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First published online 21 March 2007
doi: 10.1242/dev.02838

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Specific ablation of the transcription factor CREB in sympathetic neurons surprisingly protects against developmentally regulated apoptosis

Rosanna Parlato^*, Christiane Otto^*,, Yvonne Begus, Stephanie Stotz and Günther Schütz

Department of Molecular Biology of the Cell I, German Cancer Research Center, D-69120 Heidelberg, Germany.

View larger version (129K): [in this window] [in a new window]   Fig. 1. Generation of a CREB mutation selective for sympathetic neurons. (A) Schematic representation of the DBHiCre PAC used for transgenesis. The linear insert of 150 kb carrying the transgene construct contains approximately 100 kb upstream of the ATG of the Dbh gene. (B) Detection of DBHCre activity at E11.5 in the reporter mouse line ROSA26 by whole-mount ß-galactosidase staining in sagittal sections at different levels of the sympathetic chain, from rostral to caudal (i), (ii) and (iii). Adjacent sagittal sections from E12.5 control (Crebfl/fl) and mutant (CrebDBHCre) embryos are stained with anti-DBH (C,F), anti-Cre (D,G) and anti-CREB (E,H) antibodies. At E12.5, DBH expression characterizes sympathetic precursors in control (C) and mutant (F) embryos. In control Cre-negative precursors, CREB protein is present (D,E), whereas Cre-positive cells lose CREB immunoreactivity in the conditional mutants (G,H). Scale bar: 2 mm in B; 130 µm in C-H.

View larger version (128K): [in this window] [in a new window]   Fig. 2. CrebDBHCre mutants show no gross morphological abnormalities in the sympathetic ganglia. Sagittal sections from control (Crebfl/fl) and mutant (CrebDBHCre) embryos at E17.5 are stained with anti-CREB antibody (A,B). CREB immunoreactivity is indicated in the SCG (enclosed area) by arrows in neurons, and by arrowheads in other morphologically distinct cells. Nissl staining reveals no major differences between control (C) and mutant (D). Whole-mount immunohistochemistry with TH antibody of P2 control (E) and CrebDBHCre (F) animals reveals no major deficits in ophthalmic projections (arrows). e, eye. Scale bar: 100 µm in A,B; 50 µm in C,D; 500 µm in E,F.

View larger version (121K): [in this window] [in a new window]   Fig. 3. Defects in migration of SCG cells in Creb-null mice. Parasagittal sections of E15.5 mouse embryos immunostained with TH antibody show the SCG normally shaped and located in proximity to the inner ear (asterisk) in control (A) and CrebDBHCre; Crem-/- mutants (B), but not in Creb-/- mutants (F). The stellate ganglion is located in the thoracic region in the CrebDBHCre; Crem-/- mutant (D) and in the respective control littermate (C) as well as in the control littermate of Creb-/- (E). However, in Creb-/- it extends more rostrally (F). At E17.5 a similar pattern is found in Creb-/- showing less sympathetic neurons in the area of the SCG (H, circled area) and more in the area of the stellate ganglion in comparison with control (G). Black arrowhead indicates the tubercle of the first rib, used as a positional reference to compare the position of the stellate ganglia. Scale bar: 300 µm in A-F; 600 µm in G,H.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Survival of sympathetic neurons in absence of CREB. Immunohistochemistry with an antibody recognizing activated caspase-3 (brown) is used to analyze survival of sympathetic neurons in the SCG at E17.5, and anti-TH antibody (blue) is used to identify the region of interest in controls (A,D), Creb-/- (B,E) and CrebDBHCre; Crem-/- mutants (C,F). Representative sections are shown in A-F. Red arrows indicate examples of activated caspase-3-positive cells. (G) Quantitative analysis of apoptotic cells reveals reduced levels of apoptosis in sympathetic neurons of CrebDBHCre; Crem-/- mutants (abbreviated as M) at E17.5 in comparison with controls (abbreviated as C). At postnatal stages P0/P2 the level of apoptosis is reduced in control pups and does not change in CrebDBHCre; Crem-/- mutants. (H) The size of the SCG in CrebDBHCre; Crem-/- mutants is similar between controls and mutants at E17.5. At P0/P2 the size of the SCG in CrebDBHCre; Crem-/- mutants is significantly larger than in controls. The mean±s.e.m. for both SCG in at least four to five mice of each genotype are shown. Values are considered significantly different with *P<0.05 and ***P<0.001 compared with controls. Scale bar: 250 µm in A-C; 40 µm in D-F. Asterisk indicates the inner ear.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Neither CREM nor ATF-1 influence survival of sympathetic neurons. Quantitative analysis of apoptotic cells reveals reduced levels of apoptosis in sympathetic neurons of CrebDBHCre, CrebDBHCre; Crem+/-, CrebDBHCre; Crem-/- and CrebDBHCre; Atf-1-/- mutants at E17.5 in comparison with the respective controls. The mean±s.e.m. for both SCGs in at least three mice of each genotype are shown. Values are considered significantly different with *P<0.05 compared with controls.

View larger version (100K): [in this window] [in a new window]   Fig. 6. p75NTR expression in sympathetic neurons is dependent on CREB. Non-radioactive in situ hybridization with a riboprobe specific for the p75NTR on representative sagittal sections from control (A,C) and CrebDBHCre; Crem-/- (B,D) showing the SCG (A,B) and basal cholinergic neurons (C,D) at E17.5. Asterisk indicates the inner ear. Immunohistochemistry with an antibody recognizing the p75NTR protein is used to analyze protein expression in the SCG at E17.5 in controls (E) and in CrebDBHCre; Crem-/- (F). Scale bar: 250 µm in A-D; 40 µm in E,F; 20 µm in insets.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Schematic representation of the sympathetic neurons in wild type and in the different CREB mutants. Survival of developing neurons depends on availability of NGF produced by target organs (black dots). Neurons not receiving enough neurotrophin undergo apoptotic cell death (broken line). Black and white nuclei indicate the presence or absence of CREB. In Creb-/- mutants CREB expression is lost in the developing neurons and also in the target cells, where CREB may be required to express molecular cues prior to neurotrophin dependence. Loss of CREB in sympathetic neurons and target cells results in partial misplacement of the neurons and in increased neuronal death, possibly exacerbated by the reduced neurotrophic supply. In the conditional mutants, CREB expression is lost only in noradrenergic neurons of sympathetic ganglia, resulting in an increased number of surviving neurons. The neurotrophic supply from target organs is probably unaffected. This scenario indicates cell autonomous pro-survival mechanisms operative in the absence of CREB.