Hiroki Nishida

The larvacean Oikopleura dioica is a fast-developing chordate because of its small number of cells (∼4500 in juveniles) and rapid development to complete morphogenesis by 10 h after fertilization. Strikingly, most of its blastomeres are restricted to give rise to a single cell-type by the 32-cell stage of embryogenesis, unlike cell fate determination at the 110-cell stage in ascidians. In this study, RNA-sequencing (RNA-seq) revealed non-canonical properties of O. dioica: (1) an initial zygotic gene expression of 950 genes at the 16- to 32-cell stage; (2) 25 transcription factors (TFs) are expressed in the 32-cell stage (fewer than half of the TFs underlying gene regulatory networks in ascidian embryogenesis were lost or not expressed); (3) five maternal mRNAs localized in the vegetal-posterior blastomeres in animal and vegetal hemispheres; and (4) three maternal mRNAs localized in the small vegetal pole region of unfertilized eggs. These observations indicate that this fast-developing chordate lacks the first phase of development in ascidians: fertilization-driven ooplasmic movements that drive postplasmic RNAs toward the vegetal pole. These data have been deposited in ANISEED (https://www.aniseed.fr/) as transcriptome resources.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicateOikopleura dioicaaround the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes,Drosophila,or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation inO. dioicasuggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

Stem cell division contributes to the generation of various cell types during animal development, especially a diverse pool of neural cells in the nervous system. One example is reiterated unequal stem cell divisions, in which a large stem cell undergoes a series of oriented unequal divisions to produce a chain of small daughter cells that differentiate. We show that reiterated unequal stem cell divisions are involved in the formation of the brain in simple chordate appendicularians (larvaceans). Two large neuroblasts in the anterior and middle of the brain-forming region of hatched larvae were observed. They produced at least 30 neural cells out of 96 total brain cells before completion of brain formation at 10 hours after fertilization by reiterated unequal stem cell divisions. The daughter cells of the anterior neuroblast were postmitotic, and the number was at least 19. The neuroblast produced small daughter neural cells posteriorly every 20 min. The neural cells first moved toward the dorsal side, turned in the anterior direction, aligned in a single line according to their birth order, and showed collective movement to accumulate in the anterior part of the brain. The anterior neuroblast originated from the right-anterior blastomeres of the eight-cell embryos and the right a222 blastomere of the 64-cell embryo. The posterior neuroblast also showed reiterated unequal stem cell divisions, and generated at least 11 neural cells. Sequential unequal stem cell divisions without stem cell growth have been observed in protostomes, such as insects and annelids. The results provide the first examples of this kind of stem cell division during brain formation in non-vertebrate deuterostomes.

SUMMARY: Genes are not randomly distributed throughout chromosomes. How gene order evolves and how selective constraints act to preserve or vary gene order, both at the macrosyntenic level of whole chromosomes or microsyntenic level of gene blocks, are central questions of evolutionary biology and genomics that remain largely unsolved. Here, after sequencing several genomes of the appendicularian tunicate Oikopleura dioica from different locations around the globe, we show an unprecedented amount of genome scrambling in animals with no obvious morphological differences, consistent with cryptic speciation. Our assemblies suggest that all members of this clade possess a common 3-chromosome karyotype, and that different species largely preserve gene content, despite the presence of thousands of rearrangements in gene order. The movements of genes are largely restricted to chromosome arms and sex-specific regions, which appear to be the primary unit of macrosynteny conservation, and examples of these within-arm movements can be seen in the Hox and Fgf gene families. Our approach employing whole-genome alignments demonstrates that segments containing protein-coding elements tend to be preserved at the microsyntenic scale, consistent with strong purifying selection, with appreciably less preservation of non-coding elements. Unexpectedly, scrambling did not preserve operon structure across species, suggesting an absence of selective pressure to maintain operon structure. As well, genome scrambling does not occur uniformly across all chromosomes, as short chromosome arms possess shorter genes, smaller operons, more breakpoints, and elevated dN/dS values compared to long chromosome arms. Estimation of divergence times among the cryptic O. dioica lineages yielded an estimated breakpoint accumulation rate of 6 to 25 breakpoints per megabase per million years, which is an order of magnitude higher than the rates for other ascidian tunicates or Drosophila species. Therefore, O. dioica appears to be an attractive animal system to unravel the mechanisms that underlie gene order and synteny conservation, as well as exploring the limits of genome scrambling without an apparent impact on phenotypic evolution.

