An organogenesis network-based comparative transcriptome analysis for understanding early human development in vivo and in vitro

<p>Abstract</p> <p>Background</p> <p>Integrated networks hold great promise in a variety of contexts. In a recent study, we have combined expression and interaction data to identify a putative network underlying early human organogenesis that contains two modules, the stemness-relevant module (hStemModule) and the differentiation-relevant module (hDiffModule). However, owing to its hypothetical nature, it remains unclear whether this network allows for comparative transcriptome analysis to advance our understanding of early human development, both <it>in vivo </it>and <it>in vitro</it>.</p> <p>Results</p> <p>Based on this integrated network, we here report comparisons with the context-dependent transcriptome data from a variety of sources. By viewing the network and its two modules as gene sets and conducting gene set enrichment analysis, we demonstrate the network's utility as a quantitative monitor of the stem potential <it>versus </it>the differentiation potential. During early human organogenesis, the hStemModule reflects the generality of a gradual loss of the stem potential. The hDiffModule indicates the stage-specific differentiation potential and is therefore not suitable for depicting an extended developmental window. Processing of cultured cells of different types further revealed that the hStemModule is a general indicator that distinguishes different cell types in terms of their stem potential. In contrast, the hDiffModule cannot distinguish between differentiated cells of different types but is able to predict differences in the differentiation potential of pluripotent cells of different origins. We also observed a significant positive correlation between each of these two modules and early embryoid bodies (EBs), which are used as <it>in vitro </it>differentiation models. Despite this, the network-oriented comparisons showed considerable differences between the developing embryos and the EBs that were cultured <it>in vitro </it>over time to try to mimic <it>in vivo </it>processes.</p> <p>Conclusions</p> <p>We strongly recommend the use of these two modules either when pluripotent cell types of different origins are involved or when the comparisons made are constrained to the in <it>vivo </it>embryos during early human organogenesis (and an equivalent <it>in vitro </it>differentiation models). Network-based comparative transcriptome analysis will contribute to an increase in knowledge about human embryogenesis, particularly when only transcriptome data are currently available. These advances will add an extra dimension to network applications.</p

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas

A roadmap for the Human Developmental Cell Atlas

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors

PMCID: PMC4434585.-- et al.The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.The research was supported by the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre. Work was funded by grants from the Ministerio de Economía y Competitividad (CB07/08/0021, SAF2011-27086, PLE2009-0162 to J.F., BFU2013-41322-P to J.L.G-S.), the Andalusian Government (BIO-396 to J.L.G-S.), the Wellcome Trust (WT088566 and WT097820 to N.A.H., WT101033 to J.F.), the Manchester Biomedical Research Centre, ERC advanced starting grant IMDs (C.H-H.C. and L.V.) and the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre (L.V.). R.E.J. is a Medical Research Council clinical training fellow. The authors are grateful to C. Wright (Vanderbilt University) for zebrafish Pdx1 antiserum, J. Postlethwait (Purdue University) for a Sox9b clone, H. Sasaki (Kumamoto University) for a TEAD–EnR clone, C. Vinod and L. Abi for research nurse assistance, and clinical colleagues at Central Manchester University Hospitals NHS Foundation Trust. The authors thank J. Garcia-Hurtado for technical assistance (IDIBAPS).Peer Reviewe

Single-cell profiling for advancing birth defects research and prevention

Cellular analysis of developmental processes and toxicities has traditionally entailed bulk methods (e.g., transcriptomics) that lack single cell resolution or tissue localization methods (e.g., immunostaining) that allow only a few genes to be monitored in each experiment. Recent technological advances have enabled interrogation of genomic function at the single-cell level, providing new opportunities to unravel developmental pathways and processes with unprecedented resolution. Here, we review emerging technologies of single-cell RNA-sequencing (scRNA-seq) to globally characterize the gene expression sets of different cell types and how different cell types emerge from earlier cell states in development. Cell atlases of experimental embryology and human embryogenesis at single-cell resolution will provide an encyclopedia of genes that define key stages from gastrulation to organogenesis. This technology, combined with computational models to discover key organizational principles, was recognized by Science magazine as the “Breakthrough of the year” for 2018 due to transformative potential on the way we study how human cells mature over a lifetime, how tissues regenerate, and how cells change in diseases (e.g., patient-derived organoids to screen disease-specific targets and design precision therapy). Profiling transcriptomes at the single-cell level can fulfill the need for greater detail in the molecular progression of all cell lineages, from pluripotency to adulthood and how cell–cell signaling pathways control progression at every step. Translational opportunities emerge for elucidating pathogenesis of genetic birth defects with cellular precision and improvements for predictive toxicology of chemical teratogenesis

Identification of biomarkers of the retinoic acid signaling pathway in the zebrafish embryo model to predict human developmental toxicants

