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ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.

BackgroundAnalyzing close species with diverse developmental modes is instrumental for investigating the evolutionary significance of physiological, anatomical and behavioral features at a molecular level. Many examples of trait loss are known in metazoan populations living in dark environments. Tunicates are the closest living relatives of vertebrates and typically present a lifecycle with distinct motile larval and sessile adult stages. The nervous system of the motile larva contains melanized cells associated with geotactic and light-sensing organs. It has been suggested that these are homologous to vertebrate neural crest-derived melanocytes. Probably due to ecological adaptation to distinct habitats, several species of tunicates in the Molgulidae family have tailless (anural) larvae that fail to develop sensory organ-associated melanocytes. Here we studied the evolution of Tyrosinase family genes, indispensible for melanogenesis, in the anural, unpigmented Molgula occulta and in the tailed, pigmented Molgula oculata by using phylogenetic, developmental and molecular approaches.ResultsWe performed an evolutionary reconstruction of the tunicate Tyrosinase gene family: in particular, we found that M. oculata possesses genes predicted to encode one Tyrosinase (Tyr) and three Tyrosinase-related proteins (Tyrps) while M. occulta has only Tyr and Tyrp.a pseudogenes that are not likely to encode functional proteins. Analysis of Tyr sequences from various M. occulta individuals indicates that different alleles independently acquired frameshifting short indels and/or larger mobile genetic element insertions, resulting in pseudogenization of the Tyr locus. In M. oculata, Tyr is expressed in presumptive pigment cell precursors as in the model tunicate Ciona robusta. Furthermore, a M. oculata Tyr reporter gene construct was active in the pigment cell precursors of C. robusta embryos, hinting at conservation of the regulatory network underlying Tyr expression in tunicates. In contrast, we did not observe any expression of the Tyr pseudogene in M. occulta embryos. Similarly, M. occulta Tyr allele expression was not rescued in pigmented interspecific M. occulta × M. oculata hybrid embryos, suggesting deleterious mutations also to its cis-regulatory sequences. However, in situ hybridization for transcripts from the M. occulta Tyrp.a pseudogene revealed its expression in vestigial pigment cell precursors in this species.ConclusionsWe reveal a complex evolutionary history of the melanogenesis pathway in tunicates, characterized by distinct gene duplication and loss events. Our expression and molecular data support a tight correlation between pseudogenization of Tyrosinase family members and the absence of pigmentation in the immotile larvae of M. occulta. These results suggest that relaxation of purifying selection has resulted in the loss of sensory organ-associated melanocytes and core genes in the melanogenesis biosynthetic pathway in M. occulta.

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

Ascidians present a striking dichotomy between conserved phenotypes and divergent genomes: embryonic cell lineages and gene expression patterns are conserved between distantly related species. Much research has focused on Ciona or Halocynthia spp. but development in other ascidians remains poorly characterized. In this study, we surveyed the multipotent myogenic B7.5 lineage in Molgula spp. Comparisons to the homologous lineage in Ciona revealed identical cell division and fate specification events that result in segregation of larval, cardiac, and pharyngeal muscle progenitors. Moreover, the expression patterns of key regulators are conserved, but cross-species transgenic assays uncovered incompatibility, or 'unintelligibility', of orthologous cis-regulatory sequences between Molgula and Ciona. These sequences drive identical expression patterns that are not recapitulated in cross-species assays. We show that this unintelligibility is likely due to changes in both cis- and trans-acting elements, hinting at widespread and frequent turnover of regulatory mechanisms underlying otherwise conserved aspects of ascidian embryogenesis.

The Motor Ganglion (MG) is a small collection of neurons that control the swimming movements of the tunicate tadpole larva. Situated at the base of the tail, molecular and functional comparisons suggest that may be a homolog of the spinal cord and/or hindbrain ("rhombospinal" region) of vertebrates. Here we review the most current knowledge of the development, connectivity, functions, and unique identities of the neurons that comprise the MG, drawn mostly from studies in Ciona spp. The simple cell lineages, minimal cellular composition, and comprehensively mapped "connectome" of the Ciona MG all make this an excellent model for studying the development and physiology of motor control in aquatic larvae. Research Highlights: We review the latest research on the development, circuitry, and function of the Motor Ganglion of tunicate larvae, focused primarily on the most well‐studied species in the Ciona genus. [ABSTRACT FROM AUTHOR]

Marine larvae must sense various environmental cues to find a suitable spot where they can settle and metamorphose. New work identifies the specific neurons that transduce these cues in the larva of Ciona , a non-vertebrate chordate. [ABSTRACT FROM AUTHOR]

