ABSTRACT
The lung is a complex organ with various cell types having distinct roles. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been challenging to determine their effectiveness in each cell type due to the lack of appropriate analytical methods. We employed three distinct approaches to study silencing efficacy within different cell types. First, we used lineage markers to identify cell types in flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins CD47 or CD98. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure silencing efficacy in distinct cell types; to the best of our knowledge, this is the first time scRNA-seq has been applied to measure the efficacy of oligonucleotide therapeutics. In both approaches, fibroblasts were the most susceptible to locally delivered ASOs, with significant silencing also in endothelial cells. Third, we confirmed that the robust silencing in fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Across independent approaches, we demonstrate that intratracheally administered LNA gapmer ASOs robustly induce gene silencing in lung fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4-8 weeks after a single dose. Thus, lung fibroblasts are well aligned with ASOs as therapeutics.
Subject(s)
Endothelial Cells , Fibroblasts/drug effects , Lung/cytology , Oligonucleotides, Antisense/administration & dosage , Animals , Fibroblasts/metabolism , Gene Silencing , Lung/drug effects , Mice , Oligonucleotides/administration & dosage , Trachea/metabolismABSTRACT
Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF-Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF-Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF-Y or TALE defines genes in specific developmental processes, such that NF-Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF-Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF-Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.
Subject(s)
CCAAT-Binding Factor/metabolism , Gene Expression , Genome , Homeodomain Proteins/metabolism , Transcriptional Activation , Zebrafish/embryology , Zygote/metabolism , Animals , Cilia/genetics , Embryonic Development/genetics , Female , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Male , Zebrafish ProteinsABSTRACT
Maldevelopment of the pharyngeal endoderm, an embryonic tissue critical for patterning of the pharyngeal region and ensuing organogenesis, ultimately contributes to several classes of human developmental syndromes and disorders. Such syndromes are characterized by a spectrum of phenotypes that currently cannot be fully explained by known mutations or genetic variants due to gaps in characterization of critical drivers of normal and dysfunctional development. Despite the disease-relevance of pharyngeal endoderm, we still lack a comprehensive and integrative view of the molecular basis and gene regulatory networks driving pharyngeal endoderm development. To close this gap, we apply transcriptomic and chromatin accessibility single-cell sequencing technologies to generate a multi-omic developmental resource spanning pharyngeal endoderm patterning to the emergence of organ-specific epithelia in the developing mouse embryo. We identify cell-type specific gene regulation, distill GRN models that define developing organ domains, and characterize the role of an immunodeficiency-associated forkhead box transcription factor.
Subject(s)
Chromatin/genetics , Gene Expression Regulation, Developmental , Pharynx/embryology , Transcriptome , Animals , Chromatin/metabolism , Endoderm/embryology , Endoderm/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Organogenesis , Pharynx/metabolism , Single-Cell Analysis , Thymocytes/cytology , Thymocytes/metabolismABSTRACT
Treating premature infants with high oxygen is a routine intervention in the context of neonatal intensive care. Unfortunately, the increase in survival rates is associated with various detrimental sequalae of hyperoxia exposure, most notably bronchopulmonary dysplasia (BPD), a disease of disrupted lung development. The effects of high oxygen exposure on other developing organs of the infant, as well as the possible impact such disrupted development may have on later life remain poorly understood. Using a neonatal mouse model to investigate the effects of hyperoxia on the immature immune system we observed a dramatic involution of the thymic medulla, and this lesion was associated with disrupted FoxP3+ regulatory T cell generation and T cell autoreactivity. Significantly, administration of mesenchymal stromal cell-derived extracellular vesicles (MEx) restored thymic medullary architecture and physiological thymocyte profiles. Using single cell transcriptomics, we further demonstrated preferential impact of MEx treatment on the thymic medullary antigen presentation axis, as evidenced by enrichment of antigen presentation and antioxidative-stress related genes in dendritic cells (DCs) and medullary epithelial cells (mTECs). Our study demonstrates that MEx treatment represents a promising restorative therapeutic approach for oxygen-induced thymic injury, thus promoting normal development of both central tolerance and adaptive immunity.
Subject(s)
Extracellular Vesicles/transplantation , Hyperoxia/complications , Mesenchymal Stem Cells/metabolism , T-Lymphocytes , Thymus Gland , Animals , Animals, Newborn , Extracellular Vesicles/metabolism , Heterografts , Humans , Mice , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Thymus Gland/immunology , Thymus Gland/pathology , Umbilical CordABSTRACT
Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFß signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , RNA, Guide, Kinetoplastida/metabolism , Transcription Factors/metabolism , Cell Differentiation , Chromatin/metabolism , Endoderm/cytology , Endoderm/metabolism , Hepatocyte Nuclear Factor 3-beta/antagonists & inhibitors , Hepatocyte Nuclear Factor 3-beta/genetics , Hepatocyte Nuclear Factor 3-beta/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , RNA Interference , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolism , Signal Transduction , Single-Cell Analysis , Smad4 Protein/genetics , Smad4 Protein/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transforming Growth Factor beta/metabolismABSTRACT
We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.