Filomena Ristoratore

Cloning of ascidian homeobox genes provides evidence for a primordial chordate cluster

In order to isolate genes important in controlling embryonic development in Tunicates, a genomic library from the ascidian Ciona intestinalis was screened with a degenerate oligodeoxyribonucleotide encoding the third helix of Antennapedia-type homeoboxes. Fourteen C. intestinalis homeobox genes, corresponding to several classes of homeodomains, have been identified. Five of the isolated homeoboxes show their highest homology to members of the Vertebrate HOX clusters. mRNAs for two of the isolated homeoboxes are present in unfertilized C. intestinalis eggs.

Unusual number and genomic organization of Hox genes in the tunicate Ciona intestinalis.

Hox genes are organized in genomic clusters. In all organisms where their role has been studied, Hox genes determine developmental fate along the antero-posterior axis. Hence, these genes represent an ideal system for the understanding of relationships between the number and expression of genes and body organization. We report in this paper that the ascidian Ciona intestinalis genome appears to contain a single Hox gene complex which shows absence of some of the members found in all chordates investigated up to now. Furthermore, the complex appears to be either unusually long or split in different subunits. We speculate that such an arrangement of Hox genes does not correspond to the chordate primordial cluster but occurred independently in the ascidian lineage.

FGF/MAPK/Ets signaling renders pigment cell precursors competent to respond to Wnt signal by directly controlling Ci-Tcf transcription

FGF and Wnt pathways constitute two fundamental signaling cascades, which appear to crosstalk in cooperative or antagonistic fashions in several developmental processes. In vertebrates, both cascades are involved in pigment cell development, but the possible interplay between FGF and Wnt remains to be elucidated. In this study, we have investigated the role of FGF and Wnt signaling in development of the pigment cells in the sensory organs of C. intestinalis. This species possesses the basic features of an ancestral chordate, thus sharing conserved molecular developmental mechanisms with vertebrates. Chemical and targeted perturbation approaches revealed that a FGF signal, spreading in time from early gastrulation to neural tube closure, is responsible for pigment cell precursor induction. This signal is transmitted via the MAPK pathway, which activates the Ci-Ets1/2 transcription factor. Targeted perturbation of Ci-TCF, a downstream factor of the canonical Wnt pathway, indicated its contribution to pigment cell differentiation Furthermore, analyses of the Ci-Tcf regulatory region revealed the involvement of the FGF effector, Ci-Ets1/2, in Ci-Tcf transcriptional regulation in pigment cell precursors. Our results indicate that both FGF and the canonical Wnt pathways are involved in C. intestinalis pigment cell induction and differentiation. Moreover, we present a case of direct transcriptional regulation exerted by the FGF signaling cascade, via the MAPK-ERK-Ets1/2, on the Wnt downstream gene Ci-Tcf. Several examples of FGF/Wnt signaling crosstalk have been described in different developmental processes; however, to our knowledge, FGF-Wnt cross-interaction at the transcriptional level has never been previously reported. These findings further contribute to clarifying the multitude of FGF-Wnt pathway interactions.

Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians

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. DOI: http://dx.doi.org/10.7554/eLife.03728.001

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

A new transglutaminase‐like from the ascidian Ciona intestinalis 1

A cDNA clone encoding a transglutaminase (TGase) was isolated from a cDNA library prepared from the larval stage of Ciona intestinalis. The cDNA sequence has an open reading frame encoding a protein of 696 amino acids and is about 36% identical to 11 other TGase sequences. In addition, the critical residues thought to form the catalytic center are conserved. The Ciona TGase (CiTGase) has an extension of 39 amino acids in the NH2‐terminal region similar to that reported for keratinocyte TGases. A phylogenetic analysis among other types of TGases demonstrated that CiTGase represents a new type of the enzyme.

New Insights into the Evolution of Metazoan Tyrosinase Gene Family

Tyrosinases, widely distributed among animals, plants and fungi, are involved in the biosynthesis of melanin, a pigment that has been exploited, in the course of evolution, to serve different functions. We conducted a deep evolutionary analysis of tyrosinase family amongst metazoa, thanks to the availability of new sequenced genomes, assessing that tyrosinases (tyr) represent a distinctive feature of all the organisms included in our study and, interestingly, they show an independent expansion in most of the analyzed phyla. Tyrosinase-related proteins (tyrp), which derive from tyr but show distinct key residues in the catalytic domain, constitute an invention of chordate lineage. In addition we here reported a detailed study of the expression territories of the ascidian Ciona intestinalis tyr and tyrps. Furthermore, we put efforts in the identification of the regulatory sequences responsible for their expression in pigment cell lineage. Collectively, the results reported here enlarge our knowledge about the tyrosinase gene family as valuable resource for understanding the genetic components involved in pigment cells evolution and development.

Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis

During the development of the central nervous system (CNS), combinations of transcription factors and signalling molecules orchestrate patterning, specification and differentiation of neural cell types. In vertebrates, three types of melanin-containing pigment cells, exert a variety of functional roles including visual perception. Here we analysed the mechanisms underlying pigment cell specification within the CNS of a simple chordate, the ascidian Ciona intestinalis. Ciona tadpole larvae exhibit a basic chordate body plan characterized by a small number of neural cells. We employed lineage-specific transcription profiling to characterize the expression of genes downstream of fibroblast growth factor signalling, which govern pigment cell formation. We demonstrate that FGF signalling sequentially imposes a pigment cell identity at the expense of anterior neural fates. We identify FGF-dependent and pigment cell-specific factors, including the small GTPase, Rab32/38 and demonstrated its requirement for the pigmentation of larval sensory organs.

Natural variation of model mutant phenotypes in Ciona intestinalis.

Background The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. Methodology/Principal Findings Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. Conclusions/Significance Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity.

A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening

The introduction of new genome editing tools such as ZFNs, TALENs and, more recently, the CRISPR/Cas9 system, has greatly expanded the ability to knock-out genes in different animal models, including zebrafish. However, time and costs required for the screening of a huge number of animals, aimed to identify first founder fishes (F0), and then carriers (F1) are still a bottleneck. Currently, high-resolution melting (HRM) analysis is the most efficient technology for large-scale InDels detection, but the very expensive equipment demanded for its application may represent a limitation for research laboratories. Here, we propose a rapid and cheap method for high-throughput genotyping that displays efficiency rate similar to the HRM. In fact, using a common ViiA™7 real-time PCR system and optimizing the parameters of the melting analysis, we demonstrated that it is possible to discriminate between the mutant and the wild type melting curves. Due to its simplicity, rapidity and cheapness, our method can be used as a preliminary one-step approach for massive screening, in order to restrict the scope at a limited number of embryos and to focus merely on them for the next sequencing step, necessary for the exact sequence identification of the induced mutation. Moreover, thanks to its versatility, this simple approach can be readily adapted to the detection of any kind of genome editing approach directed to genes or regulatory regions and can be applied to many other animal models.