TALE and NF-Y co-occupancy marks enhancers of developmental control genes during zygotic genome activation in zebrafish [preprint]

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts control to the embryo at early blastula stages. ZGA is thought to be mediated by specialized maternally deposited transcription factors (TFs), but here we demonstrate that NF-Y and TALE – TFs with known later roles in embryogenesis – co-occupy unique genomic elements at zebrafish ZGA. We show that these elements are selectively associated with early-expressed genes involved in transcriptional regulation and possess enhancer activity in vivo. In contrast, we find that elements individually occupied by either NF-Y or TALE are associated with genes acting later in development – such that NF-Y controls a cilia gene expression program while TALE TFs control expression of hox genes. We conclude that NF-Y and TALE have a shared role at ZGA, but separate roles later during development, demonstrating that combinations of known TFs can regulate subsets of key developmental genes at vertebrate ZGA

Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia.

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

Combinatorial action of NF–Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish

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

Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development

Summary: 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 TGFβ 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. : Genga et al. utilize a single-cell RNA-sequencing-based CRISPR interference approach to screen transcription factors predicted to have a role in human definitive endoderm differentiation. The perturbation screen identifies an important role of TGFβ signaling-related factors. Follow-up of FOXA2 reveals genome-wide molecular changes and altered differentiation competency in endoderm. Keywords: pluripotent stem cells, endoderm, single-cell RNA-seq, CRISPRi, human development, chromatin accessibility, hepatic endoderm, dCas9-KRAB, stem cell differentiation, perturbation scree

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Restore Thymic Architecture and T Cell Function Disrupted by Neonatal Hyperoxia

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