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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.
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