Lionel Christiaen - US grants

PROJECT SUMMARY / ABSTRACT Critical physiological and pathological processes, such as wound healing, blood vessel formation and cancer metastasis, rely on directed collective cell migrations, whereby groups of cells collectively polarize and move together in an orderly fashion. The ability of cell collectives to migrate directionally is determined in part by the tissue-specific transcriptional inputs that define the complement of expressed genes and thus their competence to migrate. The long-term goal of this project is to understand how tissue-specific transcription regulators control the basic cellular processes underlying directed collective cell migration. To this aim, the simplified embryos of a chordate species, the ascidian Ciona intestinalis, will be used to study the migration of pre- cardiac mesoderm cells, called ?trunk ventral cells? (TVCs). The TVCs provide the simplest possible model of directed collective cell migration in live embryos. On each side of the embryo, only two cells migrate together and display a clear Leader-Trailer polarity aligned with the direction of migration: the leader TVC displays a broad leading edge and more conspicuous protrusions than the trailer. It was previously established that Mesp, Fibroblast growth factor (Fgf) signaling and FoxF transcriptional inputs determine the ability of TVCs to migrate. Moreover, TVCs migrate strictly between the endodermal and ectodermal germ layers, a hallmark of mesodermal cardiac progenitors. It was determine that these surrounding tissues contribute to canalizing TVCs' innate motility towards collective polarity and directed migration. The goal of the proposed research is to understand how transcriptionally-controlled intrinsic TVC properties interface with extrinsic signals to determine collective polarity and directed migration in the embryo. Preliminary studies suggested that the gene encoding the discoidin domain receptor (Ddr) is upregulated by Mesp, FGF and FoxF transcriptional inputs in the TVCs, where it promotes adhesion to the epidermis. Using newly developed quantitative imaging methods, the detailed mechanisms controlling Ddr expression, localization and activity will be analyzed. The hypothesis that a cell-autonomous antagonism between Ddr and vascular endothelial growth factor receptor (Vegfr) signaling positions the migrating TVCs between the epidermis and endoderm will be tested. Preliminary observations suggest that Ddr promotes adhesion to the epidermis by regulating vesicle trafficking. The hypothesis that Ddr acts in Rab4/Rab11-positive endosomes to promote the recycling of integrins to the plasma membrane will be tested. Finally, the functions of regulated candidate effectors of collective migration will be studied extensively using TVC-specific CRISPR/Cas9-mediated loss-of-function assays and high-content phenotypic analyses. A provisional model of the biomolecular network controlling the subcellular processes underlying TVC behavior will be built. Particular attention will be paid to the candidate modulators of Ddr, Vegfr and integrin functions. Completion of this project will illuminate the systems' level mechanisms linking intrinsic transcriptional inputs and extrinsic signals to define cell-specific behaviors.

DESCRIPTION (provided by applicant): Understanding the mechanisms directing progressive specification of heart cells from multipotent cardiovascular progenitors is essential for the development of regenerative therapies using induced pluripotent stem (iPS) cells. A simple chordate model system, the tunicate Ciona intestinalis, will be used to analyze the cellular and molecular mechanisms that determine muscle-type specification in the cardiogenic lineage. In Ciona embryos, the bilateral pairs of precardiac cells, called trunk ventral cells (TVCs), undergo stereotyped asymmetric cell divisions that distinguish the heart from the atrial siphon muscle (ASM) precursors. The latter then migrate toward the dorso-lateral atrial siphon placode. Following asymmetric divisions of the TVCs, the genes encoding the transcription factors COE and Islet are specifically up- regulated in the ASMs. In addition, COE is necessary and sufficient to block heart specification and promote the ASM fate, including expression of an ASM-specific Islet enhancer and cell migration toward the dorsal side of the larva. Finally, targeted expression of the constitutively active Notch intracellular domain using a TVC-specific enhancer is sufficient to inhibit ASM- specific expression of COE, Islet and cell migration. These observations led to the hypothesis that the initial asymmetric divisions result in heart-specific Notch signaling, which blocks ASM fate specification, possibly by inhibiting the expression of COE. In order to test this hypothesis, the cis-regulatory sequences that control ASM-specific expression of COE will be isolated and characterized, and the function of Notch signaling upstream of COE will be determined. The expression and localization patterns of endogenous regulators and effectors of Notch signaling will be documented in order to gain insight into the mechanisms that polarize the Notch signal during asymmetric TVC divisions. The effects of Notch signaling, COE and Islet on heart vs. ASM fate specification and cell migration will be analyzed using previously established assays in order to begin to characterize the epistatic relationships between these regulators. Finally, whole genome gene expression changes underlying heart vs. ASM fate specification will be documented by obtaining heart and ASM-specific transcription profiles using fluorescence activated cell sorting and microarrays. The results obtained upon completion of this project will characterize the regulation and function of COE, a novel negative regulator of heart fate specification, and illuminate the cellular and molecular mechanisms controlling muscle fate specification and cell migration in the cardiogenic lineage.

