Distinct epicardial gene regulatory programs drive development and regeneration of the zebrafish heart.

Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart fully regenerates following injury. Reactivation of cardiac developmental programs is considered key to successfully regenerating the heart, yet the regulation underlying the response to injury remains elusive. Here, we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardia. We identified epicardial enhancer elements with specific activity during development or during adult heart regeneration. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programs driving each of these processes, which were largely distinct. Loss of Hif1ab, Nrf1, Tbx2b, and Zbtb7a, central regulators of the regenerating epicardial network, in injured hearts resulted in elevated epicardial cell numbers infiltrating the wound and excess fibrosis after cryoinjury. Our work identifies differences between the regulatory blueprint deployed during epicardial development and regeneration, underlining that heart regeneration goes beyond the reactivation of developmental programs.

The SWI/SNF Complex in Neural Crest Cell Development and Disease.

While the neural crest cell population gives rise to an extraordinary array of derivatives, including elements of the craniofacial skeleton, skin pigmentation, and peripheral nervous system, it is today increasingly recognized that Schwann cell precursors are also multipotent. Two mammalian paralogs of the SWI/SNF (switch/sucrose nonfermentable) chromatin-remodeling complexes, BAF (Brg1-associated factors) and PBAF (polybromo-associated BAF), are critical for neural crest specification during normal mammalian development. There is increasing evidence that pathogenic variants in components of the BAF and PBAF complexes play central roles in the pathogenesis of neural crest-derived tumors. Transgenic mouse models demonstrate a temporal window early in development where pathogenic variants in Smarcb1 result in the formation of aggressive, poorly differentiated tumors, such as rhabdoid tumors. By contrast, later in development, homozygous inactivation of Smarcb1 requires additional pathogenic variants in tumor suppressor genes to drive the development of differentiated adult neoplasms derived from the neural crest, which have a comparatively good prognosis in humans.

Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in zebrafish.

Due to its genetic amenability coupled with advances in genome editing, zebrafish is an excellent model to examine the function of (epi)genomic elements. Here, we repurposed the Ac/Ds maize transposition system to efficiently characterise zebrafish cis-regulated elements, also known as enhancers, in F0-microinjected embryos. We further used the system to stably express guide RNAs enabling CRISPR/dCas9-interference (CRISPRi) perturbation of enhancers without disrupting the underlying genetic sequence. In addition, we probed the phenomenon of antisense transcription at two neural crest gene loci. Our study highlights the utility of Ac/Ds transposition as a new tool for transient epigenome modulation in zebrafish.

Multi-layered transcriptional control of cranial neural crest development.

The neural crest (NC) is an emblematic population of embryonic stem-like cells with remarkable migratory ability. These distinctive attributes have inspired the curiosity of developmental biologists for over 150 years, however only recently the regulatory mechanisms controlling the complex features of the NC have started to become elucidated at genomic scales. Regulatory control of NC development is achieved through combinatorial transcription factor binding and recruitment of associated transcriptional complexes to distal cis-regulatory elements. Together, they regulate when, where and to what extent transcriptional programmes are actively deployed, ultimately shaping ontogenetic processes. Here, we discuss how transcriptional networks control NC ontogeny, with a special emphasis on the molecular mechanisms underlying specification of the cephalic NC. We also cover emerging properties of transcriptional regulation revealed in diverse developmental systems, such as the role of three-dimensional conformation of chromatin, and how they are involved in the regulation of NC ontogeny. Finally, we highlight how advances in deciphering the NC transcriptional network have afforded new insights into the molecular basis of human diseases.

RNA-Responsive gRNAs for Controlling CRISPR Activity: Current Advances, Future Directions, and Potential Applications.

CRISPR-Cas9 has emerged as a major genome manipulation tool. As Cas9 can cause off-target effects, several methods for controlling the expression of CRISPR systems were developed. Recent studies have shown that CRISPR activity could be controlled by sensing expression levels of endogenous transcripts. This is particularly interesting, as endogenous RNAs could harbor important information about the cell type, disease state, and environmental challenges cells are facing. Single-guide RNA (sgRNA) engineering played a major role in the development of RNA-responsive CRISPR systems. Following further optimizations, RNA-responsive sgRNAs could enable the development of novel therapeutic and research applications. This review introduces engineering strategies that could be employed to modify Streptococcus pyogenes sgRNAs with a focus on recent advances made toward the development of RNA-responsive sgRNAs. Future directions and potential applications of these technologies are also discussed.

