Met-induced Pea3 propagation can be executed through PI3K and Src pathways. (A) ISH analysis of the establishment of Pea3 expression domain in spinal MNs . Images show brachial region of flat-mounted E12.5 spinal cords hybridised with Pea3 probes in WT, metd/d, met2P/2P, and met2S/2S embryos. Whereas Pea3 MN pool is reduced in metd/d, it appears normal in met2P/2P and met2S/2S embryos. (B) Quantifications of the lateral expansion of the Pea3 domain by signal intensity analysis: each plot represents the average signal distribution measured on the indicated number of spinal cord sides along the red dotted line as positioned in each image in (A) (WT: n=14; metd/d: n=12; met2P/2P: n=6; met2S/2S: n=6). (C): Brachial regions of flat mounted spinal cords stained with X-Gal to reveal β-galactosidase activity of the Pea3LacZ knock-in. This context with only one functional copy of Pea3 is sufficient for complete Pea3 propagation by Met2P, but is not permissive for Met2S receptor. There was a lack of propagation in metd/d: Pea3LacZ/+ as compared to met+/+:Pea3LacZ/+ spinal cords. (D) Quantifications of the lateral expansion of the Pea3/β-galactosidase-positive domain by signal intensity analysis: each plot represents the average signal distribution as measured on the indicated number of spinal cord sides (WT: n=8; metd/d: n=6; met2P/2P: n=8; met2S/2S: n=8) along the red dotted line as positioned in each image in (C). (E,F) Quantifications and statistical analyses of the sum of the signal intensities corresponding to measurements of Pea3 ISH in a Pea3+/+ context (B), or for X-gal staining in a Pea3LacZ context (D). Each dot represents the sum of intensity for one sample, the number of samples being as indicated in (B) and (D). The absence of one Pea3 functional copy in a Pea3LacZ context reveals a significant difference in Pea3 dosage requirement between met2P/2P and met2S/2S embryos.