Single-cell analysis of cell fate bifurcation in the chordate Ciona.

BACKGROUND: Inductive signaling interactions between different cell types are a major mechanism for the further diversification of embryonic cell fates. Most blastomeres in the model chordate Ciona robusta become restricted to a single predominant fate between the 64-cell and mid-gastrula stages. The deeply stereotyped and well-characterized Ciona embryonic cell lineages allow the transcriptomic analysis of newly established cell types very early in their divergence from sibling cell states without the pseudotime inference needed in the analysis of less synchronized cell populations. This is the first ascidian study to use droplet scRNAseq with large numbers of analyzed cells as early as the 64-cell stage when major lineages such as primary notochord first become fate restricted. RESULTS AND CONCLUSIONS: We identify 59 distinct cell states, including new subregions of the b-line neural lineage and the early induction of the tail tip epidermis. We find that 34 of these cell states are directly or indirectly dependent on MAPK-mediated signaling critical to early Ciona patterning. Most of the MAPK-dependent bifurcations are canalized with the signal-induced cell fate lost upon MAPK inhibition, but the posterior endoderm is unique in being transformed into a novel state expressing some but not all markers of both endoderm and muscle. Divergent gene expression between newly bifurcated sibling cell types is dominated by upregulation in the induced cell type. The Ets family transcription factor Elk1/3/4 is uniquely upregulated in nearly all the putatively direct inductions. Elk1/3/4 upregulation together with Ets transcription factor binding site enrichment analysis enables inferences about which bifurcations are directly versus indirectly controlled by MAPK signaling. We examine notochord induction in detail and find that the transition between a Zic/Ets-mediated regulatory state and a Brachyury/FoxA-mediated regulatory state is unexpectedly late. This supports a "broad-hourglass" model of cell fate specification in which many early tissue-specific genes are induced in parallel to key tissue-specific transcriptional regulators via the same set of transcriptional inputs.

Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships.

Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not equivalent genetic elements.

Brachyury controls Ciona notochord fate as part of a feed-forward network.

The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails are more effective at reprogramming other cell types than Bra alone. We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network, and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically defined master regulator.

ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates.

ANISEED (https://www.aniseed.cnrs.fr) is the main model organism database for the worldwide community of scientists working on tunicates, the vertebrate sister-group. Information provided for each species includes functionally-annotated gene and transcript models with orthology relationships within tunicates, and with echinoderms, cephalochordates and vertebrates. Beyond genes the system describes other genetic elements, including repeated elements and cis-regulatory modules. Gene expression profiles for several thousand genes are formalized in both wild-type and experimentally-manipulated conditions, using formal anatomical ontologies. These data can be explored through three complementary types of browsers, each offering a different view-point. A developmental browser summarizes the information in a gene- or territory-centric manner. Advanced genomic browsers integrate the genetic features surrounding genes or gene sets within a species. A Genomicus synteny browser explores the conservation of local gene order across deuterostome. This new release covers an extended taxonomic range of 14 species, including for the first time a non-ascidian species, the appendicularian Oikopleura dioica. Functional annotations, provided for each species, were enhanced through a combination of manual curation of gene models and the development of an improved orthology detection pipeline. Finally, gene expression profiles and anatomical territories can be explored in 4D online through the newly developed Morphonet morphogenetic browser.

Iterative and Complex Asymmetric Divisions Control Cell Volume Differences in Ciona Notochord Tapering.

The notochord of the invertebrate chordate Ciona forms a tapered rod at tailbud stages consisting of only 40 cylindrical cells in a single-file column. This tapered shape involves differences in notochord cell volume along the anterior-posterior axis. Here, we quantify sibling cell volume asymmetry throughout the developing notochord and find that there are distinctive patterns of unequal cleavage in all 4 bilateral pairs of A-line primary notochord founder cells and also in the B-line-derived secondary notochord founder cells. A quantitative model confirms that the observed patterns of unequal cleavage are sufficient to explain all the anterior-posterior variation in notochord cell volume. Many examples are known of cells that divide asymmetrically to give daughter cells of different size and fate. Here, by contrast, a series of subtle but iterative and finely patterned asymmetric divisions controls the shape of an entire organ. Quantitative 3D analysis of cell shape and spindle positioning allows us to infer multiple cellular mechanisms driving these unequal cleavages, including polarized displacements of the mitotic spindle, contributions from the shape of the mother cell, and late changes occurring between anaphase and abscission that potentially involve differential cortical contractility. We infer differential use of these mechanisms between different notochord blastomeres and also between different rounds of cell division. These results demonstrate a new role for asymmetric division in directly shaping a developing organ and point toward complex underlying mechanisms.

