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The tissues examined were the CE, the CS (to which the endothelium remained attached), and the lens. In both the CS (Fig.?3E) and the CE (Fig.?3F), Sema3A mRNA is at its highest level at E7��which is approximately 100 fold greater than at E14. Then, between E7 and E8 the CS undergoes a Adenine precipitous decrease in its Sema3A mRNA (p?see more To verify that these patterns of expression of Sema3A mRNA in the CS and CE reflect changes in the protein itself, we also performed Western blot for Sema3A in both the CS (Fig.?3H) and CE (Fig.?3I) with ��-tubulin as a loading control. The temporal patterns of these parallel those of the qRT-PCR described above. Lastly, concerning the Sema3A in the lens, both we (described earlier), and others (Chilton and Guthrie, 2003?and?Lwigale and Bronner-Fraser, 2007) have detected this by in situ hybridization. Therefore, to examine whether developmental changes occur in lens Sema3A that would fit into the patterns of innervation we examined the lens by qRT-PCR high throughput screening (Fig.?3G) and western blot (Fig.?3J). The results do not show any significant change in the level of Sema3A expression over the course of corneal innervation (see also Discussion). As the physiological action of a regulatory factor also requires the presence of an appropriate receptor in the target tissue, we examined whether during innervation developmental changes occur in the receptor for Sema3A�CNrp1. For this we analyzed Nrp1 in the OTG by in situ hybridization and qRT-PCR��performed over the same time frame as for the Sema3A. By in situ hybridization (Fig. 4A and B), from E7 through E14, Nrp1 is expressed broadly throughout the OTG. However, the expression appears to decrease in intensity. This decrease was confirmed by qRT-PCR (Fig.?4C), which showed a significant decrease in Nrp-1 mRNA (p?=?0.003) from E7 to E14.