Developmental basis of trachea-esophageal birth defects.

Trachea-esophageal defects (TEDs), including esophageal atresia (EA), tracheoesophageal fistula (TEF), and laryngeal-tracheoesophageal clefts (LTEC), are a spectrum of life-threatening congenital anomalies in which the trachea and esophagus do not form properly. Up until recently, the developmental basis of these conditions and how the trachea and esophagus arise from a common fetal foregut was poorly understood. However, with significant advances in human genetics, organoids, and animal models, and integrating single cell genomics with high resolution imaging, we are revealing the molecular and cellular mechanisms that orchestrate tracheoesophageal morphogenesis and how disruption in these processes leads to birth defects. Here we review the current understanding of the genetic and developmental basis of TEDs. We suggest future opportunities for integrating developmental mechanisms elucidated from animals and organoids with human genetics and clinical data to gain insight into the genotype-phenotype basis of these heterogeneous birth defects. Finally, we envision how this will enhance diagnosis, improve treatment, and perhaps one day, lead to new tissue replacement therapy.

Generation of esophageal organoids and organotypic raft cultures from human pluripotent stem cells.

The human and murine esophagus have some substantial differences that limit the utility of mouse as a model to study human esophagus development and disease. Due to these limitations several recent reports describe the development of methods to generate human esophageal tissues via the directed differentiation of pluripotent stem cells. Methods for differentiation are based on knowledge of years of studying embryonic development of the esophagus in vertebrate animal models. Esophageal tissues derived from human pluripotent stem cells have been used to study both development and diseases affecting the esophagus. Here, we provide a detailed protocol for the directed differentiation of human pluripotent stem cells into human esophageal organoids and organotypic raft cultures, that are highly similar, morphologically and transcriptionally, to the human esophagus epithelium. We discuss limitations of the current esophageal models and the importance of engineering more complex tissue models with muscle and enteric nerves. Moving forward, these models might be utilized for the development of personalized treatments, as well as other therapeutic solutions.

Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration.

Apoptotic cells expose Phosphatidylserine (PS), that serves as an "eat me" signal for engulfing cells. Previous studies have shown that PS also marks degenerating axonsduring developmental pruning or in response to insults (Wallerian degeneration), but the pathways that control PS exposure on degenerating axons are largely unknown. Here, we used a series of in vitro assays to systematically explore the regulation of PS exposure during axonal degeneration. Our results show that PS exposure is regulated by the upstream activators of axonal pruning and Wallerian degeneration. However, our investigation of signaling further downstream revealed divergence between axon degeneration and PS exposure. Importantly, elevation of the axonal energetic status hindered PS exposure, while inhibition of mitochondrial activity caused PS exposure, without degeneration. Overall, our results suggest that the levels of PS on the outer axonal membrane can be dissociated from the degeneration process and that the axonal energetic status plays a key role in the regulation of PS exposure.