Multiple inputs into a posterior-specific regulatory network in the Ciona notochord.

The gene regulatory networks underlying Ciona notochord fate specification and differentiation have been extensively investigated, but the regulatory basis for regionalized expression within the notochord is not understood. Here we identify three notochord-expressed genes, C11.331, C12.115 and C8.891, with strongly enriched expression in the secondary notochord cells at the posterior tip of the tail. C11.331 and C12.115 share a distinctive expression pattern that is highly enriched in the secondary notochord lineage but also graded within that lineage with the strongest expression at the posterior tip. Both genes show similar responses to pharmacological perturbations of Wnt and FGF signaling, consistent with an important role for Wnt and FGF ligands expressed at the tail tip. Reporter analysis indicates that the C11.331 cis-regulatory regions are extensively distributed, with multiple non-overlapping regions conferring posterior notochord-enriched expression. Fine-scale analysis of a minimal cis-regulatory module identifies discrete positive and negative elements including a strong silencer. Truncation of the silencer region leads to increased expression in the primary notochord, indicating that C11.331 expression is influenced by putative regulators of primary versus secondary notochord fate. The minimal CRM contains predicted ETS, GATA, LMX and Myb sites, all of which lead to reduced expression in secondary notochord when mutated. These results show that the posterior-enriched notochord expression of C11.331 depends on multiple inputs, including Wnt and FGF signals from the tip of the tail, multiple notochord-specific regulators, and yet-to-be identified regulators of regional identity within the notochord.

ANISEED 2017: extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets.

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.

Functional and evolutionary insights from the Ciona notochord transcriptome.

The notochord of the ascidian Ciona consists of only 40 cells, and is a longstanding model for studying organogenesis in a small, simple embryo. Here, we perform RNAseq on flow-sorted notochord cells from multiple stages to define a comprehensive Ciona notochord transcriptome. We identify 1364 genes with enriched expression and extensively validate the results by in situ hybridization. These genes are highly enriched for Gene Ontology terms related to the extracellular matrix, cell adhesion and cytoskeleton. Orthologs of 112 of the Ciona notochord genes have known notochord expression in vertebrates, more than twice as many as predicted by chance alone. This set of putative effector genes with notochord expression conserved from tunicates to vertebrates will be invaluable for testing hypotheses about notochord evolution. The full set of Ciona notochord genes provides a foundation for systems-level studies of notochord gene regulation and morphogenesis. We find only modest overlap between this set of notochord-enriched transcripts and the genes upregulated by ectopic expression of the key notochord transcription factor Brachyury, indicating that Brachyury is not a notochord master regulator gene as strictly defined.

Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

Stochasticity and stereotypy in the Ciona notochord.

Fate mapping with single cell resolution has typically been confined to embryos with completely stereotyped development. The lineages giving rise to the 40 cells of the Ciona notochord are invariant, but the intercalation of those cells into a single-file column is not. Here we use genetic labeling methods to fate map the Ciona notochord with both high resolution and large sample sizes. We find that the ordering of notochord cells into a single column is not random, but instead shows a distinctive signature characteristic of mediolaterally-biased intercalation. We find that patterns of cell intercalation in the notochord are somewhat stochastic but far more stereotyped than previously believed. Cell behaviors vary by lineage, with the secondary notochord lineage being much more constrained than the primary lineage. Within the primary lineage, patterns of intercalation reflect the geometry of the intercalating tissue. We identify the latest point at which notochord morphogenesis is largely stereotyped, which is shortly before the onset of mediolateral intercalation and immediately after the final cell divisions in the primary lineage. These divisions are consistently oriented along the AP axis. Our results indicate that the interplay between stereotyped and stochastic cell behaviors in morphogenesis can only be assessed by fate mapping experiments that have both cellular resolution and large sample sizes.

Quantitative and in toto imaging in ascidians: working toward an image-centric systems biology of chordate morphogenesis.

Developmental biology relies heavily on microscopy to image the finely controlled cell behaviors that drive embryonic development. Most embryos are large enough that a field of view with the resolution and magnification needed to resolve single cells will not span more than a small region of the embryo. Ascidian embryos, however, are sufficiently small that they can be imaged in toto with fine subcellular detail using conventional microscopes and objectives. Unlike other model organisms with particularly small embryos, ascidians have a chordate embryonic body plan that includes a notochord, hollow dorsal neural tube, heart primordium and numerous other anatomical details conserved with the vertebrates. Here we compare the size and anatomy of ascidian embryos with those of more traditional model organisms, and relate these features to the capabilities of both conventional and exotic imaging methods. We review the emergence of Ciona and related ascidian species as model organisms for a new era of image-based developmental systems biology. We conclude by discussing some important challenges in ascidian imaging and image analysis that remain to be solved.

