Much of our knowledge on DS comes

Much of our knowledge on DS comes from the documentation of symptoms in patients and analysis of mouse models. Although the contribution of both sources has been crucial in gaining a better understanding of the syndrome, the molecular pathways leading to the development of DS are still largely unknown. The derivation of ESCs with trisomy 21 enabled us to study the molecular processes that underlie DS in human cells and address questions that could not be addressed in other models. In recent years several studies have used both ESCs and iPSCs with trisomy 21 to study different aspects of DS such as hematopoiesis, heart development, and neural differentiation (Bosman et al., 2015; Chang et al., 2015; Chou et al., 2012; Maclean et al., 2012; Murray et al., 2015). Our analysis of DS-NPCs showed aberrant expression of key neuronal genes. In fact, two of the downregulated genes in DS-NPCs, POU3F2 and ASCL1, have been used together with MYT1L for direct conversion of fibroblasts into functional neurons, thus highlighting the developmental perturbation of DS cells (Vierbuchen et al., 2010). In recent years, the role of RUNX1 in neural development has been studied in different models. These studies suggested that RUNX1 plays an important role in the proliferation and differentiation of NPCs, the control of neurite outgrowth, and the impact on axonal pathfinding (Inoue et al., 2008; Theriault et al., 2005; Yoshikawa et al., 2015, 2016). Moreover, RUNX1 has been suggested to play a role in the peripheral and CNS development, in defining different Go 6976 compartments and in consolidation of specific neuronal identity in the developing mouse nervous system (Levanon et al., 2001; Simeone et al., 1995; Stifani et al., 2008; Zagami and Stifani, 2010). However, the role of RUNX1 in human neural development is still obscure. RUNX1 has been associated with DS in terms of its contribution to the increased risk of leukemia as seen in DS patients (De Vita et al., 2010). However, the involvement of RUNX1 in the neural phenotype of the syndrome has not been fully addressed. Based on our experimental data, we suggest that the extra copy of RUNX1 in DS-NPCs may disrupt different molecular pathways during neural development. This in turn could lead to perturbation in forebrain development and increased apoptosis as indicated by our data. In this study, we disrupted the expression of RUNX1 to demonstrate the importance of this gene in the phenotypes of DS. Ablation of RUNX1 resulted in downregulation of key developmental genes and cellular pathways related to neuron migration and cell growth, with reduced apoptosis in gene-edited DS-NPCs. These results highlight the importance of dosage balance of RUNX1 in DS cells. Our results are supported by a study based on a meta-analysis of DS, suggesting that RUNX1 is a transcription regulator that has a global dosage effect on other chromosomes, affecting genes related to CNS development and neuron differentiation (Vilardell et al., 2011). One hallmark of the facial phenotypes of DS patients is a protruding tongue and speech impediment. It was previously shown that these phenotypes of DS patients are, at least partially, the result of abnormal neuromuscular junctions in tongue muscles (Yarom et al., 1986). Interestingly, a recent study of RUNX1 demonstrated its involvement in the axonal pathfinding to specific tongue muscles (Yoshikawa et al., 2015). Our work links the roles of RUNX1 in the development of the nervous system to the neural phenotype observed in DS patients and suggests that this gene carries out a key function in the development of several of the phenotypes seen in DS. The expression patterns and role of RUNX1 in the human developing peripheral and central nervous systems should be furthered explored. Understanding the molecular processes underlying DS will help in the search for targeted therapy and provide further insights into the genetic dosage imbalance associated with this syndrome.