? DESCRIPTION (provided by applicant): Modern diagnostics and treatments for inherited and acquired cardiovascular diseases require in-depth knowledge of the mechanisms that control heart cell identity during embryonic development. In mammals, the facial and lower jaw muscles - collectively referred to as pharyngeal muscles - share a common origin with heart progenitors in the cardiopharyngeal mesoderm. This relationship is reflected in the DiGeorge syndrome, where altered Tbx1 function causes cardiovascular and craniofacial malformations. The heart vs. pharyngeal muscle fate choice is difficult to study in the early mammalian embryos, as is the cellular environment, a.k.a. niche, which determines whether cardiopharyngeal progenitor cells remain multipotent or become specified into either cardiac or pharyngeal muscles. Larvae of the ascidian Ciona intestinalis, a marine invertebrate among the closest relatives to the vertebrates, possess a simplified cardiopharyngeal lineage of cells that make successive heart vs. pharyngeal muscles choices in a simple and stereotyped manner. This can be studied with high spatiotemporal resolution using targeted molecular perturbations, confocal microscopy and lineage- specific transcription profiling that combines fluorescence activated cell sorting (FACS) and next generation RNA sequencing (RNA-seq). The ascidian cardiopharyngeal mesoderm arises from two progenitors, which produce the heart and the atrial siphon muscles (ASM) that control the exhalant opening. It was found that bipotent cardiopharyngeal progenitors undergo oriented asymmetrical cell divisions that produce distinct first and second heart precursors and ASM precursors. Molecularly, the cardiopharyngeal progenitors display multilineage transcriptional priming, i.e. they activate both early cardiac and ASM programs. These then segregate to their corresponding precursors due to regulatory cross-antagonisms: early ASM regulators inhibit the heart program in ASM precursors, while the ASM program is inhibited in the heart precursors. Here, regulatory mechanisms governing progressive ASM fate specification will be analyzed by testing the hypothesis that feedforward regulatory circuits control sequential gene activation. Next, the hypothesis that the orientation of asymmetric cell division determines differential interaction between a specific niche and the ASM vs. heart precursors will be explored. Finally, defined tissue-specific molecular perturbations, FACS and RNA-seq assays, including from single-cell samples, will define transcriptional signatures for multipotent cardiopharyngeal progenitors, first and second heart precursors and early ASM precursors. These results will characterize the regulatory properties that define cardiopharyngeal multipotency and uncover mechanisms that regulate conserved heart vs. pharyngeal muscle fate choices in chordates.

PROJECT SUMMARY This proposal is to fund the Ninth International Tunicate Meeting to be held at New York University, from July 16th to July 21st, 2017. This meeting attracts the international community of researchers using tunicates as model organisms for a broad range of studies, including cell and developmental biology, neurobiology and immunity, post-embryonic development and regeneration, genetics and genomics, ecology and evolution. Tunicates are the closest living relatives and comprise thousands of species that roam the oceans worldwide. For each one of the above topics, tunicates provide unique advantages as model organisms: for cell and developmental biology, they display simple embryos with fixed lineages that allows chordate development to be studied with unprecedented spatial and temporal resolution; for neurobiology, they possess an extremely simplified central nervous system containing only ~150 neurons. A fully characterized synaptome and simple swimming behavior; for immunity, colonial ascidians possess the only established allorecognition system outside of vertebrates; for evolution, the stereotyped early development of ascidians remained virtually unchanged in the face of over 450 million years of profound genome divergence, making them the best test-bed to study developmental systems drift; finally, tunicates are poised to provide important multidisciplinary insights into the impact of global environmental changes on marine communities. The above topics will be covered through approximately eight plenary sessions, two formal poster sessions and two keynote lectures. A scientific committee representative of the diversity of the field has been defined and will be tasked with selecting abstracts for oral presentations, paying particular attention to appropriate representation of diverse groups, especially trainees. A career panel workshop will address the particularities and opportunities using tunicates as model systems in academia. A round table discussion will be held in a town hall meeting format to discuss community-wide issue such as developing genomic resources (e.g. reagents for CRISPR/Cas9 or RNAi), defining guidelines and best practices, developing, distributing and maintaining experimental and database resources). Social functions will further encourage informal interactions and foster creative discussions. As for previous versions of the meeting, the proceedings will be published either through a single meeting report and/or by partnering with a sponsoring published to produce a dedicated journal issue made up of several papers focused on tunicates. This version of the international tunicate meeting promises to be particularly vibrant as suggested by the initial enthusiasm generated by our announcement to hold it in New York City.