A one-dimensional model of PCP signaling: polarized cell behavior in the notochord of the ascidian Ciona.

Despite its importance in development and physiology the planar cell polarity (PCP) pathway remains one of the most enigmatic signaling mechanisms. The notochord of the ascidian Ciona provides a unique model for investigating the PCP pathway. Interestingly, the notochord appears to be the only embryonic structure in Ciona activating the PCP pathway. Moreover, the Ciona notochord as a single-file array of forty polarized cells is a uniquely tractable system for the study of polarization dynamics and the transmission of the PCP pathway. Here, we test models for propagation of a polarizing signal, interrogating temporal, spatial and signaling requirements. A simple cell-cell relay cascading through the entire length of the notochord is not supported; instead a more complex mechanism is revealed, with interactions influencing polarity between neighboring cells, but not distant ones. Mechanisms coordinating notochord-wide polarity remain elusive, but appear to entrain general (i.e., global) polarity even while local interactions remain important. However, this global polarizer does not appear to act as a localized, spatially-restricted determinant. Coordination of polarity along the long axis of the notochord requires the PCP pathway, a role we demonstrate is temporally distinct from this pathway's earlier role in convergent extension and intercalation. We also reveal polarity in the notochord to be dynamic: a cell's polarity state can be changed and then restored, underscoring the Ciona notochord's amenability for in vivo studies of PCP.

Role of vaginal mesh hysteropexy for the management of advanced uterovaginal prolapse.

OBJECTIVE: To determine the role of vaginal mesh hysteropexy in the management of advanced genital organ prolapse as assessed by subjective and objective parameters. STUDY DESIGN: Retrospective case series of 77 women followed for at least 1 year after vaginal mesh hysteropexy performed for stage III or greater uterovaginal prolapse. The primary outcome was Pelvic Organ Prolapse Quantification (POP- Q) stage < II and no subjective bothersome bulge and no further interventions for prolapse. Secondary outcomes assessed were complications such as intraoperative bleeding, injuries, and postoperative complications such as mesh exposure, mesh retraction, dyspareunia, urinary incontinence, and voiding dysfunction. RESULTS: Mean follow-up was 13.7 +/- 4.1 months. Our composite success score was 85.7%. The anatomic (POP-Q) success score was 90.9%. Most failures (all but 1) were stage II with cervix as leading edge. Incidence of de novo dyspareunia was 3.7% and vaginal mesh erosion was 6.5%. Most patients 68/77 (88.3%) were discharged home voiding normally. CONCLUSION: Vaginal mesh hysteropexy offers good success; however, comparative studies are required to validate its true role.

Anterior-posterior regionalized gene expression in the Ciona notochord.

BACKGROUND: In the simple ascidian chordate Ciona, the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non-compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape, and behavior vary consistently along the anterior-posterior (AP) axis. RESULTS: Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non-uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin-like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF-ß ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL, and TGF-ß expression are evident even before the notochord cells have intercalated into a single-file column. CONCLUSIONS: We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression.

Accumulation of the transcription factor ABA-insensitive (ABI)4 is tightly regulated post-transcriptionally.

ABA-INSENSITIVE (ABI)4 is a transcription factor implicated in response to ABA in maturing seeds, and seedling responses to ABA, salt, and sugar. Previous studies have shown that ABI4 transcripts are high in seeds and in seedlings exposed to high concentrations of glucose and, to a lesser extent, osmotic agents and ABA, but that transcript levels are very low through most of vegetative growth. This study examined ABI4 protein accumulation indirectly, using transgenic lines expressing fusions to GFP and GUS. The GFP fusions were active, but undetectable visually or immunologically. Comparison of transcript and activity levels for GUS expression showed that inclusion of the ABI4 coding sequence reduced the ratio of activity to transcript ∼40-fold when driven by the CaMV 35S promoter, and nearly 150-fold when controlled by the ABI4 promoter. At least part of this discrepancy is due to proteasomal degradation of ABI4, resulting in a half-life of 5-6 h for the ABI4-GUS fusion. Comparison of the spatial localization of transcripts and fusion proteins indicated that the protein preferentially accumulated in roots such that transcript and protein distribution had little similarity. The components mediating targeting to the proteasome or other mechanisms of spatial restriction have not yet been identified, but several domains of ABI4 appear to contribute to its instability.

Direct targets of the transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors.

