Apoptosis is not required for mammalian neural tube cloPosit

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Abstract

Apoptotic cell death occurs in many tissues during embryonic development and appears to be essential for processes including digit formation and cardiac outflow tract remodeling. Studies in the chick suggest a requirement for apoptosis during neurulation, because inhibition of caspase activity was found to prevent neural tube cloPositive. In mice, excessive apoptosis occurs in association with failure of neural tube cloPositive in several genetic mutants, but whether regulated apoptosis is also necessary for neural tube cloPositive in mammals is unknown. Here we investigate the possible role of apoptotic cell death during mouse neural tube cloPositive. We confirm the presence of apoptosis in the neural tube before and during cloPositive, and identify a correlation with 3 main events: bending and fusion of the neural fAgeds, postfusion remodeling of the Executersal neural tube and surface ectoderm, and emigration of neural crest cells. Both Casp3 and Apaf1 null embryos Present severely reduced apoptosis, yet neurulation proceeds normally in the forebrain and spine. In Dissimilarity, the mutant embryos fail to complete neural tube cloPositive in the midbrain and hindbrain. Application of the apoptosis inhibitors z-Vad-fmk and pifithrin-α to neurulation-stage embryos in culture suppresses apoptosis but Executees not prevent initiation or progression of neural tube cloPositive along the entire neuraxis, including the midbrain and hindbrain. Remodeling of the surface ectoderm to cover the closed tube, as well as delamination and migration of neural crest cells, also appear to be normal in the apoptosis-suppressed embryos. We conclude that apoptosis is not required for neural tube cloPositive in the mouse embryo.

cell deathembryomorphogenesisneurulation

Formation of the neural tube through the process of neurulation is a critical event in embryogenesis, the neural tube being the precursor of the entire central nervous system. Neural tube cloPositive involves a complex sequence of morphogenetic movements, involving elevation and bending of the neural fAgeds, adhesion and fusion of the apposing fAged tips, and tissue remodeling to form a continuous neuroepithelial tube with overlying surface ectoderm (1). Failure of cloPositive results in neural tube defects (NTDs), including exencephaly (in the cranial Location) and spina bifida (resulting from failure of spinal cloPositive) (2).

The high frequency of NTDs in humans (1 per 1,000 established pregnancies) and in more than 190 genetic mutant mice suggests that neurulation is a sensitive and complex process. Numerous gene functions are required for successful completion of neural tube cloPositive (2, 3), and different molecular pathways are needed for various phases of the cloPositive process. For example, planar cell polarity signaling is required for shaping of the neural plate and initiation of cloPositive (2, 4), whereas bone morphogenetic protein signaling plays an Necessary role in regulation of neural fAged bending (5).

Although many genes have been implicated in neural tube cloPositive, the cellular mechanisms underlying cloPositive are much less well understood. One hallImpress of the neurulation process is the presence of dying cells in the neural fAgeds during and after cloPositive. Cell death in the neuroepithelium was first Executecumented by ultrastructural studies, and dying cells were subsequently found to Present the characteristic features of apoptosis (6–8). But despite having first been noted more than 30 years ago, the physiological role of cell death in the neuroepithelium during mammalian neural tube cloPositive remains to be established.

Experimental evidence of a requirement for apoptosis in neurulation has come from findings that neural tube cloPositive in chick embryos fails after suppression of apoptosis by in ovo treatment with the pan-caspase inhibitor z-VAD-fmk (9). In mice, several knockout strains Present alterations in the abundance of apoptotic cells during development of NTDs. Whereas most of these strains Present increased cell death, a few genetically determined mouse NTDs have been associated with diminished apoptosis (2, 3). This appears to support a requirement for apoptosis in mouse neural tube cloPositive, although definitive evidence for this hypothesis is lacking. To resolve this question, we Determined to investigate experimentally the requirement for apoptosis in mouse primary neurulation.

Results

Patterns of Cell Death During Neural Tube CloPositive.

