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First published online 11 August 2004
doi: 10.1242/dev.01313

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The CES-2-related transcription factor E4BP4 is an intrinsic regulator of motoneuron growth and survival

Dirk Junghans^*, Sophie Chauvet, Emmanuelle Buhler, Keith Dudley, Toby Sykes and Christopher E. Henderson

INSERM UMR623, Developmental Biology Institute of Marseille (IBDM), CNRS – INSERM – Université Méditerranée, Campus de Luminy – Case 907, 13288 MARSEILLE Cedex 09, France

View larger version (22K): [in a new window]   Fig. 5. E4BP4 is not regulated by survival signalling pathways in motoneurons. Semi-quantitative RT-PCR and western-blot analysis of E4BP4 expression in cultured motoneurons in the presence or absence of trophic support 6 hours after plating. (A) Radioactive Southern-blot analysis of RT-PCR amplified transcripts. (B) Western blots showing E4BP4 and -tubulin expression. (C) Relative band intensities of RT-PCR and Western-blot analysis. Each band was scanned and its intensity analysed by Image J software. Each single E4BP4 band was normalised with respect to its corresponding actin or tubulin control band and then the values for parallel cultures with or without trophic factors were expressed as an intensity ratio (see Materials and methods). Mean values are plotted (mean±s.e.m.; n=30 independent RT-PCR experiments and n=10 western blots).

View larger version (107K): [in a new window]   Fig. 1. Expression of E4BP4 during motoneuron development. (A) Whole-mount in situ hybridisation for E4BP4 on a flat-mounted E14 rat spinal cord using a mouse probe shows prominent labelling in the lumbar (L) region (arrow), weaker labelling at thoracic levels (T) and no detectable signal at sacral levels (S). (B) In situ hybridisation on sections of E9 chicken embryos using a chicken probe shows prominent labelling at limb-innervating levels (arrow). (C) Higher-magnification picture of the ventral horn of E9 lumbar spinal cord. The major motor columns defined by ISL1 labelling of an adjacent section (not shown) are indicated. MMC, medial motor column; LMCm and LMCl, medial and lateral subdivisions of the lateral motor column. (D) RT-PCR on freshly-purified E14 rat motoneurons (left panel) and chicken embryo spinal cord at indicated ages (right panel). (E) Western blot of chicken E7 spinal cord using an anti-mouse E4BP4 immune serum, performed as described (Ikushima et al., 1997).

View larger version (18K): [in a new window]   Fig. 2. Survival-promoting activity of E4BP4 over-expressed in motoneurons. (A) Effects on motoneuron survival following trophic deprivation. E14 rat motoneurons electroporated with a plasmid encoding GFP (control) or with GFP and E4BP4 were seeded in the absence or the presence (not shown) of the neurotrophic factor BDNF. Survival values were expressed as a percentage of the value in BDNF at the same stage (see text). E4BP4 provided nearly complete protection against cell death at 24 hours or 48 hours after seeding. In long-term cultures (5 days in vitro, 5 d), the survival promoting effect was barely significant. Values are means ± s.e.m.; n=3-5 independent experiments. Statistical significance was calculated by Student's t-test (two-tailed). (B) Protection by E4BP4 against cell death triggered by Fas death receptor. Motoneurons electroporated with control and E4BP4 plasmids were exposed to soluble Fas ligand (sFasL) in the presence of BDNF. Survival is expressed as a percentage of the value in controls without sFasL (mean±s.e.m., n=3). (C) Effects of the PI3K inhibitor LY294002 (LY) on motoneuron survival induced by E4BP4. Survival is expressed as a percentage of the value for control neurons in BDNF. Mean±s.e. m.; n=4.

View larger version (14K): [in a new window]   Fig. 3. E4BP4 enhances cell body growth. (A) Histograms for cell body area from a single experiment. Motoneurons electroporated with GFP were cultured without trophic factors (control), or with neurotrophic factor (BDNF). Motoneurons expressing E4BP4 were cultured without BDNF. Cell body area for 80-200 neurons in each condition was measured 1 day later and expressed in arbitrary pixel units. There is a clear shift to larger sizes in motoneurons expressing E4BP4. (B) Median cell body areas in each condition (mean±s.e.m.; n=5 independent experiments). Statistical significance was calculated by Student's t-test (two-tailed). The addition of BDNF to E4BP4 electroporated motoneurons did not further increase cell body size (7.3±0.3 pixels; n=3).

View larger version (55K): [in a new window]   Fig. 4. E4BP4 potently stimulates axonal outgrowth. (A) Typical images of control and E4BP4-expressing motoneurons after 1 day in culture, as used for subsequent image analysis. Scale bar: 20 µm. (B) Cumulative plot of length of longest neurite from one representative experiment. For each value of neurite length, the percentage of the total number of neurons having a neurite at least that length is plotted. A shift of the curve to the right thus indicates increased axonal growth. Values from control neurons (black line), control neurons with BDNF (broken line) and E4BP4-expressing neurons without BDNF (grey line) are shown. (C) Median values for length of the longest neurite, expressed relative to control neurons with BDNF, fixed as 1 (mean±s.e.m.; n=5 independent experiments).

View larger version (63K): [in a new window]   Fig. 6. E4BP4 reduces programmed cell death of motoneurons in vivo. (A) Scheme showing the electroporation protocol and areas used for TUNEL quantification. (B) High-magnification view of an E5 chicken embryo expressing GFP in ventral roots (arrows), used as a criterion for selecting embryos in which motoneurons were successfully electroporated. (C) Images of one representative spinal cord co-electroporated with GFP and E4BP4, and viewed at E7 using transmission (left panel) or fluorescence (centre panel) optics. In the right panel, the zone of GFP expression has been traced onto the transmission image. The spinal cord has been opened and dorsal regions appear at the left and right of the images, while the midline runs vertically through the tissue. The position of the ventral horns is visualised by the TUNEL signal, as only motoneurons undergo PCD at this stage. Within the area of GFP expression, three zones are apparent. In zone 1, E4BP4 is expressed dorsally to motoneurons and does not affect the TUNEL signal (asterisk). In zone 2, E4BP4 expressed in the motor columns reduces the number of TUNEL-positive cells compared with the contralateral side (compare black and open arrowheads). In zone 3, E4BP4 expressed in motoneurons not already undergoing cell death does not trigger apoptosis (arrow). (D) In each area indicated in A, numbers of TUNEL-positive neurons at E7 were expressed as a percentage of the value on the contralateral side in each spinal cord. Values are shown for embryos electroporated with GFP alone (green bars) or with GFP and E4BP4 (orange bars). Means±s.e.m. are indicated; numbers of embryos analysed are indicated above the bars. Overexpression of E4BP4 reduces the number of dying motoneurons by 45%, whereas GFP alone has no effect.