The plant hormone abscisic acid (ABA) is a key regulator of seed development. In addition to promoting seed maturation, ABA inhibits seed germination and seedling growth. Many components involved in ABA response have been identified, including the transcription factors ABA insensitive (ABI)4 and ABI5. The genes encoding these factors are expressed predominantly in developing and mature seeds, and are positive regulators of ABA mediated inhibition of seed germination and growth. The direct effects of ABI4 and ABI5 in ABA response remain largely undefined. To address this question, plants over-expressing ABI4 or ABI5 were used to allow identification of direct transcriptional targets. Ectopically expressed ABI4 and ABI5 conferred ABA-dependent induction of slightly over 100 genes in 11 day old plants. In addition to effector genes involved in seed maturation and reserve storage, several signaling proteins and transcription factors were identified as targets of ABI4 and/or ABI5. Although only 12% of the ABA- and ABI-dependent transcriptional targets were induced by both ABI factors in 11 day old plants, 40% of those normally expressed in seeds had reduced transcript levels in both abi4 and abi5 mutants. Surprisingly, many of the ABI4 transcriptional targets do not contain the previously characterized ABI4 binding motifs, the CE1 or S box, in their promoters, but some of these interact with ABI4 in electrophoretic mobility shift assays, suggesting that sequence recognition by ABI4 may be more flexible than known canonical sequences. Yeast one-hybrid assays demonstrated synergistic action of ABI4 with ABI5 or related bZIP factors in regulating these promoters, and mutant analyses showed that ABI4 and these bZIPs share some functions in plants.

Molecular aspects of seed dormancy.

Seed dormancy provides a mechanism for plants to delay germination until conditions are optimal for survival of the next generation. Dormancy release is regulated by a combination of environmental and endogenous signals with both synergistic and competing effects. Molecular studies of dormancy have correlated changes in transcriptomes, proteomes, and hormone levels with dormancy states ranging from deep primary or secondary dormancy to varying degrees of release. The balance of abscisic acid (ABA):gibberellin (GA) levels and sensitivity is a major, but not the sole, regulator of dormancy status. ABA promotes dormancy induction and maintenance, whereas GA promotes progression from release through germination; environmental signals regulate this balance by modifying the expression of biosynthetic and catabolic enzymes. Mediators of environmental and hormonal response include both positive and negative regulators, many of which are feedback-regulated to enhance or attenuate the response. The net result is a slightly heterogeneous response, thereby providing more temporal options for successful germination.

Targets of the Gal4 transcription activator in functional transcription complexes.

Although biochemical and genetic methods have detected many activator-transcription factor interactions, the direct functional targets of most activators remain undetermined. For this study, photo-cross-linkers positioned within the Gal4 C-terminal acidic activating region were used to identify polypeptides in close physical proximity to Gal4 during transcription activation in vitro. Of six specifically cross-linked polypeptides, three (Tra1, Taf12, and Gal11) are subunits of four complexes (SAGA, Mediator, NuA4, and TFIID) known to play a role in gene regulation. These cross-linking targets had differential effects on activation. SAGA was critical for activation by Gal4, Gal11 contributed modestly to activation, and TFIID and NuA4 were not important for activation under our conditions. Tra1, Taf12, and Gal11 have also been identified as cross-linking targets of the Gcn4 acidic central activating region. Our results demonstrate that two unrelated acidic activators converge on the same set of functional targets.

Activator-independent functions of the yeast mediator sin4 complex in preinitiation complex formation and transcription reinitiation.

RNA polymerase II (Pol II) Mediator plays an essential role in both basal and activated transcription. Previously, subunits of the Sin4 Mediator complex (Sin4, Pgd1, Gal11, and Med2) have been implicated in both positive and negative transcriptional regulation. Furthermore, it was proposed that this subcomplex constitutes an activator-binding domain. A yeast nuclear-extract system was used to investigate the biochemical role of the Sin4 complex. In contrast to previous findings, we found at least two general activator-independent roles for the Sin4 complex. First, mutations in sin4 and pgd1 destabilized the Pol II-Med complex, leading to a reduced rate and extent of preinitiation complex (PIC) formation both in the presence and absence of activators. Although reduced in amount compared with the wild type, PICs that are formed lacking the Sin4 complex are stable and can initiate transcription normally. Second, mutation of pgd1 causes partial disruption of the Sin4 complex and leads to a defect in transcription reinitiation. This defect is caused by dissociation of mutant Mediator from promoters after initiation, leading to nonfunctional Scaffold complexes. These results show that function of the Sin4 complex is not essential for transcription activation in a crude in vitro system but that it plays key roles in the general transcription mechanism.