Xenacoelomorph-Specific Hox Peptides: Insights into the Phylogeny of Acoels, Nemertodermatids, and Xenoturbellids

Xenacoelomorpha has recently been proposed as an animal taxon that includes acoels, nemertodermatids, and xenoturbellids. Their flattened bodies are very simple and lack discrete organs. The Acoela and Nemertodermatida (which comprise Acoelomorpha) were traditionally regarded as early-diverged extant orders of the class Turbellaria of the phylum Platyhelminthes. Recent anatomical studies and molecular phylogenetic studies demonstrate that the two groups belong to the phylum Xenacoelomorpha together with Xenoturbellida. However, debate remains in regard to whether Xenacoelomorpha is monophyletic, and whether xenacoelomorphs are sisters to all other bilaterians or have close affinity to ambulacrarians. The present study addresses the first question by examining the presence or absence of diagnostic peptide sequences shared by the three taxa. Hox genes have been used to investigate the phylogenetic relationships of metazoans. It has been shown that lophotrochozoans, rotifers, and chaetognaths share diagnostic peptide sequences in the C-terminal region of the Lox5 (Hox5/6/7) homeodomain proteins, which supports the clustering of these taxa. Examination of the decoded genome of the acoel Praesagittifera naikaiensis and reported xenacoelomorph Hox genes revealed that acoels share a peptide NLK(S/T)MSQ(V/I)D, which starts immediately after the homeodomain sequence of the central Hox4/5/6. In addition, we found another diagnostic peptide, KEGKL, in the C-terminal region of the anterior Hox1, which is shared by all the three groups of xenacoelomorphs, but not other bilaterians. Furthermore, two acoels, Praesagittifera naikaiensis and Symsagittifera roscoffensis, share another peptide SG(A/P)-PGM in the posterior Hox9/11/13. These results support the designation of the phylum Xenacoelomorpha, in which Acoela is a discrete group

A draft nuclear-genome assembly of the acoel flatworm Praesagittifera naikaiensis

Background:Acoels are primitive bilaterians with very simple soft bodies, in which many organs, including the gut, are not developed. They provide platforms for studying molecular and developmental mechanisms involved in the formation of the basic bilaterian body plan, whole-body regeneration, and symbiosis with photosynthetic microalgae. Because genomic information is essential for future research on acoel biology, we sequenced and assembled the nuclear genome of an acoel, Praesagittifera naikaiensis.Findings:To avoid sequence contamination derived from symbiotic microalgae, DNA was extracted from embryos that were free of algae. More than 290x sequencing coverage was achieved using a combination of Illumina (paired-end and mate-pair libraries) and PacBio sequencing. RNA sequencing and Iso-Seq data from embryos, larvae, and adults were also obtained. First, a preliminary ∼17–kilobase pair (kb) mitochondrial genome was assembled, which was deleted from the nuclear sequence assembly. As a result, a draft nuclear genome assembly was ∼656 Mb in length, with a scaffold N50 of 117 kb and a contig N50 of 57 kb. Although ∼70% of the assembled sequences were likely composed of repetitive sequences that include DNA transposons and retrotransposons, the draft genome was estimated to contain 22,143 protein-coding genes, ∼99% of which were substantiated by corresponding transcripts. We could not find horizontally transferred microalgal genes in the acoel genome. Benchmarking Universal Single-Copy Orthologs analyses indicated that 77% of the conserved single-copy genes were complete. Pfam domain analyses provided a basic set of gene families for transcription factors and signaling molecules.Conclusions:Our present sequencing and assembly of the P. naikaiensis nuclear genome are comparable to those of other metazoan genomes, providing basic information for future studies of genic and genomic attributes of this animal group. Such studies may shed light on the origins and evolution of simple bilaterians

Transcriptomic evidence for Brachyury expression in the caudal tip region of adult Ptychodera flava (Hemichordata)

Most metazoans have a single copy of the T-box transcription factor gene Brachyury. This gene is expressed in cells of the blastopore of late blastulae and the archenteron invagination region of gastrulae. It appears to be crucial for gastrulation and mesoderm differentiation of embryos. Although this expression pattern is shared by most deuterostomes, Brachyury expression has not been reported in adult stages. Here we show that Brachyury of an indirect developer, the hemichordate acorn worm Ptychodera flava, is expressed not only in embryonic cells, but also in cells of the caudal tip (anus) region of adults. This spatially restricted expression, shown by whole-mount in situ hybridization, was confirmed by Iso-Seq RNA sequencing and single-cell RNA-seq (scRNA-seq) analysis. Iso-Seq analysis showed that gene expression occurs only in the caudal region of adults, but not in anterior regions, including the stomochord. scRNA-seq analysis showed a cluster that contained Brachyury-expressing cells comprising epidermis- and mesoderm-related cells, but which is unlikely to be associated with the nervous system or muscle. Although further investigation is required to examine the roles of Brachyury in adults, this study provides important clues for extending studies on Brachyury expression involved in development of the most posterior region of deuterostomes.journal articl

Regeneration in the Hemichordate Ptychodera flava

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worm's body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.Zoological Science Award 2011(日本動物学会2011年度論文賞)受賞論

Ancestral Stem Cell Reprogramming Genes Active in Hemichordate Regeneration

Hemichordate enteropneust worms regenerate extensively in a manner that resembles the regeneration for which planaria and hydra are well known. Although hemichordates are often classified as an extant phylogenetic group that may hold ancestral deuterostome body plans at the base of the deuterostome evolutionary line leading to chordates, mammals, and humans, extensive regeneration is not known in any of these more advanced groups. Here we investigated whether hemichordates deploy functional homologs of canonical Yamanaka stem cell reprogramming factors, Oct4, Sox2, Nanog, and Klf4, as they regenerate. These reprogramming factors are not expressed during regeneration of limbs, fins, eyes or other structures that represent the best examples of regeneration in chordates. We first examined Ptychodera flava EST libraries and identified Pf-Pou3, Pf-SoxB1, Pf-Msxlx, and Pf-Klf1/2/4 as most closely related to the Yamanaka factors, respectively. In situ hybridization analyses revealed that all these homologs are expressed in a distinct manner during head regeneration. Furthermore, Pf-Pou3 partially rescued the loss of endogenous Oct4 in mouse embryonic stem cells in maintaining the pluripotency gene expression program. Based on these results, we propose that hemichordates may have co-opted these reprogramming factors for their extensive regeneration or that chordates may have lost the ability to mobilize these factors in response to damage. The robustness of these pluripotency gene circuits in the inner cell mass and in formation of induced pluripotent stem cells from mammalian somatic cells shows that these programs are intact in humans and other mammals and that these circuits may respond to as yet unknown gene regulatory signals, mobilizing full regeneration in hemichordates