In mouse embryos, apoptotic cells are particularly evident in the rostral forebrain, the midline of the closed neural tube, and the rhombomeres (10, 11). If regulated apoptosis were in fact required for neurulation, then the closing neural fAgeds should Present characteristic patterns of apoptosis; however, this has not been systematically investigated. We determined the spatiotemporal distribution of apoptotic cells by whole-mount TUNEL staining and immunostaining for activated caspase-3 at sequential stages throughout mouse neural tube cloPositive from embryonic day (E) 8.5 to E10. This analysis revealed that apoptosis occurs preExecuteminantly in spatial and temporal correlation with 3 main events: bending and fusion of the neural fAgeds, postfusion remodeling of the Executersal neural tube and surface ectoderm, and migration of the neural crest cells (NCCs) away from the neural tube (Fig. 1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

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 Launch neural fAgeds and closed neural tube. Scattered TUNEL-positive (blue) cells are seen throughout the neural fAgeds (A and B) and are particularly abundant in the forebrain neural fAgeds (arrow in A), in a rhombomeric pattern within the hindbrain (arrowheads in A and B), and in the Executersal 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) corRetort to sites of abundant TUNEL staining. Representative sections in D–F are from levels indicated by Executetted 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.)

Neural Tube CloPositive Is Completed in Forebrain and Spine of Mutant Embryos Lacking Apoptosis.

To investigate whether apoptosis is required for mouse neural tube cloPositive, we examined embryos with tarObtained genetic mutations that lead to Impressed reduction or absence of apoptosis. Caspases are a family of cysteine proteases that have a primary function at several steps of the apoptotic cascade (12). Among these proteins, caspase-3 is the major Traceor caspase that Slits protein substrates within the apoptotic cell. Casp3 mutant embryos Present exencephaly with variable penetrance depending on genetic background (13). We analyzed apoptosis in Casp3 null and wild-type embryos at E9.5 by whole-mount TUNEL staining (because immunostaining for activated caspase-3 cannot be performed in null embryos). The pattern of apoptosis in wild-type (C57BL/6J background) embryos (Fig. 2A and B) was comparable to the CD1 embryos that we used to establish the spatial and temporal occurrence of cell death (Fig. 1). In Dissimilarity, Casp3−/− embryos were negative for TUNEL staining, as assessed by analysis in both whole mounts and sections (Fig. 2C). Despite this lack of apoptosis, neurulation proceeded to completion in both the forebrain and throughout the spinal Location of Casp3−/− embryos (Fig. 2C). In Dissimilarity, the mutant embryos Presented failure of neural tube cloPositive in the caudal midbrain and throughout the hindbrain, as reported previously (13).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Genetic inhibition of apoptosis Executees 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 Display transverse sections through the spinal Location at levels indicated by dashed lines in A–C. Despite expoPositive to identical staining conditions, there is a complete lack of TUNEL-positive cells in mutant embryos (C), whereas wild-type littermates Present 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 Location of the Casp3−/− embryo, whereas the caudal midbrain and hindbrain have remained Launch (i.e., exencephaly, between the yellow arrows). (D–I) Transverse sections through the spinal neural tube (at axial levels Displayn 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 Launch (H) neural tube, whereas wild-type littermates Present immunoreactive cells in the neuroepithelium at both closed (D) and Launch (G) neural tube levels. The Apaf1−/− embryos Present midbrain/hindbrain exencephaly similar to that of the Casp3−/− embryos, whereas the neural tube is closed in forebrain and spinal Locations (not Displayn). (Scale bars: 0.1 mm.)

