Essential role for survivin in early brain development

Abstract

Apoptosis is an essential process during normal neuronal development. Approximately one-half of the neurons produced during neurogenesis die before completion of CNS maturation. To characterize the role of the inhibitor of apoptosis gene, survivin, during neurogenesis, we used the Cre-loxP-system to generate mice lacking survivin in neuronal precursor cells. Conditional deletion of survivin starting at embryonic day 10.5 leads to massive apoptosis of neuronal precursor cells in the CNS. Conditional mutants were born at the expected Mendelian ratios; however, these died shortly after birth from respiratory insufficiency, without primary cardiopulmonary pathology. Newborn conditional mutants showed a marked reduction in the size of the brain associated with severe, mutifocal apoptosis in the cerebrum, cerebellum, brainstem, spinal cord, and retina. Caspase-3 and caspase-9 activities in the mutant brains were significantly elevated, whereas bax expression was unchanged from controls. These results show that survivin is critically required for the survival of developing CNS neurons, and may impact on our understanding of neural repair, neural development, and neurodegenerative diseases. Our study is the first to solidify a role for survivin as an antiapoptotic protein during normal neuronal development in vivo.

Figure 1.

Brain malformation in nestin-cre;survivin^lox/lox mice. a, Whole mounts of E17.5 mice showing the abnormal head morphology of a mutant (MUT) compared with a wild-type (WT) littermate. b, Dissected brain from mutant and wild-type embryos at E17.5. c, Sagittal H&E-stained sections of mice at P0 showing the brain structures of survivin mutant and wild-type animals. The cerebral cortex (CC) and cerebellum (CB) were absent in the mutant brains. The brainstem (BS) was relatively preserved but smaller in the mutant than in the wild-type animals.

Figure 2.

H&E-stained transverse sections at E13.5 showing multifocal destruction of brain architecture within the developing brain of the mutant embryos. Scale bars, 50 μm. WT, Wild type; MUT, mutant; A, anterior; P, posterior.

Figure 3.

H&E-stained sections from pial (top) to ventricular (bottom) surface at E9.5-E12.5 showing progressive destruction of cortical architecture within the developing cerebral cortex of the mutant embryos. Scale bars, 500 μm. WT, Wild type; MUT, mutant.

Figure 4.

Top, TUNEL assays showing progressive increases in apoptotic cell death within the cerebral cortex of the mutant embryos, starting at E10.5. Scale bars, 500 μm. Bottom, A histogram depicts the average number of TUNEL-positive cells per area for the wild-type (WT) and mutant (MUT) animals. ^*Statistically significant difference (p = 0.01). Error bars indicate SD.

Figure 5.

Top, PCNA staining showing small differences in the number of proliferating cells within the cerebral cortex between mutant and wild-type animals. Scale bars, 500 μm. Bottom, A histogram depicts the average number of PCNA-positive cells per area for the wild-type (WT) and mutant (MUT) animals. No statistical differences were observed. Error bars indicate SD.

Figure 6.

Top, PHH3 expression showing a moderate decrease in the number of cells in G₂/M in the cerebral cortex of the mutant embryos. Scale bars, 500 μm. Bottom, A histogram depicts the average number of PHH3-positive cells per area for the wild-type (WT) and mutant (MUT) animals. ^*Statistically significant difference (p = 0.02). Error bars indicate SD.

Figure 7.

Activated caspase expression in the developing cortex of nestin-cre;survivin^lox/lox embryos. Top, Sections from the cerebral cortex from E9.5 through E12.5 were stained with activated caspase-3 antibodies. A progressive increase in expression of activated caspase-3 is seen, starting at E10.5. Scale bars, 50 μm. Bottom, A histogram depicts the average number of caspase-3-positive cells per area for the wild-type (WT) and mutant (MUT) animals. ^*Statistically significant difference (p < 0.001). Error bars indicate SD.

Figure 8.

Functional caspase activity in the developing cortex of nestin-cre;survivin^lox/lox embryos. Developing cortical tissues were evaluated for functional caspase-3/7, -8, and -9 activities at E10.5 and E12 using a luciferase-based chemical assay system (Caspase-Glo; Promega). Relative luciferase units (RLU) are proportional to functional caspase activity, as indicated by the manufacturer. Gray columns represent the samples from wild-type animals, and black columns represent the samples from the mutants. Three animals in each group were used (n = 3). ^*Statistical significance (p < 0.001 for all). Error bars indicate SD.

Figure 9.

Effects of a lack of survivin within the developing spinal cord. a, Sections of the developing spinal cord from E12 embryos were stained with H&E. Neuronal disorganization and cell death is observed. DRG, Dorsal root ganglia; D, dorsal; V, ventral. Scale bars, 100 μm. b, Sections of spinal cords from mutant (MUT) mice and wild-type (WT) littermates at P0 were stained with GFAP. Giant cells observed in the mutant animals expressed GFAP, suggesting they are of an astrocytic lineage. Scale bars, 25 μm.

Figure 10.

Retinal defects in nestin-cre;survivin^lox/lox mice. TUNEL assays show increased apoptosis in the retina at E17.5 in the nestin-cre;survivin^lox/lox embryos. Apoptotic nuclei appear dark red. Scale bars, 50 μm. WT, Wild type; MUT, mutant.