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. 2017 Sep 19;6:e30647. doi: 10.7554/eLife.30647

Figure 4. The BMPs do not act as morphogens either in vivo or in vitro.

(A–I) Chicken spinal cords were electroporated at HH stage 15 with Gfp (A, D, G), [low] Bmp4 (B, E, H) or [high] Bmp4 (C, F, I) under the control of the CAG enhancer, and incubated until HH stage 25. Thoracic transverse sections were labeled with antibodies against pSmad1/5/8 (red, A–C), Lhx2/9 (red, D–F), Isl (green, D–F), Lhx1/5 (red, G–I) and Pax2 (green, G–I). (A–C, J) R-Smad activation increases over a range of ~20% to~130% as the level of Bmp4 misexpression increases. 5 ng/μl CAG::Bmp4, n = 27 sections from 3 embryos, p<0.008 similar to control; 25 ng/ul CAG::Bmp4, n = 14 sections from 2 embryos, p<4.4×10−5; 50 ng/ul CAG::Bmp4, n = 23 sections from 3 embryos, p<2.4×10−8; 500 ng/ul CAG::Bmp4, n = 19 sections from 3 embryos, p<3.6×10−7; Gfp control: n = 27 sections from 3 embryos, p>0.47. (D–I, J) The concentration of BMP4 determines the efficiency of dI1-3 specification in chicken embryos. Thus, high levels of Bmp4 expression direct more Lhx2/9+ dI1s (F, n = 28 sections from 3 embryos, p<1.8×10−16), Lhx1/5+ dI2s (I, n = 22 sections from 3 embryos, p<7.6×10−11) and Isl1+ dI3s (F, n = 28 sections from 3 embryos, p<6.3×10−7) compared to lower concentrations of Bmp4 expression (E, Lhx2/9: n = 29 sections from 3 embryos, p<7.6×10−14; Isl1: n = 29 sections from 3 embryos, p<0.006; H, Lhx1/5: n = 26 sections from 3 embryos, p<5.1×10−7). Expression of Gfp had no effect (Lhx2/9: D, n = 28 sections from 3 embryos, p>0.88; Isl1: D, n = 29 sections from 3 embryos, p>0.89; Lhx1/5: G, n = 29 sections from 3 embryos, p>0.28). (K) Similarly, increasing the concentration of a given BMP improves its ability to direct mESCs towards a specific dorsal fate, as measured by increased gene expression. BMP4: n = 4 independent experiments; BMP5: n = 3 experiments; BMP6: n = 2 experiments; BMP7: n = 2 experiments. Samples were run in triplicate within each experiment. Probability of similarity between control and experimental groups, *p<0.05, **p<0.005, ***p<0.0005, Student’s t-test or Mann Whitney test. Scale bar: 80 µm.

Figure 4—source data 1. The BMPs do not act as morphogens either in vivo or in vitro (experimental data).
elife-30647-fig4-data1.xlsx (126.8KB, xlsx)
DOI: 10.7554/eLife.30647.016

Figure 4.

Figure 4—figure supplement 1. Temporal effect of the BMPs on dorsal IN identity in vitro.

Figure 4—figure supplement 1.

(A–H) Mouse ESCs were treated with BMPs and collected every 3 days to determine how the profile of transcription factor expression changes in response to different concentrations of BMPs over time. In all cases, neither changing the duration of exposure to BMP recombinant proteins, or altering the BMP concentration changes dorsal cell identity as predicted by morphogen models (n = 2 independent experiments/BMP). Probability of similarity between control and experimental groups, *p<0.05, **p<0.005, Student’s t-test.