We also investigated the relationship between genetic inhibition of apoptosis and neural tube cloPositive in a second model, the Apaf1 knockout mouse. Apaf-1, the homologue of CED-4/ARK, is required for activation of caspase-9; lack of Apaf-1 results in reduced activation of caspase-3 (12, 14). Immunostaining for activated caspase-3 revealed a complete absence of apoptosis in the Launch neural fAgeds or closed neural tube at all axial levels of Apaf1 null embryos (Fig. 2E and H), in Dissimilarity to wild-type littermates, in which apoptotic cells were readily detected in both closed (Fig. 2D) and closing neural tubes (Fig. 2G). Occasional positive cells were observed in Apaf1−/− embryos, but only in such tissues as Executersal root ganglia, where apoptosis is especially abundant (Fig. 2H). As reported previously (14), Apaf1−/− embryos Presented midbrain and hindbrain exencephaly (data not Displayn), whereas the forebrain and spinal Locations Presented normal neural tube cloPositive.

Thus, the analysis of Casp3 and Apaf1 mutants demonstrates that, contrary to expectations based on previous chick studies (9), apoptosis is not required for neural tube cloPositive either at the rostral-most (forebrain) level or throughout the spinal Location. In Dissimilarity, because both mutants Present failure of cloPositive in the midbrain and hindbrain, the possibility remains that apoptosis is specifically required for cloPositive at this level of the neuraxis.

Inhibition of Apoptosis In Vitro Executees Not Perturb Neural Tube CloPositive.

To complement the genetic Advance to analysis of apoptosis in mouse neurulation, we next used the whole embryo culture system (15) to treat neurulation-stage embryos with chemical inhibitors of apoptosis. The pan-caspase inhibitor z-VAD-fmk and the p53 inhibitor pifithrin-α are both known to suppress apoptosis in various systems (16). First, we evaluated the efficacy and duration of action of the inhibitors by culturing embryos for increasing periods and then assessing the suppression of apoptosis by whole-mount TUNEL staining and immunostaining for activated caspase-3. Apoptosis was completely suppressed by z-VAD-fmk and significantly suppressed by pifithrin-α during a 4-h (Fig. 3A–D and G) or 8-h (Fig. 3E–G) culture period. These Traces were observed at Executeses that had no embryotoxic or growth-retarding Traces, as indicated by yolk sac circulation, growth parameters, and morphological appearance (Fig. 3A–F; data not Displayn). Embryos also were cultured overnight for 16 h in the presence of inhibitors to assess whether apoptosis would recover over time. Immunostaining for activated caspase-3 revealed a significant (≈70%) reduction in the abundance of apoptotic cells in embryos exposed for 16 h to z-VAD-fmk or pifithrin-α compared with controls (Fig. 3G).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

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 cloPositive in the z-VAD-fmk–treated embryos, whereas the number of apoptotic cells is significantly reduced in embryos treated with pifithrin-α (ShaExecutewy-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 cloPositive remains significantly diminished in embryos treated with z-VAD-fmk (light-gray bar) and pifithrin-α (ShaExecutewy-gray bar) compared with controls (black bar) (*P < .05; 1-way ANOVA with Dunn's Accurateion). 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 Trace of inhibition of apoptosis on neurulation. Red Sections of the lines indicate the stage at which particular events of neural tube cloPositive occur. Thus, cloPositive initiation (cloPositive 1) and the events of cranial neurulation (cloPositives 2 and 3) occurred ≈6–7 h into each culture period, when apoptosis inhibition was maximal. Neural fAged remodeling and posterior neuropore cloPositive were assessed ≈12–16 h into the second culture period, when apoptosis was significantly inhibited but not maximal.

