Extended Data Fig. 2: Effect of morphogen dosages on R-C patterning of µNTLS.
From: A patterned human neural tube model using microfluidic gradients
a. Representative stitched confocal micrographs showing R-C patterned µNTLS on day 7 under different concentrations of CHIR as indicated, stained for OTX2, HOXB1, HOXB4, and HOXC9. Concentrations of RA and FGF8 were kept the same as 500 nM and 200 ng mL−1, respectively. Zoom-in views of boxed regions are shown on the right. Intensity maps depict relative mean values of indicated markers as a function of relative R-C position in µNTLS. For 1.5 µM CHIR, nOTX2 = 12, nHOXB1 = 30, nHOXB4 = 30, nHOXC9 = 18, and nexperiment = 3; For 3 µM CHIR, nOTX2 = 12, nHOXB1 = 22, nHOXB4 = 22, nHOXC9 = 12, and nexperiment = 3; For 4.5 µM CHIR, nOTX2 = 14, nHOXB1 = 28, nHOXB4 = 28, nHOXC9 = 14, and nexperiment = 3. b. Representative stitched confocal micrographs showing R-C patterned µNTLS on day 7 under different concentrations of FGF8 as indicated, stained for OTX2, HOXB1, HOXB4, and HOXC9. Concentrations of CHIR and RA were kept the same as 3 µM and 500 nM, respectively. Zoom-in views of boxed regions are shown on the right. Intensity maps depict relative mean values of indicated markers as a function of relative R-C position in µNTLS. For without FGF8, nOTX2 = 13, nHOXB1 = 25, nHOXB4 = 25, nHOXC9 = 12, and nexperiment = 3; For 200 ng mL−1 FGF8, nOTX2 = 12, nHOXB1 = 22, nHOXB4 = 22, nHOXC9 = 12, and nexperiment = 3; For 400 ng mL−1 FGF8, nOTX2 = 10, nHOXB1 = 20, nHOXB4 = 20, nHOXC9 = 10, and nexperiment = 2. c. Representative stitched confocal micrographs showing R-C patterned µNTLS on day 7 treated with or without RA as indicated, stained for OTX2, HOXB1, HOXB4, and HOXC9. Concentrations of CHIR and FGF8 were kept the same as 3 µM and 200 ng mL−1, respectively. Zoom-in views of boxed regions are shown on the right. Intensity maps depict relative mean values of indicated markers as a function of relative R-C position in µNTLS. For without RA, nOTX2 = 14, nHOXB1 = 39, nHOXB4 = 39, nHOXC9 = 25, and nexperiment = 3; For 500 nM RA, nOTX2 = 12, nHOXB1 = 22, nHOXB4 = 22, nHOXC9 = 12, and nexperiment = 3. d. Plot showing relative R-C positions of OTX2+, HOXB1+, HOXB4+, and HOXC9+ domains in R-C patterned µNTLS on day 7 under indicated conditions. Rostral and caudal ends of µNTLS are designated as 0 and 1, respectively. Error bars represent mean ± s.e.m. n values are provided in a-c. In a-c, nuclei were counterstained with DAPI. Scale bars, 400 µm.
