Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling

We directed mESCs towards the dorsal sensory interneurons (dIs) using RA-/+ BMP4 and tested the effects of an axon guidance molecule netrin1 on dI differentiation in vitro. Using bulk-RNA seq analysis, we investiagted when the addition of netrin 1 will affect the transcriptome of differentiating dIs. For this analysis, we used three timewindows (conditions 1-3) for netrin addition (1) condition 1 netrin 1 was added together with RA+BMP4 at day 4, and RNA seq was conducted at day 5, (2) netrin1 was added after the RA+BMP4 mediated patterning at day 5 and RNA seq was conducted at day 6, and (3) netrin1 was added for the extended duration between day 5-day9 to investigate the netrin1 effects throughout the dI differentiation and RNA seq was conducted at day 9. Through this analysis we identified that netrin1 downregulate BMP signaling pathway, possibly through modlulating the mRNA processing of key mediator of BMP signaling such as Id genes and BMP signaling inhibitor Smad7 and affect dI differentiation. This is the first report to show that netrin1 regulate dI fate specification in the dorsal spinal cord. Overall design: 3- replicate samples were collected at day 5 (condition 1), and day 6 (condition 2), while 2 replicates passed the quality control for day 9 RNA seq (condition 3), Total 16 samples were collected from 3-independent differentiations.