Spatiotemporal analysis of apoptosis in the neuroepithelium. TUNEL (A–C) and activated caspase-3 (D–F) staining of CD1 embryos at E8.5 (A and D), E9.0 (B and E), and E9.5 (C and F) reveals the presence of apoptotic cells at several sites in the open neural folds and closed neural tube. Scattered TUNEL-positive (blue) cells are seen throughout the neural folds (A and B) and are particularly abundant in the forebrain neural folds (arrow in A), in a rhombomeric pattern within the hindbrain (arrowheads in A and B), and in the dorsal midline of the closed neural tube (black arrows in B and C). The locations of cells positive for activated caspase-3 (brown staining, arrowheads in D–F) correspond to sites of abundant TUNEL staining. Representative sections in D–F are from levels indicated by dotted lines in A–C, respectively. Drawings (G–I) summarize the main sites of apoptosis (in red) seen at E8.5 (G), E9.0 (H), and E9.5 (I). At least 8 embryos per stage were analyzed. (Scale bar: 0.1 mm.)

Genetic inhibition of apoptosis does not prevent neurulation in forebrain or spine. (A–C) Whole-mount TUNEL staining of Casp3 wild-type (A and B) and null (C) embryos. Insets show transverse sections through the spinal region at levels indicated by dashed lines in A–C. Despite exposure to identical staining conditions, there is a complete lack of TUNEL-positive cells in mutant embryos (C), whereas wild-type littermates exhibit plentiful apoptosis (white arrowheads in A and B), in a pattern comparable to that seen in CD1 embryos (see Fig. 1). Note that the neural tube has closed in the forebrain and spinal region of the Casp3^−/− embryo, whereas the caudal midbrain and hindbrain have remained open (i.e., exencephaly, between the yellow arrows). (D–I) Transverse sections through the spinal neural tube (at axial levels shown in F and I) of Apaf1 wild-type (D and G) and null (E and H) embryos immunostained for activated caspase-3. In the mutant embryos, no staining is evident in either closed (E) or open (H) neural tube, whereas wild-type littermates exhibit immunoreactive cells in the neuroepithelium at both closed (D) and open (G) neural tube levels. The Apaf1^−/− embryos exhibit midbrain/hindbrain exencephaly similar to that of the Casp3^−/− embryos, whereas the neural tube is closed in forebrain and spinal regions (not shown). (Scale bars: 0.1 mm.)

Pifithrin-α and z-VAD-fmk suppress apoptosis in cultured embryos. (A–F) TUNEL staining of DMSO-treated control (A, C, and E), pifithrin-α–treated (B), and z-VAD-fmk–treated (D and F) E8.5 embryos after 4 h (A–D) and 8 h (E and F) of culture. TUNEL-positive cells are clearly visible in the control embryos (e.g., arrowhead in C) but are absent from the treated embryos. (G) After 6 h of culture, there are no activated caspase-3–positive cells in the spinal neural tube immediately rostral to the site of closure in the z-VAD-fmk–treated embryos, whereas the number of apoptotic cells is significantly reduced in embryos treated with pifithrin-α (dark-gray bar) compared with controls (black bar). After 16 h of culture, the number of activated caspase-3–positive cells in the spinal neural tube rostral to the site of closure remains significantly diminished in embryos treated with z-VAD-fmk (light-gray bar) and pifithrin-α (dark-gray bar) compared with controls (black bar) (*P < .05; 1-way ANOVA with Dunn's correction). Ten sections were counted from each of 2 embryos in each experimental group. (H) Time line of embryo cultures in the study. Numbers indicate somite stages, and yellow lines (divided into hourly intervals) indicate the duration of cultures designed to evaluate the effect of inhibition of apoptosis on neurulation. Red portions of the lines indicate the stage at which particular events of neural tube closure occur. Thus, closure initiation (closure 1) and the events of cranial neurulation (closures 2 and 3) occurred ≈6–7 h into each culture period, when apoptosis inhibition was maximal. Neural fold remodeling and posterior neuropore closure were assessed ≈12–16 h into the second culture period, when apoptosis was significantly inhibited but not maximal.

Inhibition of apoptosis does not adversely affect spinal neurulation. After overnight culture in the presence of apoptosis inhibitors, dimensions of the posterior neuropore indicate the progression of spinal neurulation. No differences in posterior neuropore length (A) or width (B) were noted between the experimental groups at each somite stage analyzed. Note the progressive decline in both parameters with increasing somite stage. A minimum of 3 embryos per experimental group were assessed at each somite stage. Compared with control embryos (C), culture in the presence of pifithrin-α (D) or z-VAD-fmk (E) had no apparent effect on mitotic activity, as indicated by immunostaining for phospho-histone H3 (pink-stained cells in C–E). (F) Mitotic index, calculated for the 5 sections rostral to the site of neural tube closure (3 embryos per experimental group), did not differ between treatment groups (P >.05; 1-way ANOVA). Graph bars represent mean ± SE.

Apoptosis is not required for neural fold remodeling postfusion. Transverse sections through the spinal neural tube of stage-matched (19 somites) control (A, D, and G), pifithrin-α–treated (B, E, and H), and z-VAD-fmk–treated (C, F, and I) embryos, immunostained for activated caspase-3 (A–C) or E-cadherin (D–I). Note the lack of apoptosis in the inhibitor-treated embryos (B and C) ≈12 h into the culture period compared with the control embryos, in which caspase-3–positive cells are seen both in the midline (arrow in A) and elsewhere. At an axial level immediately rostral to the site of neural fold fusion, the E-cadherin–positive surface ectoderm (arrowhead in E) is already intact in all treatment groups (D–F), indicating completion of neural fold remodeling. The midline dorsal indentation indicates the close proximity of the section to the point of neural fold remodeling. The surface ectoderm also is intact at a more rostral level, where remodeling occurred earlier (G–I). Breaks in the surface ectoderm lateral to the neural tube represent histological processing artifacts. Abbreviations: g, hindgut; n, neural tube. (Scale bar: 0.1 mm.)

Neural crest migration from the hindbrain does not appear to be affected by inhibition of apoptosis. Whole-mount in situ hybridization for the neural crest marker cadherin-6 in control (A), pifithrin-α–treated (B), and z-VAD-fmk–treated (C) embryos reveals no differences in the pattern of NCC emigration. (D–F) High-magnification views of the white boxed areas shown in A–C. Two distinct streams of NCC (I and II; arrows in A) are seen to migrate toward the first and second branchial arches. In the inhibitor-treated embryos (B, C, E, and F), the separation of migration streams is retained, and there is no obvious reduction in NCC number. A minimum of 3 embryos per experimental group were analyzed. Because z-VAD-fmk treatment causes slight growth retardation, some embryos were cultured for an additional 4 h, to allow comparison with stage-matched controls (inset in C, with concurrently hybridized control shown in the inset in A). (Scale bar: 0.2 mm.)