In tunicates, several species in the Molgulidae family have convergently lost the tailed, swimming larval body plan, including the morphogenesis of the notochord, a major chordate-defining trait. Through the comparison of tailless M. occulta and a close relative, the tailed species M. oculata , we show that notochord-specific expression of the Collagen Type I/II Alpha (Col1/2a) gene appears to have been lost specifically in the tailless species. Using CRISPR/Cas9-mediated mutagenesis in the tailed laboratory model tunicate Ciona robusta , we demonstrate that Col1/2a plays a crucial role in the convergent extension of notochord cells during tail elongation. Our results suggest that the expression of Col1/2a in the notochord, although necessary for its morphogenesis in tailed species, is dispensable for tailless species. This loss is likely a result of the accumulation of cis -regulatory mutations in the absence of purifying selective pressure. More importantly, the gene itself is not lost, likely due to its roles in other developmental processes, including during the adult stage. Our study further confirms the Molgulidae as an interesting family in which to study the evolutionary loss of tissue-specific expression of indispensable genes. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.11.006 Byline: Lionel Christiaen (a)(b), Alberto Stolfi (a), Michael Levine (a) Keywords: TGF-beta; Threshold response; Feedback loop; Ascidian; Kernel Abstract: Gene regulatory networks control the progressive specification of cell types and govern morphogenetic events during development. However, how morphogenetic events reciprocally affect gene expression remains poorly understood. Here, we analyzed the function of BMP signaling and expression of downstream target genes during cell migration of the precardiac mesoderm (trunk ventral cells, TVCs) in ascidian embryos. Our results indicate that migrating TVCs experience increasing BMP signaling as they migrate towards the ventral trunk epidermis, which expresses sustained levels of Bmp2/4. This increasing signaling intensity allows the successive activation of GATAa, Tolloid, Bmp2/4 and NK4. Initial activation of GATAa, Tolloid and Bmp2/4 contribute to a positive feedback loop involving cell migration, chordin inhibition and BMP ligand production. Sustained levels of BMP signaling become sufficient to activate NK4 expression, which in turn contributes to a negative feedback loop inhibiting Bmp2/4 and Tolloid expression. In addition, NK4 appears to inhibit cell migration thus providing a 'transcriptional brake' to stop TVC migration. Our observations led us to propose a model for the coordination of cell migration and gene expression based on the temporal unfolding of a gene regulatory sub-network in a relevant developmental context. Author Affiliation: (a) Center for Integrative Genomics, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA (b) Department of Biology, New York University, 1009 Silver Building, 100 Washington Square East, New York, NY 10003-6688, USA Article History: Received 7 July 2009; Revised 3 November 2009; Accepted 4 November 2009 Article Note: (footnote) [star] This article was accepted in 2009 to celebrate the 50th anniversary of Developmental Biology.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.01.033 Byline: Lionel Christiaen, Alberto Stolfi, Brad Davidson, Michael Levine Keywords: Ascidian; Heart; Endoderm; Muscles; Maternal determinants; Transcription Abstract: Mesp encodes a bHLH transcription factor required for specification of the cardiac mesoderm in Ciona embryos. The activities of Macho-1 and [beta]-catenin, two essential maternal determinants, are required for Mesp expression in the B7.5 blastomeres, which constitute the heart field. The T-box transcription factor Tbx6 functions downstream of Macho-1 as a direct activator of Mesp expression. However, Tbx6 cannot account for the restricted expression of Mesp in the B7.5 lineage since it is expressed throughout the presumptive tail muscles. Here we present evidence that the LIM-homeobox gene Lhx3, a direct target of [beta]-catenin, is essential for localized Mesp expression. Lhx3 is expressed throughout the presumptive endoderm and B7.5 blastomeres. Thus, the B7.5 blastomeres are the only cells to express sustained levels of the Tbx6 and Lhx3 activators. Like mammalian Lhx3 genes, Ci-Lhx3 encodes two isoforms with distinct N-terminal peptides. The Lhx3a isoform appears to be expressed both maternally and zygotically, while the Lhx3b isoform is exclusively zygotic. Misexpression of Lhx3b is sufficient to induce ectopic Mesp activation in cells expressing Tbx6b. Injection of antisense morpholino oligonucleotides showed that the Lhx3b isoform is required for endogenous Mesp expression. Mutations in the Lhx3 half-site of Tbx6/Lhx3 composite elements strongly reduced the activity of a minimal Mesp enhancer. We discuss the delineation of the heart field by the synergistic action of muscle and gut determinants. Author Affiliation: Department of Molecular & Cell Biology, Division of Genetics, Genomics and Development, Center for Integrative Genomics, University of California Berkeley, CA, 94720-3200, USA Article History: Received 18 November 2008; Revised 16 January 2009; Accepted 23 January 2009

Many species in the tunicate family Molgulidae have independently lost their swimming larval form and instead develop as tailless, immotile larvae. These larvae do not develop structures that are essential for swimming such as the notochord, otolith, and tail muscles. However, little is known about neural development in these nonswimming larvae. Here, we studied the patterning of the Motor Ganglion (MG) of Molgula occulta, a nonswimming species. We found that spatial patterns of MG neuron regulators in this species are conserved, compared with species with swimming larvae, suggesting that the gene networks regulating their expression are intact despite the loss of swimming. However, expression of the key motor neuron regulatory gene Ebf (Collier/Olf/EBF) was reduced in the developing MG of M. occulta when compared with molgulid species with swimming larvae. This was corroborated by measuring allele‐specific expression of Ebf in hybrid embryos from crosses of M. occulta with the swimming species M. oculata. Heterologous reporter construct assays in the model tunicate species Ciona robusta revealed a specific cis‐regulatory sequence change that reduces expression of Ebf in the MG, but not in other cells. Taken together, these data suggest that MG neurons are still specified in M. occulta larvae, but their differentiation might be impaired due to reduction of Ebf expression levels. Research highlights: We report the larval motor neuron‐specific loss of expression of the transcription factor‐encoding gene, Ebf, in the tunicate Molgula occulta, which has undergone evolutionary loss of the swimming larval life cycle stage. We identified cis‐regulatory sequence differences between M. occulta and the closely related M. oculata, which has retained the swimming larval stage, that likely affect the transcription of Ebf specifically in larval motor neurons, but not other neurons or tissues. [ABSTRACT FROM AUTHOR]