Figure 5. TGF-β signaling is essential for the development of midbrain DA neurons in vivo. Chick embryos were treated with a function-blocking specific anti-TGF-β antibody from E2–7, E4–7, or E6–10, thereafter processed for TH immunohistochemistry, and TH-ir neurons were counted in the midbrain (n = 4), diencephalon (n = 3), and hindbrain (locus coeruleus;n = 3) as described in Materials and Methods. Representative frontal sections are shown, taken at the same level from control embryos, treated with vehicle alone (A, D, G), or embryos treated with neutralizing antibody from E2 to E7 (B, E, H). The small insets in A and B demarcate regions shown at higher magnification in the second, larger inset in the top right corner of the representative pictures. In the absence of TGF-β, there are only a few TH-positive neurons left in the midbrain, whereas in the diencephalon and locus coeruleus there is no significant change. Note that the nucleus tegmenti-pedunculo-pontinus of the chicken is the homolog of the mammalian substantia nigra. v, Ventricle. Scale bar: A, B, D, E, G, H,50 μm. C, F, I, K, L, Quantitation of these experiments is shown. Results are expressed in absolute numbers and given as mean ± SEM. *p < 0.05, ***p < 0.001.