Fig. 1. Loss of midbrain dopaminergic neurons in engrailed double mutant embryo by apoptosis. (A) E12 whole-mount preparation of isolated neural tube. TH-positive neurons are located in the mesencephalic flexure (arrow) of wild-type (A) and mutant (A') embryo. The TH domain in the mutant is smaller than the wild type and there are no axons heading in rostral direction. (B) Midsagittal sections of E14 embryos. In the wild type (B), mDA neurons have continued to differentiate and start to form the SNC and VTA (arrow). In the mutant embryos (B'), no TH-positive cells are detectable in the ventral midbrain. Additionally, the anlage for the cerebellum (Cb), inferior colliculus (IC) and superior colliculus (SC) are absent. (C-E) Transverse sections of E12 En1⁺/tlz;En2^–/– ventral midbrain immunostained against TH (green) and the En1 reporter, ß-gal (red). (E) Merged image of C and D. The majority of TH-positive cells do not express En1. (F-H) 48 hours later at E14 at the same level, almost all TH (green)-positive cells express the En1 reporter (red). (H) Merged image of F and G. (I-K) Coronal section of ventral midbrain of E13.5 En1^–/–;En2^–/– embryo. A rounded TH-positive cell body is detectable (I, arrow). This cell is positive for activated caspase 3 (J, arrow) and exhibits a condensed and fragmented nucleus (K, arrow) revealed by DAPI staining. (K, inset) Magnification of the pyknotic nucleus (arrow). A,B rostral is towards the right; C-K is dorsal towards the top. Scale bars: 200 µm in A,B; 50 µm in C-K.