Rescue of Neural Tube Defects in Pax-3-Deficient Embryos by P53 Loss of Function: Implications for Pax-3- Dependent Development and Tumorigenesis

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RESEARCH COMMUNICATION

opment (Kioussi et al. 1995). However, regulation of Rescue of neural tube defects these genes by Pax-3 may be indirect, as none of the in Pax-3-deficient embryos genes that have been found to be over- or underexpressed as a function of Pax-3 have high-affinity Pax-3 binding by p53 loss of function: sites, with the exception of one element on the MyoD implications for Pax-3- promoter (Phelan and Loeken 1998). We showed previously that in Sp/Sp embryos, NTDs dependent development are associated with neuroepithelial apoptosis (Phelan et al. 1997). This suggested that disruption of a Pax-3-de- and tumorigenesis pendent developmental program may cause the malformed structures to undergo apoptosis. An alternative Lydie Pani,1,2 Melissa Horal,1 1–3 explanation is that Pax-3 directly or indirectly inhibits and Mary R. Loeken apoptosis. Several other studies lend support to the latter interpretation. For example, apoptosis is prevalent in 1Section on Cellular and Molecular Physiology, Joslin somites of Splotch embryos (Borycki et al. 1999), and Diabetes Center, 2Department of Medicine, Harvard Medical inhibition of Pax-3 expression with antisense oligo- School, Boston, Massachusetts 02215, USA nucleotides, or expression of an engineered PAX-3 fused Pax-3 is a transcription factor that is expressed in the to a transcriptional repressor domain, causes apoptosis in cultured presomitic mesoderm, pediatric rhabdomyo- neural tube, neural crest, and dermomyotome. We pre- sarcoma (RMS), and melanoma (Barr et al. 1993; Galili et viously showed that apoptosis is associated with neural al. 1993; Shapiro et al. 1993; Bernasconi et al. 1996; Bo- tube defects (NTDs) in Pax-3-deficient Splotch (Sp/Sp) rycki et al. 1999; Scholl et al. 2001). A question that is embryos. Here we show that p53 deficiency, caused by raised by these observations is whether inhibition of germ-line mutation or by pifithrin-␣, an inhibitor of p53- apoptosis is an essential, or even the sole, function of dependent apoptosis, rescues not only apoptosis, but also Pax-3 during development or transformation. NTDs, in Sp/Sp embryos. Pax-3 deficiency had no effect The product of the p53 tumor suppressor gene medi- on p53 mRNA, but increased p53 protein levels. These ates apoptosis in response to many genotoxic stresses results suggest that Pax-3 regulates neural tube closure (Appella and Anderson 2001). p53-dependent apoptosis is by inhibiting p53-dependent apoptosis, rather than by responsible for elimination of transformed cells and sup- pression of tumor growth in vivo; loss of p53 function is inducing neural tube-specific gene expression. associated with poor clinical prognosis of many human Received December 12, 2001; revised version accepted malignancies (Fisher 1994). Very few studies have inves- January 31, 2002. tigated whether apoptosis during embryogenesis is p53- mediated. Notably, deficiency of XRCC4 or DNA ligase IV, both of which participate in nonhomologous end- Pax-3 encodes a DNA-binding transcription factor that is joining DNA double-strand break repair and V(D)J re- expressed in neuroepithelium, presomitic mesoderm, combination, causes embryonic lethality and massive and neural crest (Goulding et al. 1991; Chalepakis et al. neuronal apoptosis, and these effects are rescued by p53 1994). Homozygous Sp/Sp embryos carry loss-of-func- deficiency (Frank et al. 2000; Gao et al. 2000). This sug- tion Pax-3 alleles and develop open neural tube defects gests that XRCC4 and DNA ligase IV participate in DNA (NTDs), specifically, exencephaly, spina bifida, or both, repair during normal embryogenesis, and that in the ab- with 100% penetrance, and die midgestation (Auerbach sence of DNA repair, affected structures undergo p53- 1954; Epstein et al. 1993). The embryonic lethality is dependent apoptosis. However, involvement of p53 in caused by defective cardiac neural crest migration and apoptosis involving DNA strand breaks is consistent consequent malformation of cardiac outflow tracts (Con- with its response to genotoxic stress. Whether apoptosis way et al. 1997; Epstein et al. 2000). Heterozygous Sp/+ associated with malformations caused by other mecha- embryos are viable, but manifest white patches of fur on nisms, such as Pax-3 deficiency, is p53-mediated has not a dark coat background, caused by defective neural crest- been determined. derived melanocyte development. Because of the failure of Pax-3-expressing structures to properly form in Results and Discussion Splotch embryos, it has been accepted that Pax-3 regulates expression of differentiation-specific genes during Inactivation of p53 rescues Pax-3-deficient embryos development of these structures. In support of this hy- from neural tube defects pothesis, Pax-3 has been shown to be upstream of myogenic gene expression, for example, of the genes Myf-5 To investigate the involvement of p53 in apoptosis and +/− and MyoD, during skeletal muscle development (Maroto NTD caused by Pax-3 deficiency, Sp/+ and p53 mice et al. 1997; Tajbakhsh et al. 1997) and to inhibit expres- were crossed, and then double heterozygous progeny sion of myelin basic protein during Schwann cell devel- were mated to introduce a variable p53 genotype onto Sp/Sp embryos. When examined on embryonic day 10.5 (E10.5), as expected, all of the Sp/Sp embryos that were [Key Words: Pax-3; P53; neural tube; apoptosis] p53 3 wild type at the locus had developed open NTDs Corresponding author. (Table 1). Remarkably, loss of both p53 alleles prevented E-MAIL [email protected]; FAX (617) 732-2541. −/− Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ NTDs in all Sp/Sp p53 embryos. Even p53 heterozy- gad.969302. gosity was sufficient to prevent NTD in 42% of Sp/Sp

