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First published online 2 June 2004
doi: 10.1242/dev.01128

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Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons

Department of Neuroanatomy, Interdisciplinary Center of Neuroscience, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany

View larger version (85K): [in a new window]   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.

View larger version (57K): [in a new window]   Fig. 2. Axonal outgrowth and survival in vitro. Dissociated E12 ventral midbrain of control (mixture of En1+/–;En2–/– or En2–/–) and En double mutant in 3D collagen matrix (A), on coverslips coated with laminin (B) and a membrane carpet derived from wild-type E12 midbrain (C) after 1 day in culture stained against TH. For the first 24 hours in vitro, dissociated En double mutant mDA neurons are viable and extend axonal processes. (D) The mean length of the TH-positive processes was the same for mutant and littermate controls. The difference was not statistically significant (Student’s t-test, P=0.35). Error bars are not shown, as axonal outgrowth varied between 0.3 and 78 µm. n=number of cells measured out of four independent experiments for each genotype. (E) Average number of TH-positive En double mutant cells counted at several time points. After dissociation, disappearance of mDA neurons was arrested for 24 hours. Thereafter, the mutant cells follow their in vivo counterparts such that almost no TH-positive cells are left 72 hours post dissociation. n=5 independent cell culture experiments for each time point. Error bars indicate s.d. (F-I) Cell culture at about 48 hours post dissociation stained against TH (F,F', green) and activated caspase 3 (G,G', red). Each sample was counterstained with DAPI (H-I') to identify cell nucleus. Mutant mDA neurons (arrows) retract their processes, round up and are positive for activated caspase 3. An additional sign for apoptosis is the pyknotic nuclei. (F'-I') By contrast, TH-positive cells derived from littermate controls possess elongated processes and show no signs of apoptosis (arrows). (I,I') Magnification of H,H'. Scale bars: 20 µm for A-C,F-H; 10 µm in I.

View larger version (67K): [in a new window]   Fig. 3. Cell-mixing experiment. (A-C) After multiple intraperitoneal BrdU injections, almost all cells derived from En double mutants were positive for BrdU (B) when attached to a coverslip after 1 hour. (C) Example of double-labelled TH-positive neurons (arrow) (TH, green; BrdU, red). (D-F) In the mixture of En double mutant cells and control, no TH-positive, BrdU-labelled cells were present after 3 days. (G) Number of TH-positive cells after 24 hours and 72 hours in vitro: control (wild type), mixture of heterozygote (En1+/–;En2–/–) and homozygote (En2–/–). In controls, the number of mDA neurons decreased only slightly between 24 hours and 72 hours. Regardless of culture conditions, the numbers of TH-positive cells derived from engrailed double mutants decreased almost to zero after 72 hours from a baseline of an average of 45 TH-positive mutant cells at 24 hours. n=12 for each bar (Student’s t-test, P<0.001). Error bars indicate s.d. Scale bars: 20 µm.

View larger version (61K): [in a new window]   Fig. 4. RNA interference on primary cell cultures derived from En2–/– ventral midbrain. (A) Numerical chart of RNA interference normalised against mock-transfected control. Only the transfection with En1-specific siRNA oligo duplexes reduced the numbers of TH-positive cells after 96 hours. n=10 independent experiments for each transfection (Student’s t-test, P=0.001 for mock control versus En1 transfection). Error bars indicate s.d. (B) Time course for the loss of mDA neurons after transfection with En1 oligos normalised against mock-transfected controls. Twelve hours after transfection, there were no changes in the numbers of TH-positive neurons; however, a significant proportion of En1-negative cells were present. Number of TH-positive cells gradually decreased until 96 hours when the amount of DA neurons stabilised. Maximum amount of TH-positive and En1-negative cells was detectable at 48 hours. (C-F) Immunohistochemistry on En1 transfected cultures after 48 hours. The transfected TH-positive cells (arrow) are round, have no processes, are En1 negative (red) and possess a fragmented, pyknotic nucleus (DAPI). (C'-F') The mock-transfected control cells (double arrowhead) maintained their neurites and are En1 positive. (F,F') Magnification of DAPI staining in E,E'. (G-J) Confocal image of En1 transfected culture. Three TH-positive neurons are present in this field; one (arrow) has a rounded cell body, is En1 negative and stained positive for activated caspase 3 (blue). Two others (double arrowheads) have spindle shaped cell bodies with attached neurites, are En1 positive and caspase 3 negative. (K-M) Forty-eight hours after transfection with Pbx1-specific RNA duplexes, Pbx1 expression is silenced in some mDA neurons (arrow) but is preserved in the majority (double arrowhead). Regardless of Pbx1 expression, the cells maintained their processes and the shapes of their cell bodies. (N) The degree of silencing 48 hours after transfection with Pbx1- and En1-specific RNA oligos. The same proportion of TH-positive cells, 12%, is negative for Pbx1 and En1. However, the number of TH-positive cells is reduced only after En1 transfection (increase in number after Pbx1 transfection is not statistically significant). Scale bar: 20 µm in C-E,G-M; 10 µm in F.

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