Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona

The CRISPR/Cas9 system has emerged as an important tool for a wide variety of genome engineering applications, including reverse genetic screens. Previously, we described the implementation of the CRISPR/Cas9 system to induce tissue-specific mutations at targeted locations in the genome of the sea squirt Ciona (STOLFI et al. 2014). In the present study, we designed 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues in the Ciona embryo and measured their mutagenesis efficacy rates by massively parallel indel detection at the targeted loci using highthroughput sequencing. We show that the combined activity of two highly active sgRNAs allows us to generate large (>3 kbp) deletions of intervening genomic DNA in somatic cells of electroporated embryos, permitting tissue-specific gene knockouts. Additionally, we employed L1-regularized regression modeling to develop an optimal sgRNA design algorithm (TuniCUT), based on correlations between target sequence features and mutagenesis rates. Using this algorithm, we have predicted mutagenesis rates for sgRNAs targeting all 4,853,589 sites in the Ciona genome, which we have compiled into a "CRISPR/Cas9-induced Ciona Knock-Out" (Ci2KO) sgRNA sequence library. Finally, we describe a new method for the assembly of sgRNA expression cassettes using a simple one-step overlap PCR (OSO-PCR) protocol. These cassettes can be electroporated directly into Ciona embryos as unpurified PCR products to drive sgRNA expression, bypassing the need for time-consuming cloning and plasmid DNA preparations. We anticipate that this method will be used in combination with genome-wide sgRNA predictions to systematically investigate tissue-specific gene functions in Ciona

Ring Finger 149-Related Is an FGF/MAPK-Independent Regulator of Pharyngeal Muscle Fate Specification

During embryonic development, cell-fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors of both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study cardiopharyngeal fate specification with cellular resolution, as only two bilateral pairs of multipotent cardiopharyngeal progenitors give rise to the heart and to the pharyngeal muscles (also known as atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented and asymmetric divisions. Here, we identify the primed gene ring finger 149 related (Rnf149-r), which later becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss of Rnf149-r function impairs atrial siphon muscle morphogenesis, and downregulates Tbx1/10 and Ebf, two key determinants of pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of the loss of FGF/MAPK signaling in the cardiopharyngeal lineage, and an integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations has identified a significant overlap between candidate FGF/MAPK and Rnf149-r target genes. However, functional interaction assays suggest that Rnf149-r does not directly modulate the activity of the FGF/MAPK/Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF/MAPK signaling on shared targets, as well as on FGF/MAPK-independent targets through (a) separate pathway(s).publishedVersio

A modular cis-regulatory system controls isoform-specific pitx expression in ascidian stomodæum

AbstractPituitary homeobox (pitx) genes have been identified in vertebrates as critical molecular determinants of various craniofacial ontogenetic processes including pituitary organogenesis. Accordingly, a prominent conserved feature of pitx genes in chordates is their early expression in the anterior neural boundary (ANB) and oral ectoderm, also known as the stomodæum. Here we used the ascidian model species Ciona intestinalis to investigate pitx gene organization and cis-regulatory logic during early stages of oral development. Two distinct Ci-pitx mRNA variants were found to be expressed in mutually exclusive embryonic domains. Ci-pitx and vertebrate pitx2 genes display remarkably similar exon usage and organization, suggesting ancestry of the pitx transcriptional unit and regulation in chordates. We next combined phylogenetic footprinting, transient transgenesis, and confocal imaging methods to study the Ci-pitx cis-regulatory system, with special emphasis on the regulation of isoform-specific ANB/stomodæal expression. Among 10 conserved noncoding sequences (CNSs) interspersed in C. intestinalis and Ciona savignyi pitx loci, we identified two separate cis-regulatory modules (CRMs) that drive ANB/stomodæal expression in complementary spatiotemporal patterns. We discuss the developmental relevance of these data that provide an entry point to investigate the gene regulatory networks (GRNs) that position and shape oral structures in chordates

Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue-scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.publishedVersio

Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona

The CRISPR/Cas9 system has emerged as an important tool for a wide variety of genome engineering applications, including reverse genetic screens. Previously, we described the implementation of the CRISPR/Cas9 system to induce tissue-specific mutations at targeted locations in the genome of the sea squirt Ciona (STOLFI et al. 2014). In the present study, we designed 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues in the Ciona embryo and measured their mutagenesis efficacy rates by massively parallel indel detection at the targeted loci using highthroughput sequencing. We show that the combined activity of two highly active sgRNAs allows us to generate large (>3 kbp) deletions of intervening genomic DNA in somatic cells of electroporated embryos, permitting tissue-specific gene knockouts. Additionally, we employed L1-regularized regression modeling to develop an optimal sgRNA design algorithm (TuniCUT), based on correlations between target sequence features and mutagenesis rates. Using this algorithm, we have predicted mutagenesis rates for sgRNAs targeting all 4,853,589 sites in the Ciona genome, which we have compiled into a "CRISPR/Cas9-induced Ciona Knock-Out" (Ci2KO) sgRNA sequence library. Finally, we describe a new method for the assembly of sgRNA expression cassettes using a simple one-step overlap PCR (OSO-PCR) protocol. These cassettes can be electroporated directly into Ciona embryos as unpurified PCR products to drive sgRNA expression, bypassing the need for time-consuming cloning and plasmid DNA preparations. We anticipate that this method will be used in combination with genome-wide sgRNA predictions to systematically investigate tissue-specific gene functions in Ciona