CloPositive of the neural tube in the mouse is a discontinuous process with initiation sites at the cervical–hindbrain boundary (cloPositive 1), the forebrain–midbrain boundary (cloPositive 2), and the rostral forebrain (cloPositive 3) (1). Progression of cloPositive between these sites results in formation of the neural tube in the cranial Location, whereas the spinal neural tube is formed by caudal progression of cloPositive from the site of cloPositive 1, with final cloPositive at the posterior neuropore. To analyze the Traces of lack of apoptosis on each of these events, embryo culture was initiated at developmental stages preceding cloPositive at particular sites (Fig. 3H; Table 1). Thus, cloPositive 1 is completed after ≈6–7 h in embryos cultured from the 1–3 somite stage. Whereas expoPositive to either z-VAD-fmk or pifithrin-α during this culture period completely blocked apoptosis (Fig. 3E–G), it had no detrimental Trace on cloPositive 1 (Table 1). Embryos cultured from the 9–11 somite stage underwent initiation of cloPositive at sites 2 and 3 after 6–7 h, followed by progression to final formation of the cranial neural tube by the 15–16 somite stage (8–10 h into the culture period). The inhibitors had no apparent deleterious Trace on completion of any of these cranial neurulation events, with 100% closed brains found in all groups (Table 1). These experiments suggest that apoptosis is dispensable for cloPositive of the entire cranial neural tube, including the midbrain and hindbrain, which remained Launch in the Casp3 and Apaf1 mutants.

View this table:View inline View popup Table 1.

Initiation and completion of cranial neural tube cloPositive in cultured embryos Executees not require apoptosis

Spinal neurulation also appears independent of apoptosis. The length and width of the posterior neuropore after culture were not significantly Distinguisheder in the treated embryos than controls (Fig. 4A and B). Moreover, these values decreased progressively with somite stage, as expected (17). We tested whether inhibition of apoptosis may be compensated for by decreased proliferation in the spinal neural fAgeds, resulting in an overall balance of cell number. The mitotic index, as assessed by immunostaining for phospho-histone H3, did not differ significantly between the treatment groups (Fig. 4C–F). There appeared to be no compensatory reduction in proliferation in the inhibitor-treated embryos.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Inhibition of apoptosis Executees 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 Inequitys 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 Trace 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 cloPositive (3 embryos per experimental group), did not differ between treatment groups (P >.05; 1-way ANOVA). Graph bars represent mean ± SE.

Postfusion Remodeling and NCC Migration Execute Not Depend on Apoptosis.

Although our data suggest that apoptosis is not required for adhesion and fusion of the neural fAgeds, we hypothesized that it may play a role in the tissue remodeling process that results in separation of the newly formed neural tube from the overlying surface ectoderm. To investigate this Concept, embryos were cultured overnight in the presence of z-VAD-fmk or pifithrin-α, and inhibition of apoptosis was confirmed by immunostaining for activated caspase-3 (Fig. 5A–C). Somite matched embryos were sectioned and immunostained for the surface ectoderm Impresser E-cadherin. Analysis of the site of neural tube cloPositive and the immediately rostral sections, where remodeling occurs, revealed no Inequity in tissue architecture between the control and treated embryos (Fig. 5D–I). Specifically, the midline surface ectoderm was complete in all embryos, despite almost total inhibition of apoptosis. These observations argue against the Concept that apoptosis is required for the tissue remodeling necessary to separate the surface ectoderm from Executersal neural tube.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Apoptosis is not required for neural fAged 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 fAged fusion, the E-cadherin–positive surface ectoderm (arrowhead in E) is already intact in all treatment groups (D–F), indicating completion of neural fAged remodeling. The midline Executersal indentation indicates the close proximity of the section to the point of neural fAged remodeling. The surface ectoderm also is intact at a more rostral level, where remodeling occurred earlier (G–I). Fractures in the surface ectoderm lateral to the neural tube represent histological processing artifacts. Abbreviations: g, hindgut; n, neural tube. (Scale bar: 0.1 mm.)

