The results of these experiments suggest that the reduction in migrating CNC cells in Pax3-deficient embryos is due apoptosis which is evident by TUNEL reaction approximately 24 hours after the onset of Pax3
expression. Loss-of-function germline mutation of p53, or administration of an inhibitor of p53 nuclear and mitochondrial translocation, blocked apoptosis, rescued CNC migration, and rescued cardiac outflow tract septation in Pax3-deficient embryos. These results indicate that, in CNC cells, Pax3 is not required to regulate expression of genes which control migration and cardiac outflow tract septation, but it is required to inhibit p53-dependent processes leading to apoptosis and consequent defective outflow tract development. While a simple explanation for a p53-dependent process leading to apoptosis would be transcriptional regulation of pro-apoptotic genes, p53 controls other processes, such as cell cycle arrest, migration, or angiogenesis, as well (Dameron et al., 1994
; Kamijo et al., 1998
; Linke et al., 1996
; Roger et al., 2006
). Thus, it is possible that, in the absence of Pax3, one or more of these p53-dependent processes is activated in CNC, which directly or indirectly results in apoptosis.
The mechanism by which Pax3 inhibits p53 is not well understood. We have previously shown that Pax3 has no effect on p53
mRNA levels, but that steady state p53 protein levels are increased in Pax3Sp/Sp
embryos compared to w.t. embryos (Pani et al., 2002
), suggesting that Pax3 inhibits p53 protein synthesis or stability. Similar observations have been made in mouse embryonic stem cells, in which Pax3
expression has been induced either by transfection of a Pax3
expression vector or differentiation of the embryonic stem cells to neuronal precursors (Morgan and Loeken, unpublished results). Further investigation is necessary in order to further understand how Pax3 regulates p53 protein on a molecular level.
Although it was not the focus of this study, we noted that Pax3 did not appear to regulate apoptosis in somitic mesoderm and derivatives. While somites were poorly formed in Pax3LacZLacZ embryos, and the hypoglossal cord was absent (compare and ), there were no TUNEL-positive cells at the locations of these tissues, and blocking p53 did not rescue these structures. Moreover, limb musculature failed to develop in E 18.5 Pax3LacZ/LacZ p53−/− fetuses, and they were not viable, presumably due to failure of the diaphragm to develop. This indicates that Pax3 is required for developmental processes in somitic derivatives that are different from the role that it plays during development of neuroepithelial and neural crest derivatives.
Migrating CNC appeared beyond the somites at the same stage of development in Pax3LacZ/+ and Pax3LacZ/LacZ embryos, suggesting that the timing of initiation of CNC migration and the migratory speed do not seem to be impaired by Pax3 deficiency. Because inactivation of p53 completely rescued CNC migration, this suggests that Pax3 is not required to initiate or to pace CNC migration. Moreover, because TUNEL-positive cells were observed beyond the somites, located where CNC are found at the same stage of development in Pax3LacZ/+ embryos, this suggests that the CNC cells did migrate on time, but that their numbers are decreased because of attrition resulting from apoptosis. It was not possible to verify that the TUNEL-positive cells detectable on E 9.5 were CNC cells, because dead cells would not express cell-specific markers, but since the pattern, location, and numbers of TUNEL-positive cells were comparable to the pattern, location, and number of β-galactosidase-positive cells in Pax3-sufficient embryos, and β-galactosidase was present when p53-dependent apoptosis was inhibited, this strongly suggests that the TUNEL-positive cells were CNC cells.
