A cis-regulatory change underlying the motor neuron-specific loss of Ebf expression in immotile tunicate larvae.

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.

Transcriptional regulation of Rab32/38, a specific marker of pigment cell formation in Ciona robusta.

Through a myriad of pigments stored in different cells, animal pigmentation represents a crucial process to face disparate environmental and ecological challenges. In vertebrates, the small GTPase Rab32 and Rab38 have a conserved role in the transport of key melanogenic enzymes, as tyrosinase (tyr) and tyrosinase-related protein (tyrp), to the melanosomes in formation. We provide a survey on Rab32/38 evolution and its regulatory logics during pigment cell formation in Ciona robusta. Our phylogeny supports the existence of a single Rab32/38 gene in tunicates, which is probably the unique transporter for tyrosinase family members in this clade. Different deletions allow us to identify the minimal cis-regulatory element able to recapitulate the endogenous gene expression during pigment cell development in C. robusta. In this conserved region, we identified two putative binding sites for the transcription factor Mitf, which is known for its role as regulator of pigmentation in vertebrates. Mutational analysis revealed that both Mitf binding sites are essential for the activity of this regulatory region and we demonstrated that Mitf misexpression is able to induce ectopic activation of the Rab32/38 regulatory region in vivo. Our results strongly indicate that Mitf is involved in the regulation of Rab32/38 activity during Ciona pigment cell development.

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.

Purification of Fluorescent Labeled Cells from Dissociated Ciona Embryos.

Genome-wide studies in Ciona often require highly purified cell populations. In this methods chapter, we introduce multi-channel combinatorial fluorescence activated cells sorting (FACS) and magnetic-activated cell sorting (MACS) as two sensitive and efficient tools for isolating lineage-specific cell populations from dissociated Ciona embryos and larvae. We present isolation of trunk ventral cell (TVC) progeny as the test case most commonly used in our laboratory. These approaches may also be applied to purify other cell populations with the proper combination of tissue-specific reporters.

Mutation studies in ascidians: a review.

Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.

Auto and cross regulatory elements control Onecut expression in the ascidian nervous system.

The expression pattern of Onecut genes in the central and peripheral nervous systems is highly conserved in invertebrates and vertebrates but the regulatory networks in which they are involved are still largely unknown. The presence of three gene copies in vertebrates has revealed the functional roles of the Onecut genes in liver, pancreas and some populations of motor neurons. Urochordates have only one Onecut gene and are the closest living relatives of vertebrates and thus represent a good model system to understand its regulatory network and involvement in nervous system formation. In order to define the Onecut genetic cascade, we extensively characterized the Onecut upstream cis-regulatory DNA in the ascidian Ciona intestinalis. Electroporation experiments using a 2.5kb genomic fragment and of a series of deletion constructs identified a small region of 262bp able to reproduce most of the Onecut expression profile during embryonic development. Further analyses, both bioinformatic and in vivo using transient transgenes, permitted the identification of transcription factors responsible for Onecut endogenous expression. We provide evidence that Neurogenin is a direct activator of Onecut and that an autoregulatory loop is responsible for the maintenance of its expression. Furthermore, for the first time we propose the existence of a direct connection among Neurogenin, Onecut and Rx transcription factors in photoreceptor cell formation.

GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicate Ciona, which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream of tbx1 to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

Disruption of nuclear envelope integrity as a possible initiating event in tauopathies.

The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.

Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices.

During embryogenesis, chromatin accessibility profiles control lineage-specific gene expression by modulating transcription, thus impacting multipotent progenitor states and subsequent fate choices. Subsets of cardiac and pharyngeal/head muscles share a common origin in the cardiopharyngeal mesoderm, but the chromatin landscapes that govern multipotent progenitors competence and early fate choices remain largely elusive. Here, we leveraged the simplicity of the chordate model Ciona to profile chromatin accessibility through stereotyped transitions from naive Mesp+ mesoderm to distinct fate-restricted heart and pharyngeal muscle precursors. An FGF-Foxf pathway acts in multipotent progenitors to establish cardiopharyngeal-specific patterns of accessibility, which govern later heart vs. pharyngeal muscle-specific expression profiles, demonstrating extensive spatiotemporal decoupling between early cardiopharyngeal enhancer accessibility and late cell-type-specific activity. We found that multiple cis-regulatory elements, with distinct chromatin accessibility profiles and motif compositions, are required to activate Ebf and Tbx1/10, two key determinants of cardiopharyngeal fate choices. We propose that these 'combined enhancers' foster spatially and temporally accurate fate choices, by increasing the repertoire of regulatory inputs that control gene expression, through either accessibility and/or activity.

New molecular insights on the response of the green alga Tetraselmis suecica to nitrogen starvation.