Congenital heart defects affect ~1% of newborn infants and are often associated with craniofacial defects as is the case in the DiGeorge/CardioVeloFacial syndrome (DGS/CVFS). The latter is often caused by spontaneous de novo 22q11.2 deletions, which result in TBX1 haploinsufficiency, a major cause of the disease. TBX1 is thought to function in multipotent progenitors for the second heart field and branchiomeric/pharyngeal head muscles in early embryos, and thus combined cardiac and craniofacial malformations emerge from early defects in the cardiopharyngeal mesoderm. The existence of multiple interacting genetic modifiers of DGS penetrance hints at the complex gene regulatory network (GRN) controlling early cardiopharyngeal development. Despite progress in identifying key genetic determinants, the relative complexity of vertebrate embryos has hindered progress in modeling cardiopharyngeal networks. The tunicate Ciona is a tractable model where early cardiopharyngeal development can be studied with unprecedented spatial and temporal resolution in chordates, using functional genomics methods. In previous studies, comprehensive gene expression and chromatin accessibility profiles were obtained by lineage-specific whole genome assays, including single cell RNA-seq (scRNA-seq) and ATAC-seq. A method combining sample barcoding, CRISPR/Cas9-mediated mutagenesis, and scRNA-seq was developed to systematically interrogate the function of coding and non-coding genetic elements using high-content scRNA-seq assays. First, this approach will be used to profile loss-of-function perturbations for candidate transcription regulators expressed in the cardiopharyngeal lineage. Ciona and available mouse datasets will be integrated into cross-species models to jointly learn conserved and divergent features of cardiopharyngeal GRNs. Next, perturbations of accessible non-coding elements for selected transcription regulators (plus high-content scRNA-seq assays and new computational methods) will permit the explicit integration of non-coding elements in the context of our GRN models. Finally, perturbing regulators of cardiopharyngeal-specific chromatin accessibility followed by lineage-specific ATAC-seq will further disentangle the impact of transcription regulators on accessibility vs. activity. Integrating these datasets into lasting and evolving GRN models will support comprehensive understanding of early cardiopharyngeal development and the etiology of congenital cardio-craniofacial syndromes.

SUMMARY Prevalent congenital diseases, including the Di George/22q11DS, Noonan and related syndromes, present with both cardiac defects and craniofacial dysmorphism. The 22q11 Deletion Syndrome arises from eponymous deletions that cause TBX1 haploinsufficiency, while Noonan and related syndromes are RASopathies that affect the RAS-MAPK signaling pathway. However, understanding the etiology of combined cardio-craniofacial defects requires insights into the cellular and developmental contexts of gene function. In early amniote embryos, the second heart field (SHF) and branchiomeric/pharyngeal head muscles emerge from a common population of multipotent progenitors in the cardiopharyngeal mesoderm. TBX1 is thought function in cardiopharyngeal progenitors, to control both pharyngeal myogenesis and SHF development, and interact with Fibroblast Growth Factor (FGF)-MAPK signaling during heart development, highlighting the importance of studying Tbx1 and FGF-MAPK signaling in the cellular context of early cardiopharyngeal development. The tunicate Ciona emerged as a simple and powerful chordate model to study early cardiopharyngeal development, with high spatial and temporal resolution. In Ciona, four multipotent cardiopharyngeal progenitors undergo stereotyped migration and cell divisions, producing distinct first and second cardiac, and pharyngeal muscle lineages that deploy gene networks conserved with vertebrates. Leveraging the unique strengths of the Ciona system, and extensive previous work using lineage-specific perturbations, including CRISPR/Cas9-mediated mutagenesis, quantitative imaging, and multiplexed single cell genomics methods, this proposal will first explore the establishment of spatial patterns. The proposed work will address how a dynamic cardiopharyngeal niche helps polarize MAPK signaling and Tbx1/10 activation; how an intrinsic determinant of mitotic spindle positioning, the RhoGAP Depdc1, helps progenitors orient their divisions with regards to the niche, and analyze the molecular basis for the antagonism between MAPK signaling and the early heart program. Second, this proposal will explore the temporal dynamics underlying transitions between cardiopharyngeal states, by studying how de novo gene expression and fate choices are coupled with cell divisions, and how transcriptome changes in maturing progenitors determine the competence of cardiopharyngeal progenitors to form heart and pharyngeal muscle precursors. Completion of this ambitious proposal will yield far-reaching insights into emerging concepts of broad significance for cardiovascular developmental and stem cell biology.