endothelin receptor br Experimental Procedures br Author Con

Experimental Procedures
Author Contributions
Acknowledgments Our thanks go to Gioia Polidori Francisco for training and discussions, Kate Watt and Yvonne Turnbull for technical and laboratory managerial support, Kadri Oras and Laura Ferguson for experimental support, Po-Lin So and Bruce Conklin (Gladstone Institutes) for providing their unpublished protocols, and Yukio Nakamura for discussion. This research is supported by the British Heart Foundation (PG/12/75/29851) and the Institute of Medical Sciences. A.S.B. was supported by the British Heart Foundation (FS/12/37/29516).
Introduction Down syndrome (DS) is the leading genetic cause of mental impairment (Pulsifer, 1996), resulting from an extra copy of human chromosome 21. Individuals with DS display various phenotypes that affect multiple tissues (Korenberg et al., 1994), the most prevalent of which include cognitive defects, premature Alzheimer\'s disease, aging, and distinct dysmorphic facial features (Briggs et al., 2013; Galdzicki et al., 2001; Roizen and Patterson, 2003). It is thought that the pathologies of DS result from dosage sensitivity of several endothelin receptor that play a role in the development of different tissues, and from inter- and intra-chromosomal regulatory interactions (Briggs et al., 2013). Although chromosome 21 harbors about 350 genes, only a minimal region of about 50 genes within the chromosome is responsible for most of the phenotypes associated with DS. This region, which localizes to the long arm of chromosome 21, is considered the “DS-critical region”, and a third copy of this region is sufficient to cause most of the phenotypes of DS (Briggs et al., 2013; Delabar et al., 1993; Dierssen, 2012; Korenberg et al., 1994; McCormick et al., 1989; Mégarbané et al., 2009; Rahmani et al., 1989). Genes within the DS-critical region also play an important transcriptional regulatory role in different developmental processes. Thus, the effect of the dosage imbalance is not limited to genes on chromosome 21 alone, but also extends to target genes found on other chromosomes. Mouse models for DS have been the primary tool for studying this disorder in past years. The most complex mouse models developed to study DS are either mice containing a third copy of three chromosomal regions orthologous to human chromosome 21, or mice carrying the complete human chromosome 21 as an extra copy (O\'Doherty et al., 2005; Yu et al., 2010). These and other mouse models have proved to be very useful in understanding different aspects of the disorder. However, several DS phenotypes are not recapitulated due to limitations of genetic engineering or inter-species differences (Dierssen, 2012; Olson et al., 2004). The use of embryonic stem cells (ESCs) for disease modeling has enabled the study of numerous human disorders that could not have been modeled in animals due to a lack of relevant phenotypes, appearance of different phenotypes, or even embryonic lethality (Avior et al., 2016; Halevy and Urbach, 2014). In contrast to induced pluripotent stem cells (iPSCs), which are reprogrammed from adult cells, ESC models for human disorders are derived from early embryos that were found to carry a mutation or a chromosomal aberration by preimplantation genetic diagnosis (PGD) or preimplantation genetic screening (PGS), respectively. This difference is important in modeling syndromes such as DS, as only a small fraction of trisomy-21 embryos survive to term (Morris et al., 1999; Spencer, 2001). By analyzing ESCs derived from early-stage embryos, we can study the molecular pathways altered by the presence of a third copy of chromosome 21 more faithfully, as well as the ways in which this chromosomal aberration may affect embryonic development. We have previously isolated three PGS-derived ESC lines with trisomy 21, and suggested that ESCs carrying a third copy of chromosome 21 can be used as an in vitro model for DS (Biancotti et al., 2010). We have further demonstrated by global gene-expression analysis that the third copy of chromosome 21 is actively transcribed in DS-ESCs (Biancotti et al., 2010). In this study, we analyzed neural differentiation of five individual DS-ESC lines to identify molecular and cellular pathways involved in the development of this disease. Our data point to RUNX1, a gene that resides within the DS-critical region, as a key transcriptional regulator in DS neural progenitor cells (DS-NPCs). The contribution of this gene to the molecular phenotype of DS was further validated by its disruption via gene editing.