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona

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

The use of gestures in the conversations of people with aphasia

Background: Gestures are spontaneous hand and arm movements that frequently accompany speech and play an important role in everyday communication. When communication is impaired by aphasia, gestures are affected as well. It is important to find out how people with aphasia (PWA) use gesture as an accompaniment to speech, as a compensatory modality, and during lexical retrieval. This novel study examined the use and functions of gesture in conversation and investigated parameters (i.e., conversation partner, topic, and participant factors) that could have an influence on gesture production. Methodology: Language and conversation data of 20 PWA and 21 neurologically healthy participants (NHP) were collected. Participants took part in conversations with two conversation partners (familiar and unfamiliar) and two conversation topics (narrative and procedural). Video samples were analysed for gesture production, speech production, and word-finding difficulties (WFD). Results: The two groups of participants produced a similar number of gestures (t (37) = -1.060, p = .296). Gesture type was not examined statistically but showed some marginal differences between groups. Unfamiliar conversation partners elicited significantly more gestures than familiar conversation partners (F (1, 37) = 24.358, p < .001). Additionally, participants produced significantly more gestures in procedural than in narrative topics (F (1, 37) =44.807, p < .001). While all participants experienced a similar number of WFD, there was a difference between PWA and NHP regarding gesture production and resolution of WFD: NHP resolved the majority of all WFD, independent of a co-occurring gesture. Nevertheless, for PWA and NHP, there was a significant relationship between gesture production and the resolution of the WFD (X2 (1) = 12.356, p < .01 for PWA and X2 (1) = 40.657, p < .01 for NHP), indicating that WFD that occurred with gestures were more likely to be resolved than WFD that occurred without gesture production. Participants used gestures with different functions, such as facilitative gestures to resolve WFD or augmentative gestures to supplement speech. For PWA, different participant factures, such as fluency of speech (rs (17) = .487, p = .035), lexical production skills (rs (17) = .584, p = .009), and cognition (rs (17) = .582, p = .009) were linked to gesture production. Conclusions: These findings shed light on gesture processing and the different functions gestures can serve within conversation. Furthermore, they highlight the importance of pragmatic influence, such as conversation topic and conversation partner on the production of gestures. The significant relationships between participant factors and gesture production in aphasia extend the understanding of relevant skills needed to successfully employ gestures in conversation. Next to language skills, such as speech fluency and lexical retrieval, cognitive skills affected gesture production as well

A conserved regulatory program drives emergence of the lateral plate mesoderm

Cardiovascular cell lineages emerge with kidney, smooth muscle, and limb skeleton progenitors from the lateral plate mesoderm (LPM). How the LPM emerges during development and how it has evolved to form key lineages of the vertebrate body plan remain unknown. Here, we captured LPM formation by transgenic in toto imaging and lineage tracing using the first pan-LPM enhancer element from the zebrafish gene draculin (drl). drl LPM enhancer-based reporters are specifically active in LPM-corresponding territories of several chordate species, uncovering a universal LPM-specific gene program. Distinct from other mesoderm, we identified EomesA, FoxH1, and MixL1 with BMP/Nodal-controlled Smad activity as minimally required factors to drive drl-marked LPM formation. Altogether, our work provides a developmental and mechanistic framework for LPM emergence and the in vitro differentiation of cardiovascular cell types. Our findings suggest that the LPM may represent an ancient cell fate domain that predates ancestral vertebrates

A conserved regulatory program drives emergence of the lateral plate mesoderm

Cardiovascular cell lineages emerge with kidney, smooth muscle, and limb skeleton progenitors from the lateral plate mesoderm (LPM). How the LPM emerges during development and how it has evolved to form key lineages of the vertebrate body plan remain unknown. Here, we captured LPM formation by transgenic in toto imaging and lineage tracing using the first pan-LPM enhancer element from the zebrafish gene draculin (drl). drl LPM enhancer-based reporters are specifically active in LPM-corresponding territories of several chordate species, uncovering a universal LPM-specific gene program. Distinct from other mesoderm, we identified EomesA, FoxH1, and MixL1 with BMP/Nodal-controlled Smad activity as minimally required factors to drive drl-marked LPM formation. Altogether, our work provides a developmental and mechanistic framework for LPM emergence and the in vitro differentiation of cardiovascular cell types. Our findings suggest that the LPM may represent an ancient cell fate domain that predates ancestral vertebrates