net of parenchymal muscles in the anterior region (B dashed line). Scale bars are 50 µm in all aspects.</p

Expression of mesodermal genes in <i>I. pulchra</i> neoblasts.

<p>Expression of mesodermal genes in <i>I. pulchra</i> neoblasts.</p