This dissertation falls within the context of the paradigm shift in regulatory toxicology testing which promotes using a mechanistic-based approach based on in vitro tests instead of traditional animal testing to predict chemical hazards to human such as developmental toxicity. The novel research expands the understanding of developmental toxicity pathways by studying chemically-induced gene expression changes related to the perturbation of the retinoic acid signaling pathway (RA-SP) in a vertebrate embryo model. By using the zebrafish embryo (ZE) model it was possible to take advantage of the conservation of this biological pathway across vertebrate taxa, to predict potential human developmental toxicity. The ZE model is not new, however it has been primarily used and optimized for its morphology readout due to the transparent eggs enabling morphological observations during chemical exposure. However, improvements and harmonization are necessary to utilize this model with a reliable molecular level readout, to reveal relevant changes in gene expression. In chapter 2, the protocol design was refined to identify gene expression (GE) changes in the ZE. This was done by investigating the optimal exposure duration to study such changes due to the perturbation of the RA-SP. An exposure of ZE to the RA-SP agonist all-trans retinoic acid (ATRA) was performed using 6 different exposure durations, ranging from 2-117 hrs. These results identified that 4h exposure was the optimal exposure duration to study chemically-induced GE regulation specifically related to the RA-SP perturbation, thereby optimizing the ZE protocol for GE analysis. In chapters 3 and 4, the optimized ZE-GE protocol was employed to identify GE biomarker candidates for maldevelopment. After exposing ZE to two teratogenic compounds known to perturb the RA-SP (ATRA and Valproic Acid, VPA) and one non-teratogenic control compound (Folic Acid, FA), the chemically-induced perturbation of the RA-SP was explored using a whole genome scale GE analysis approach (RNAseq). The 3 test compounds each showed a specific mRNA expression profile, with 248 genes commonly regulated by both teratogenic compounds (ATRA and VPA) but not by FA. These 248 genes were implicated in several developmental processes. 62 differentially expressed genes (DEGs) were associated with nervous system development and were further examined in Chapter 3. These 62 genes were identified as potential biomarkers of early neurodevelopmental toxicity. In chapter 4, the perturbation of RA-SP on the GE associated with development of mesoderm derived tissues was investigated using bioinformatics methods. The investigation identified gene ontology (GO)-terms related to 47 DEGs. Literature indicates that these genes were normally expressed among 3 mesodermal sections (paraxial, intermediate, and lateral plate section) and 6 mesodermal tissues (somites, striated muscle, bone, kidney, circulatory system, and blood). These 47 DEGs were identified as potential biomarkers of early mesodermal maldevelopment or novel potential biomarkers for specific mesodermal organs. These proposed biomarker candidates advance the knowledge on the retinoic acid-mediated developmental toxicity mechanism. As their responses become even more broadly characterized by exploring different exposure regimes and the profiles of different chemicals, these biomarkers could contribute to predictive tools in animal-free chemical hazard and risk assessment

A Comprehensive Model and Modulation of Cellular Signaling Involved in Early Mammary Development and Aggressive Cancer Using a Novel Recombinant Protein of the G3 Domain of Laminin-5

The mammary gland is a unique and specialized epidermal organ; mammary organogenesis begins in the embryo but is not fully complete until puberty. As such, formation of the mammary gland depends on temporally and spatially regulated developmental steps that require coordination of multiple biological and cell signaling processes; many of which have parallels with cancer development. Research describing the events that occur between birth and puberty is lacking and little is known about human breast development of youth. Since mammary gland development requires a coordinated balance between cell growth, proliferation, and apoptosis, it is critical to understand which signaling pathways are utilized to relay developmental signals, and how these pathways and their targets interact and cooperate with age. Additionally, interactions between integrin molecules and their laminin ligands, especially Laminin-5 (Ln-5; also known as Laminin-332), regulate multiple facets of both embryonic development and tumor growth, invasion, and metastasis. α6β4 integrin serves as a marker to detect distant metastases in the early stages of specific malignancies and β4 integrin overexpression has been found in basal-like breast cancers, correlating with aggressiveness to institute a prognostic β4 signature that increases with tumor grade. The mechanism α6β4 integrin utilizes to modulate oncogenic signaling through association with Ln-5 molecules in the ECM is the basis for the recombinant protein (rG3, the third of five G domains of Ln-5) produced for the work reported in this dissertation. Here, it is shown there are specific transcriptional differences and a unique interaction of a gene set over time that contributes to postnatal mammary gland development, and this model clearly shares similarities and signaling pathways with oncogenic development. Especially important are pathways of the adaptive and innate immunities, ECM remodeling and integrin interactions, and extrinsic and intrinsic TP53-mediated apoptosis, greater understanding of which could lead to early detection of potential tumorigenic growth and identification of potential treatment avenues. Presented is a comprehensive model of early mammary development along with several panels of biomarkers that possess a role in normal mammary development, are involved in aggressive cancers, and are affected by apoptosis induced by rG3 treatment. rG3 has proven to be a valuable tool to study apoptotic pathways and the crosstalk among those pathways