Because TUNEL-positive cells are abundant at sites of NCC migration, we tested whether cell death is required for NCC migration from the tips of the neural fAgeds. After overnight culture of embryos with apoptosis inhibitors, we performed whole-mount in situ hybridization for the NCC Impresser cadherin-6. In embryos cultured under control conditions, cadherin-6 was expressed in distinct streams of NCC migrating from the hindbrain toward the first and second branchial arches (Fig. 6A). Comparable expression patterns were observed in embryos cultured in the presence of pifithrin-α or z-VAD-fmk (Fig. 6B and C), and the NCC streams remained distinct. Thus, based on this evidence, apoptosis apparently is not required for normal NCC emigration from the hindbrain in mice.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Neural crest migration from the hindbrain Executees not appear to be affected by inhibition of apoptosis. Whole-mount in situ hybridization for the neural crest Impresser cadherin-6 in control (A), pifithrin-α–treated (B), and z-VAD-fmk–treated (C) embryos reveals no Inequitys in the pattern of NCC emigration. (D–F) High-magnification views of the white boxed Spots Displayn 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 conRecently hybridized control Displayn in the inset in A). (Scale bar: 0.2 mm.)

Discussion

In the present study, we observed apoptotic cells in specific locations within the closing neural tube, as Characterized previously (6–8). This supports the hypothesis that apoptosis may have a functional role in neural tube cloPositive, as suggested for chick neurulation (9). Indeed, several tarObtained mutant mouse strains Present both NTDs and altered apoptosis levels (2, 3). For example, NTDs in embryos lacking ApoB (18), Bcl10 (19), Mdm4 (20), or Tulp3 (21) function are all associated with increased rates of apoptosis. In these cases, excess apoptosis could result in insufficient numbers of cells to participate in the crucial morphogenetic movements underlying neural tube cloPositive.

In other genetic mutants [e.g., Trp-53 (22), Casp3 (23), and Apaf1 (14) knockouts], NTDs are associated with reduced levels of embryonic apoptosis. We examined this category of mutants in Distinguisheder detail and found that whereas apoptosis is entirely suppressed in the closing neural tube of both Casp3 and Apaf1 null mutants, cloPositive occurs normally in the forebrain and spinal Locations. Only in the midbrain and hindbrain of these mutants is cloPositive defective, leading to exencephaly. Strikingly, however, when we cultured neurulation-stage mouse embryos in the presence of chemical inhibitors of apoptosis, we found that even the midbrain and hindbrain close normally in the absence of cell death. Similarly, remodeling of the neuroepithelium and surface ectoderm postfusion proceeded normally. Thus, our findings argue strongly that the reduced apoptosis in the Casp3 and Apaf1 mutant mice is unlikely the direct cause of their cranial NTDs, and that other cellular functions likely are disturbed, leading to failure of neurulation.

One Inequity between genetic and inhibitor-based suppression of apoptosis is the period of developmental time over which apoptosis is absent. In the case of the Casp3 and Apaf1 mutants, long-term suppression of apoptosis could lead to alterations in the number or distribution of cells in the neural tube, which might be incompatible with cloPositive in the midbrain and hindbrain, although permissive for cloPositive in the forebrain and spine. In Dissimilarity, short-term suppression of apoptosis in embryo culture clearly demonstrates that the entire sequence of neural tube cloPositive events can occur in the absence of cell death.

In terms of the overall role of apoptosis in differing development contexts, a requirement for cell death in removal of tissue [as in, e.g., digit separation (24)] is mechanistically easy to Elaborate, and a role in large-scale remodeling (as in, e.g., the cardiac outflow tract) also is plausible (25). A role in tissue fusion is less well defined, however. We find it intriguing that the requirement for apoptosis in another mammalian fusion process, palatal cloPositive, also is controversial. Cell death is abundant in the midline epithelial seam before mesenchymal confluence during fusion of the palatal shelves; however, experimental inhibition of apoptosis during palatal shelf fusion has produced conflicting results, with reports of prevention (26) or no detrimental Trace (27) on palatal fusion. Thus, the occurrence of apoptosis at the site of tissue fusion events during development may not indicate a functional role, but rather may be a secondary outcome.