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

Cellular plasticity in the neural crest and cancer.

In vertebrates, neural crest cells (NCCs) are a multipotent embryonic population generating both neural/neuronal and mesenchymal derivatives, and thus the neural crest (NC) is often referred to as the fourth germ layer. NC development is a dynamic process, where NCCs possess substantial plasticity in transcriptional and epigenomic profiles. Recent technical advances in single-cell and low-input sequencing have empowered fine-resolution characterisation of NC development. In this review, we summarise the latest models underlying NC-plasticity acquirement and cell-fate restriction, outline the connections between NC plasticity and NC-derived cancer and envision the new opportunities in studying NC plasticity and its link to cancer.

A blood-based miRNA signature with prognostic value for overall survival in advanced stage non-small cell lung cancer treated with immunotherapy.

Immunotherapies have recently gained traction as highly effective therapies in a subset of late-stage cancers. Unfortunately, only a minority of patients experience the remarkable benefits of immunotherapies, whilst others fail to respond or even come to harm through immune-related adverse events. For immunotherapies within the PD-1/PD-L1 inhibitor class, patient stratification is currently performed using tumor (tissue-based) PD-L1 expression. However, PD-L1 is an accurate predictor of response in only ~30% of cases. There is pressing need for more accurate biomarkers for immunotherapy response prediction. We sought to identify peripheral blood biomarkers, predictive of response to immunotherapies against lung cancer, based on whole blood microRNA profiling. Using three well-characterized cohorts consisting of a total of 334 stage IV NSCLC patients, we have defined a 5 microRNA risk score (miRisk) that is predictive of overall survival following immunotherapy in training and independent validation (HR 2.40, 95% CI 1.37-4.19; P < 0.01) cohorts. We have traced the signature to a myeloid origin and performed miRNA target prediction to make a direct mechanistic link to the PD-L1 signaling pathway and PD-L1 itself. The miRisk score offers a potential blood-based companion diagnostic for immunotherapy that outperforms tissue-based PD-L1 staining.

Single-cell atlas of early chick development reveals gradual segregation of neural crest lineage from the neural plate border during neurulation.

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harboring neural crest and cranial placode progenitors. Here, we a generate single-cell atlas of the developing chick epiblast from late gastrulation through early neurulation stages to define transcriptional changes in the emerging 'neural plate border' as well as other regions of the epiblast. Focusing on the border territory, the results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridization. Developmental trajectory analysis infers that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined neural crest and placode lineages. The data also can be mined to reveal changes throughout the developing epiblast.

Evidence from oyster suggests an ancient role for Pdx in regulating insulin gene expression in animals.

Hox and ParaHox genes encode transcription factors with similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. Here, we report evidence indicating that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is a likely direct target of Pdx in Pacific oyster adults. We show that one insulin-related gene, cgILP, is co-expressed with cgPdx in oyster digestive tissue. Transcriptomic comparison suggests that this tissue plays a similar role to the vertebrate pancreas. Using ATAC-seq and ChIP, we identify an upstream regulatory element of the cgILP gene which shows binding interaction with cgPdx protein in oyster hepatopancreas and demonstrate, using a cell culture assay, that the oyster Pdx can act as a transcriptional activator through this site, possibly in synergy with NeuroD. These data argue that a classic homeodomain-target gene interaction dates back to the origin of Bilateria.

Adipocyte-like signature in ovarian cancer minimal residual disease identifies metabolic vulnerabilities of tumor-initiating cells.

Similar to tumor-initiating cells (TICs), minimal residual disease (MRD) is capable of reinitiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multiregion transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells showed the same phenotype and were dependent on fatty acid oxidation (FAO) for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD.

Rapid and efficient enhancer cloning and in vivo screening using the developing chick embryo.

Here, we describe a highly efficient, medium-throughput strategy for cloning and in vivo screening of putative enhancers using the chick embryo. By incorporating 48 unique nanotags for use in NanoString nCounter® across three different fluorescent reporters and developing a rapid and efficient digestion/ligation type IIs restriction enzyme-based cloning protocol, we develop a multiplexed approach for rapidly identifying enhancer activity. For complete details on the use and execution of this protocol, please see Williams et al. (2019).

Dissociation of chick embryonic tissue for FACS and preparation of isolated cells for genome-wide downstream assays.

