A Single-Cell Transcriptomic Atlas of Thymus Organogenesis Resolves Cell Types and Developmental Maturation.

Thymus development is critical to the adaptive immune system, yet a comprehensive transcriptional framework capturing thymus organogenesis at single-cell resolution is still needed. We applied single-cell RNA sequencing (RNA-seq) to capture 8 days of thymus development, perturbations of T cell receptor rearrangement, and in vitro organ cultures, producing profiles of 24,279 cells. We resolved transcriptional heterogeneity of developing lymphocytes, and genetic perturbation confirmed T cell identity of conventional and non-conventional lymphocytes. We characterized maturation dynamics of thymic epithelial cells in vivo, classified cell maturation state in a thymic organ culture, and revealed the intrinsic capacity of thymic epithelium to preserve transcriptional regularity despite exposure to exogenous retinoic acid. Finally, by integrating the cell atlas with human genome-wide association study (GWAS) data and autoimmune-disease-related genes, we implicated embryonic thymus-resident cells as possible participants in autoimmune disease etiologies. This resource provides a single-cell transcriptional framework for biological discovery and molecular analysis of thymus organogenesis.

Integration of single-cell transcriptomes and chromatin landscapes reveals regulatory programs driving pharyngeal organ development.

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.

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.

Differentiation of human pluripotent stem cells toward pharyngeal endoderm derivatives: Current status and potential.

The pharyngeal apparatus, a transient embryological structure, includes diverse cells from all three germ layers that ultimately contribute to a variety of adult tissues. In particular, pharyngeal endoderm produces cells of the inner ear, palatine tonsils, the thymus, parathyroid and thyroid glands, and ultimobranchial bodies. Each of these structures and organs contribute to vital human physiological processes, including central immune tolerance (thymus) and metabolic homeostasis (parathyroid and thyroid glands, and ultimobranchial bodies). Thus, improper development or damage to pharyngeal endoderm derivatives leads to complicated and severe human maladies, such as autoimmunity, immunodeficiency, hypothyroidism, and/or hypoparathyroidism. To study and treat such diseases, we can utilize human pluripotent stem cells (hPSCs), which differentiate into functionally mature cells in vitro given the proper developmental signals. Here, we discuss current efforts regarding the directed differentiation of hPSCs toward pharyngeal endoderm derivatives. We further discuss model system and therapeutic applications of pharyngeal endoderm cell types produced from hPSCs. Finally, we provide suggestions for improving hPSC differentiation approaches to pharyngeal endoderm derivatives with emphasis on current single cell-omics and 3D culture system technologies.

The Kinesin-3 motor, KLP-4, mediates axonal organization and cholinergic signaling in Caenorhabditis elegans.

Microtubule plus-end directed trafficking is dominated by kinesin motors, yet kinesins differ in terms of cargo identity, movement rate, and distance travelled. Functional diversity of kinesins is especially apparent in polarized neurons, where long distance trafficking is required for efficient signal transduction-behavioral response paradigms. The Kinesin-3 superfamily are expressed in neurons and are hypothesized to have significant roles in neuronal signal transduction due to their high processivity. Although much is known about Kinesin-3 motors mechanistically in vitro, there is little known about their mechanisms in vivo. Here, we analyzed KLP-4, the Caenorhabditis elegans homologue of human KIF13A and KIF13B. Like other Kinesin-3 superfamily motors, klp-4 is highly expressed in the ventral nerve cord command interneurons of the animal, suggesting it might have a role in controlling movement of the animal. We characterized an allele of klp-4 that contains are large indel in the cargo binding domain of the motor, however, the gene still appears to be expressed. Behavioral analysis demonstrated that klp-4 mutants have defects in locomotive signaling, but not the strikingly uncoordinated movements such as those found in unc-104/KIF1A mutants. Animals with this large deletion are hypersensitive to the acetylcholinesterase inhibitor aldicarb but are unaffected by exogenous serotonin. Interestingly, this large klp-4 indel does not affect gross neuronal development but does lead to aggregation and disorganization of RAB-3 at synapses. Taken together, these data suggest a role for KLP-4 in modulation of cholinergic signaling in vivo and shed light on possible in vivo mechanisms of Kinesin-3 motor regulation.

Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen.

BACKGROUND: The obesity pandemic is associated with multiple major health concerns. In addition to diet and lifestyle, there is increasing evidence that environmental exposures to chemicals known as obesogens also may promote obesity. OBJECTIVES: We investigated the massive environmental contamination resulting from the Deepwater Horizon (DWH) oil spill, including the use of the oil dispersant COREXIT in remediation efforts, to determine whether obesogens were released into the environment during this incident. We also sought to improve the sensitivity of obesogen detection methods in order to guide post-toxicological chemical assessments. METHODS: Peroxisome proliferator-activated receptor gamma (PPARγ) transactivation assays were used to identify putative obesogens. Solid-phase extraction (SPE) was used to sub-fractionate the water-accommodated fraction generated by mixing COREXIT, cell culture media, and DWH oil (CWAF). Liquid chromatography-mass spectrometry (LC-MS) was used to identify components of fractionated CWAF. PPAR response element (PPRE) activity was measured in PPRE-luciferase transgenic mice. Ligand-binding assays were used to quantitate ligand affinity. Murine 3T3-L1 preadipocytes were used to assess adipogenic induction. RESULTS: Serum-free conditions greatly enhanced the sensitivity of PPARγ transactivation assays. CWAF and COREXIT had significant dose-dependent PPARγ transactivation activities. From SPE, the 50:50 water:ethanol volume fraction of CWAF contained this activity, and LC-MS indicated that major components of COREXIT contribute to PPARγ transactivation in the CWAF. Molecular modeling predicted several components of COREXIT might be PPARγ ligands. We classified dioctyl sodium sulfosuccinate (DOSS), a major component of COREXIT, as a probable obesogen by PPARγ transactivation assays, PPAR-driven luciferase induction in vivo, PPARγ binding assays (affinity comparable to pioglitazone and arachidonic acid), and in vitro murine adipocyte differentiation. CONCLUSIONS: We conclude that DOSS is a putative obesogen worthy of further study, including epidemiological and clinical investigations into laxative prescriptions consisting of DOSS. CITATION: Temkin AM, Bowers RR, Magaletta ME, Holshouser S, Maggi A, Ciana P, Guillette LJ, Bowden JA, Kucklick JR, Baatz JE, Spyropoulos DD. 2016. Effects of crude oil/dispersant mixture and dispersant components on PPARγ activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS #577-11-7) as a probable obesogen. Environ Health Perspect 124:112-119; http://dx.doi.org/10.1289/ehp.1409672.