The larvaceanOikopleura dioicais a planktonic chordate and an emerging model organism with a short life cycle of 5 days that belongs toTunicata (Urochordata), the sister clade of vertebrates. It is characterized by the rapid development of a tadpole-shaped body. Organ formation in the trunk proceeds within 7 h after the hatching of the tailbud larvae at 3 h after fertilization (hpf) and is completed at 10 hpf, giving rise to fully functional juveniles as miniature adult form. Serial block face scanning electron microscopy was used to acquire ~ 2000 serial transverse section images of a 3 hpf larva and a 10 hpf juvenile to characterize the structures and cellular composition of the trunk and organs using 3D images and movies. Germ cells were found to fuse and establish a central syncytial cell in the gonad as early as 10 hpf. Larval development gave rise to functional organs after several rounds of cell division through trunk morphogenesis. The feature would makeO. dioicaideal for analyzing cellular behaviors during morphogenetic processes using live imaging. The detailed descriptions of the larvae and juveniles provided in this study can be utilized as the start and end points of organ morphogenesis in this rapidly developing organism.

Ascidians with very similar embryos but highly divergent genomes are thought to have undergone extensive developmental system drift. We compared, in four species (CionaandPhallusiafor Phlebobranchia,MolgulaandHalocynthiafor Stolidobranchia), gene expression and gene regulation for a network of six transcription factors regulating peripheral nervous system (PNS) formation inCiona. All genes, but one inMolgula, were expressed in the PNS with some differences correlating with phylogenetic distance. Cross-species transgenesis indicated strong levels of conservation, except inMolgula, in gene regulation despite lack of sequence conservation of the enhancers. Developmental system drift in ascidians is thus higher for gene regulation than for gene expression and is impacted not only by phylogenetic distance, but also in a clade-specific manner and unevenly within a network. Finally, considering thatMolgulais divergent in our analyses, this suggests deep conservation of developmental mechanisms in ascidians after 390 My of separate evolution.

Mouth formation involves the processes of mouth opening, formation of the oral cavity, and the development of associated sensory organs. In deuterostomes, the surface ectoderm and the anterior part of the archenteron are reconfigured and reconnected to make a mouth opening. This study of the larval development of the larvacean, Oikopleura dioica, investigates the cellular organization of the oral region, the developmental processes of the mouth, and the formation of associated sensory cells. O. dioica is a simple chordate whose larvae are transparent and have a small number of constituent cells. It completes organ morphogenesis in 7 h, between hatching 3 h after fertilization and the juvenile stage at 10 h, when it attains adult form and starts to feed. It has two types of mechanosensory cell embedded in the oral epithelium, which is a single layer of cells. There are twenty coronal sensory cells in the circumoral nerve ring and two dorsal sensory organ cells. Two bilateral lip precursor cells (LPCs), facing the anterior surface, divide dorsoventrally and make a wedge-shaped cleft between the two daughter cells named the dorsal lip cell (DLC) and the ventral lip cell (VLC). Eventually, the DLC and VLC become detached and separated into dorsal and ventral lips, triggering mouth opening. This is an intriguing example of cell division itself contributing to morphogenesis. The boundary between the ectoderm and endoderm is present between the lip cells and coronal sensory cells. All oral sensory cells, including dorsal sensory organ cells, were of endodermal origin and were not derived from the ectodermal placode. These observations on mouth formation provide a cellular basis for further studies at a molecular level, in this simple chordate.

Larvaceans are chordates with a tadpole-like morphology. In contrast to most chordates of which early embryonic morphology is bilaterally symmetric and the left–right (L–R) axis is specified by the Nodal pathway later on, invariant L–R asymmetry emerges in four-cell embryos of larvaceans. The asymmetric cell arrangements exist through development of the tailbud. The tail thus twists 90° in a counterclockwise direction relative to the trunk, and the tail nerve cord localizes on the left side. Here, we demonstrate that larvacean embryos have nonconventional L–R asymmetries: 1) L- and R-cells of the two-cell embryo had remarkably asymmetric cell fates; 2) Ca²⁺oscillation occurred through embryogenesis; 3)Nodal, an evolutionarily conserved left-determining gene, was absent in the genome; and 4) bone morphogenetic protein gene (Bmp) homologBmp.ashowed right-sided expression in the tailbud and larvae. We also showed that Ca²⁺oscillation is required forBmp.aexpression, and that BMP signaling suppresses ectopic expression of neural genes. These results indicate that there is a chordate species lackingNodalthat utilizes Ca²⁺oscillation andBmp.afor embryonic L–R patterning. The right-sideBmp.aexpression may have arisen via cooption of conventional BMP signaling in order to restrict neural gene expression on the left side.