Microalgae are currently considered one of the most promising resources for biofuel production, aquaculture feedstock and new pharmaceuticals. Among them, green algae of the genus Tetraselmis are extensively studied for their lipid accumulation in nutrient-starvation conditions. In this paper, we present the full-transcriptome of Tetraselmis suecica and differential expression analysis between nitrogen-starved and -repleted conditions (at stationary phase) focusing not only on lipid metabolism but giving new insights on nutrient starvation responses. Transcripts involved in signal transduction pathways, stress and antioxidant responses and solute transport were strongly up-regulated when T. suecica was cultured under nitrogen starvation. On the contrary, transcripts involved in amino acid synthesis, degradation of sugars, secondary metabolite synthesis, as well as photosynthetic activity were down-regulated under the same conditions. Among differentially expressed transcripts, a polyketide synthase and three lipoxygenases (involved in the synthesis of secondary metabolites with antipredator, anticancer and anti-infective activities) were identified, suggesting the potential synthesis of bioactive compounds by this microalga. In addition, the transcript for a putative nitrilase, enzyme used in nitrile bioremediation, is here reported for the first time for T. suecica. These findings give new insights on T. suecica responses to nutrient starvation and on possible biotechnological applications for green algae.

A conserved regulatory program initiates lateral plate mesoderm emergence across chordates.

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

An FGF-driven feed-forward circuit patterns the cardiopharyngeal mesoderm in space and time.

In embryos, multipotent progenitors divide to produce distinct progeny and express their full potential. In vertebrates, multipotent cardiopharyngeal progenitors produce second-heart-field-derived cardiomyocytes, and branchiomeric skeletal head muscles. However, the mechanisms underlying these early fate choices remain largely elusive. The tunicate Ciona emerged as an attractive model to study early cardiopharyngeal development at high resolution: through two asymmetric and oriented divisions, defined cardiopharyngeal progenitors produce distinct first and second heart precursors, and pharyngeal muscle (aka atrial siphon muscle, ASM) precursors. Here, we demonstrate that differential FGF-MAPK signaling distinguishes between heart and ASM precursors. We characterize a feed-forward circuit that promotes the successive activations of essential ASM determinants, Hand-related, Tbx1/10 and Ebf. Finally, we show that coupling FGF-MAPK restriction and cardiopharyngeal network deployment with cell divisions defines the timing of gene expression and permits the emergence of diverse cell types from multipotent progenitors.

Evolutionary loss of melanogenesis in the tunicate Molgula occulta.

BACKGROUND: Analyzing 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. RESULTS: We 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. CONCLUSIONS: We 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.

Evolutionary recruitment of flexible Esrp-dependent splicing programs into diverse embryonic morphogenetic processes.

Epithelial-mesenchymal interactions are crucial for the development of numerous animal structures. Thus, unraveling how molecular tools are recruited in different lineages to control interplays between these tissues is key to understanding morphogenetic evolution. Here, we study Esrp genes, which regulate extensive splicing programs and are essential for mammalian organogenesis. We find that Esrp homologs have been independently recruited for the development of multiple structures across deuterostomes. Although Esrp is involved in a wide variety of ontogenetic processes, our results suggest ancient roles in non-neural ectoderm and regulating specific mesenchymal-to-epithelial transitions in deuterostome ancestors. However, consistent with the extensive rewiring of Esrp-dependent splicing programs between phyla, most developmental defects observed in vertebrate mutants are related to other types of morphogenetic processes. This is likely connected to the origin of an event in Fgfr, which was recruited as an Esrp target in stem chordates and subsequently co-opted into the development of many novel traits in vertebrates.

Expression of a single prominin homolog in the embryo of the model chordate Ciona intestinalis.

Prominins are a family of pentaspan transmembrane glycoproteins, expressed in various types of cells, including stem and cancer stem cells in mammals. Prominin-1 is critical in generating and maintaining the structure of the photoreceptors in the eye since mutations in the PROM1 gene are associated with retinal and macular degeneration in human. In this study, we identified a single prominin homolog, Ci-prom1/2, in the model chordate the ascidian Ciona intestinalis and characterized Ci-prom1/2 expression profile in relation to photoreceptor differentiation during Ciona embryonic development. In situ hybridization experiments show Ci-prom1/2 transcripts localized in the developing central nervous system, predominantly in photoreceptor cell precursors as early as neurula stage and expression is maintained through larva stage in photoreceptor cells around the simple eye. We also isolated the regulatory region responsible for the specific spatio-temporal expression of the Ci-prom1/2 in photoreceptor cell lineage. Collectively, we report that Ci-prom1/2 is a novel molecular marker for ascidian photoreceptor cells and might represent a potential source to enlarge the knowledge about the function of prominin family in photoreceptor cell 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.

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.