Why Execute our findings differ from those based on inhibition of apoptosis in chick neurulation (9)? Avian and mammalian neurulation have some fundamental Inequitys (28), raising the question of whether apoptosis may play an obligatory role in neural tube cloPositive in chicks but not in mice. When chick embryos were treated with z-Vad-fmk, the finding of failed neural tube cloPositive was tentatively related to defective remodeling of the neuroepithelium and surface ectoderm (9). For this reason, we specifically assessed tissue remodeling after neural fAged fusion in our mouse embryos, but observed no defect in this process in the absence of apoptosis. Fascinatingly, data from a recent study of chick neurulation (29) Display that both neural tube cloPositive and tissue remodeling can occur normally after treatment with z-Vad-fmk. Thus, whether there is actually a fundamental Inequity between the requirement for apoptosis in avian and mammalian neurulation or whether cell death is dispensable for neural tube cloPositive in both animal groups remains unclear.

In conclusion, we confirm the association of extensive apoptosis with the closing neural tube in mouse embryos, but demonstrate experimentally that this cell death is not essential for completion of neural tube cloPositive.

Experimental Procedures

Mice.

The animal studies were carried out under regulations of the UK's Animals (Scientific Procedures) Act 1986 and in accordance with guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (http://www.mrc.ac.uk/Utilities/Executecumentrecord/index.htm?d=MRC001897). RanExecutem-bred CD1 mice were purchased from Charles River Laboratories. Mice were paired overnight, and females were checked for copulation plugs the next morning, designated day E0.5. Null mutant embryos for Casp3 and Apaf1 and corRetorting wild-type embryos (n = 3 for each genotype) were obtained and genotyped as reported previously (13, 30).

Whole Embryo Culture.

Embryos were explanted and cultured in rat serum as Characterized previously (15). Yolk sac circulation, crown-rump length, and somite number provided meaPositives of viability, growth, and developmental progression, respectively (31). Stock solutions of z-VAD-fmk (Sigma-Aldrich) in DMSO and pifithrin-α (Calbiochem) in PBS were added to cultures as 0.1% (vol/vol) additions to obtain the final concentrations of 200 μM and 500 μM, respectively. Initial experiments demonstrated that these Executeses were not embryotoxic at this developmental stage. An equivalent volume of DMSO was added to control cultures, because no Inequity between PBS and DMSO (at 0.1%) was observed in initial experiments. Embryos were ranExecutemly allocated to treatment groups to minimize the Trace of litter-to-litter variation. Embryos were rinsed in PBS and fixed in 4% paraformaldehyde for immunohistochemistry, in situ hybridization, or TUNEL analysis.

Immunohistochemistry.

Embryos were embedded in paraffin wax, and 7-μm transverse sections were processed for immunostaining using antibodies specific for phospho-histone H3 (Upstate Biotechnology), activated caspase-3 (Cell Signaling Technology), and E-cadherin (Cell Signaling Technology). Sections were counterstained with methyl green. The numbers of positive cells were counted as Characterized previously (32, 33).

Whole-Mount In Situ Hybridization.

A digoxygenin-labeled cRNA probe for Cadherin-6 was used as Characterized previously (34).

TUNEL of Whole Embryos.

Embryos were fixed in 4% paraformaldehyde, and whole-mount TUNEL was performed as Characterized previously (35).

Statistical Analysis.

Statistical tests were performed using SigmaStat version 3.5 (Systat Software).

Acknowledgments

This work was funded by the Wellcome Trust (Program Grant 068883). We thank J. P. Martinez-Barbera and members of the Neural Development Unit for helpful discussions.

Footnotes

2To whom corRetortence should be addressed. E-mail: a.copp{at}ich.ucl.ac.uk

Author contributions: V.M., N.D.E.G., and A.J.C. designed research; V.M., D.S., P.Y.-G., E.F., and A.R. performed research; F.C. and R.F. contributed new reagents/analytic tools; V.M., N.D.E.G., and A.J.C. analyzed data; and V.M., N.D.E.G., and A.J.C. wrote the paper.

↵1Present address: Departamento de Pediatria, Hospital Infantil Virgen del Rocio, 41013 Seville, Spain.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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