In order to process samples by fluorescence-activated cell sorting (FACS), it is essential to obtain a single-cell suspension of dissociated cells. Numerous protocols and commercial reagents are available; however, each requires optimization for specific tissue types. Here, we describe an optimized protocol for dissociating dissected chick embryos across a broad span of developmental stages. We also provide protocols for processing targeted cell populations isolated using FACS for ATAC-seq, RNA-seq, and chromatin immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Ling and Sauka-Spengler (2019) and Williams et al. (2019).

Ex ovo electroporation of early chicken embryos.

The chick embryo is a favored model for developmental studies owing to its accessibility and ease of manipulation. Ex ovo electroporation provides a highly efficient method for screening perturbation phenotypes using a variety of reagents, including CRISPR and morpholinos. Additionally, the chick system lends itself well to rapid medium-throughput enhancer screening. Constructs facilitating tissue-specific protein pull-down can also be transfected using this protocol. Furthermore, bilateral electroporation with control and experimental reagents provides a robust assay for accurately interpreting functional perturbations. For complete details on the use and execution of this protocol, please refer to Williams et al. (2019).

The Cranial Neural Crest in a Multiomics Era.

Neural crest ontogeny plays a prominent role in craniofacial development. In this Perspective article, we discuss recent advances to the understanding of mechanisms underlying the cranial neural crest gene regulatory network (cNC-GRN) stemming from omics-based studies. We briefly summarize how parallel considerations of transcriptome, interactome, and epigenome data significantly elaborated the roles of key players derived from pre-omics era studies. Furthermore, the growing cohort of cNC multiomics data revealed contribution of the non-coding genomic landscape. As technological improvements are constantly being developed, we reflect on key questions we are poised to address by taking advantage of the unique perspective a multiomics approach has to offer.

Mechanistic Drivers of Müllerian Duct Development and Differentiation Into the Oviduct.

The conduits of life; the animal oviducts and human fallopian tubes are of paramount importance for reproduction in amniotes. They connect the ovary with the uterus and are essential for fertility. They provide the appropriate environment for gamete maintenance, fertilization and preimplantation embryonic development. However, serious pathologies, such as ectopic pregnancy, malignancy and severe infections, occur in the oviducts. They can have drastic effects on fertility, and some are life-threatening. Despite the crucial importance of the oviducts in life, relatively little is known about the molecular drivers underpinning the embryonic development of their precursor structures, the Müllerian ducts, and their successive differentiation and maturation. The Müllerian ducts are simple rudimentary tubes comprised of an epithelial lumen surrounded by a mesenchymal layer. They differentiate into most of the adult female reproductive tract (FRT). The earliest sign of Müllerian duct formation is the thickening of the anterior mesonephric coelomic epithelium to form a placode of two distinct progenitor cells. It is proposed that one subset of progenitor cells undergoes partial epithelial-mesenchymal transition (pEMT), differentiating into immature Müllerian luminal cells, and another subset undergoes complete EMT to become Müllerian mesenchymal cells. These cells invaginate and proliferate forming the Müllerian ducts. Subsequently, pEMT would be reversed to generate differentiated epithelial cells lining the fully formed Müllerian lumen. The anterior Müllerian epithelial cells further specialize into the oviduct epithelial subtypes. This review highlights the key established molecular and genetic determinants of the processes involved in Müllerian duct development and the differentiation of its upper segment into oviducts. Furthermore, an extensive genome-wide survey of mouse knockout lines displaying Müllerian or oviduct phenotypes was undertaken. In addition to widely established genetic determinants of Müllerian duct development, our search has identified surprising associations between loss-of-function of several genes and high-penetrance abnormalities in the Müllerian duct and/or oviducts. Remarkably, these associations have not been investigated in any detail. Finally, we discuss future directions for research on Müllerian duct development and oviducts.

Characterising open chromatin in chick embryos identifies cis-regulatory elements important for paraxial mesoderm formation and axis extension.

Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior-posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.

Tissue-Specific In Vivo Biotin Chromatin Immunoprecipitation with Sequencing in Zebrafish and Chicken.

Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods. For complete details on the use and execution of this protocol, please see Lukoseviciute et al. (2018) and Ling and Sauka-Spengler (2019).

Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder.

Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

Insights into olfactory ensheathing cell development from a laser-microdissection and transcriptome-profiling approach.

Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridization, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific (that is, not expressed by Schwann cells). One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.