Conway, et al. did not observe increased TUNEL-positive CNC cells in homozygous Splotch2H
) embryos on E 9.5 (Conway et al., 2000
). This may be due to differences between the Splotch2H
mutations that may differentially affect severity. Indeed, the effect of the Splotch2H
mutation on outflow tract defects is only 60–85% penetrant (Conway et al., 2000
; Conway et al., 1997a
), whereas the Splotch
defect is 100% penetrant (Auerbach, 1954
). Alternatively, the fluorescent TUNEL assay used here may be more sensitive than the assay employed by Conway, et al. They concluded that there was reduced expansion of CNC progenitors in Pax3Sp2H/Sp2H
embryos because there were fewer Wnt-1
-expressing cells on E 8.5 in the dorsal edge of the neural folds (Conway et al., 2000
is expressed in neural crest progenitors (Parr et al., 1993
) and appears to be essential for neural crest cell expansion prior to emigration from the neural tube (Dorsky et al., 1998
; Ikeya et al., 1997
). Thus, a deficiency of Wnt-1
-expressing cells in Pax3-deficient embryos could indicate that there was reduced expansion of CNC progenitors. On the other hand, cells expressing Wnt-3a
, which is also expressed in neural crest progenitors, and appears to be required for initial neural crest expansion (Dorsky et al., 1998
; Ikeya et al., 1997
; Parr et al., 1993
) were not reduced (Conway et al., 2000
). Thus, it is possible that Pax3 positively regulates Wnt-1
gene expression, and a lack of Wnt-1
expression could result from deficient transcriptional regulation by the mutant Pax3.
p53 regulates cell cycle arrest as well as apoptosis (Kamijo et al., 1998
; Linke et al., 1996
). It is possible that Pax3 is required to override p53-induced cell cycle arrest to maintain neural crest cells, or their progenitors, in a proliferative mode. Thus, Pax3-deficient cells may undergo cell cycle arrest, and eventually undergo apoptosis, unless p53 is inactivated. It is possible that p53 also plays a role to inhibit migration of CNC as it may during tumor metastasis (Roger et al., 2006
). Although the distance that CNC had migrated in Pax3-sufficient and Pax3-deficient embryos with w.t. p53 was not different, Pax3 might also play a role to override inhibition of migration.
The disappearance of β-galactosidase-positive CNC in Pax3LacZ/LacZ
embryos and appearance of TUNEL-positive cells occurred more than 24 hours after the onset of Pax3
expression, and, based on timing of responsiveness to PFT-α, approximately 20–28 hours after the time period in which p53 needed to be inhibited. Furthermore, the loss of cells did not occur all at once, as if in response to a synchronized event, but progressively fewer cells were observed all along the CNC path in successively developing embryos. DNA strand breakage, which is detected by TUNEL assay, is a relatively late event in the course of apoptotic cell death. The time between the initiation of apoptosis and the morphological detection of DNA damage depends on the cell type, and is on the order of 3–4, and up to 14, hours (Bursch et al., 1990
; Rodriguez and Schaper, 2005
). Thus, this is consistent with a requirement for Pax3 to prevent a p53-dependent process within the first approximately 4 hours of Pax3
expression, and if this process is not prevented, cells will eventually undergo apoptosis while migrating toward the cardiac field.
We noticed that, while homozygous germ line p53 loss of function completely normalized CNC migration in Pax3LacZ/LacZ
embryos, the rescue by p53 haploinsufficiency or PFT-α was variable. In contrast, there was no intermediate effect of p53 haploinsufficiency or PFT-α on neural tube defects; either neural tube closure was completely normal, or there was exencephaly and/or spina bifida, which was indistinguishable from Pax3Sp/Sp
embryos (Pani et al., 2002
). This suggests that the dose-dependency for p53 to activate apoptotic pathways in CNC and neuroepithelium may be different. On the other hand, we did not study migration of neuroepithelium before neural tube closure in Pax3Sp/Sp
embryos with different p53
genotypes, and so, it is possible that there is a variable effect of p53 cellular concentration during neuroepithelial migration, but that there is simply a critical mass of neuroepithelium or CNC that is necessary to give rise to a normal neural tube or outflow tracts.
In summary, the data reported here show that Pax3 is required for CNC migration and outflow tract septation because it inhibits p53-dependent processes which culminate in apoptosis. Investigating how Pax3 regulates p53 on a biochemical level will be important in order to have a more detailed understanding of how CNC development is controlled.