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Pax-3 and p53 during neural tube closure

Table 1. p53 deficiency suppresses NTD in Sp/Sp embryos whereas those with NTD were indistinguishable from Sp/Sp embryos. The all-or-none effect of p53 deficiency Genotype Normal NTD on neural tube development suggests that there is a Sp/Sp p53+/+ 03minimum threshold of p53 necessary to activate a pro- Sp/Sp p53+/− 20 28 gram leading to an NTD, and that below this threshold, Sp/Sp 53−/− 50normal neural tube development ensues. +/Sp p53+/+ 90 +Sp p53+/− 54 5 Inactivation of p53 rescues Pax-3-deficient embryos +Sp p53−/− 51from apoptosis +/+ p53+/+ 30 +/+ p53+/− 22 1 To determine whether p53 loss of function prevented +/+ p53−/− 31apoptosis as well as NTD, Sp/Sp embryos were assayed for apoptosis by a whole-mount TUNEL procedure. As Embryos from Sp/+ p53+/− matings were obtained on day 10.5 shown in Figure 1, numerous apoptotic cells were ob- and scored for NTD (exencephaly and/or spina bifida). Yolk sac served at defective sites of Sp/Sp p53+/+ embryos. How- DNA was used for PCR at the Pax-3, p53, Zfy, and Nfl loci. ever, Sp/Sp p53−/− and wild-type embryos were indistin- There was no difference in the gender distribution in defec- guishable, and neuroepithelial apoptosis was not ob- tive p53−/− or p53+/− embryos. There was a significant effect of served. Apoptotic cells were detectable along the apical Splotch genotype in p53+/+ embryos (P < 0.0006); in Sp/Sp em- ectodermal ridge of the limb buds in the Sp/Sp p53−/− bryos, the effect of p53 mutation to suppress NTD was statis- embryos as well as the wild-type embryos, indicating tically significant (P <0.02);inSp/− embryos and in embryos that the apoptosis leading to digit formation is not p53- that were wild-type at the Splotch locus, there was no signifi- dependent. To quantitatively compare severity of apo- cant effect of p53 mutation. ptosis in embryos with variable p53 function, neuroepithelial apoptosis in Sp/Sp embryos was scored blindly on a scale of 1–10. As shown in Figure 2A, the apoptotic embryos. All Sp/Sp embryos whose NTDs were pre- index of embryos with NTD was greater than in normal vented by loss of one or both p53 alleles were indistin- +/− embryos, and high apoptosis scores were only observed guishable from wild-type embryos, whereas Sp/Sp p53 in malformed embryos with one or two wild-type p53 embryos that were malformed were indistinguishable alleles. Similar results were obtained upon TUNEL from Sp/Sp embryos on a wild-type p53 background. analysis of embryos in which p53 had been inhibited There was no significant effect of heterozygous or homo- with pifithrin-␣ (Fig. 2B). zygous p53 mutation in Sp/+ embryos or in embryos that These results show that loss of p53 function, by ge- were wild type at the Splotch locus. netic or chemical means, prevented both apoptosis and To further test the involvement of p53 in NTD caused NTD caused by Pax-3 deficiency. Given that neural tube by Pax-3 deficiency, the effects of a p53 inhibitor, pifi- development was normal in p53-deficient embryos de- thrin-␣, were tested. Pifithrin-␣ inhibits p53-dependent spite the absence of Pax-3, this profoundly alters the con- transcription and apoptosis (Komarov et al. 1999). The cept by which Pax-3 controls neural tube development. precise mechanisms are not known, but given that These observations indicate that the apoptosis and NTD nuclear accumulation of p53 is reduced, this suggests in Sp/Sp embryos do not result from the failure of a Pax- that pifithrin-␣ stimulates nuclear export, inhibits 3-dependent neural tube-specific program. Rather, neu- nuclear import, or decreases p53 stability. Pregnant Sp/+ ral tube morphogenesis occurs by a mechanism that is females that had been mated with Sp/+ males were ad- Pax-3-independent, and Pax-3 simply keeps the cells ministered pifithrin-␣ during formation of the neural alive until the program is complete by directly or indi- tube (E8.5 and E9.5). As shown in Table 2, pifithrin-␣ rectly inhibiting apoptosis by a p53-dependent mecha- prevented NTD in 55% of Sp/Sp embryos, whereas all nism. Sp/Sp embryos whose mothers had been injected with saline developed NTD. As with Sp/Sp embryos whose NTDs were prevented by mutant p53 alleles, pifithrin- Pax-3 down-regulates p53 protein, but not mRNA ␣ -treated embryos without NTD were indistinguishable PAX-5, as well as its paralogs PAX-2 and PAX-8, inhibits Splotch from embryos that were wild type at the locus, p53 gene expression, an effect that is mediated by PAX-5 binding to the p53 promoter (Stuart et al. 1995). Thus, in Table 2. p53 inhibitor suppresses NTD in Sp/Sp embryos human astrocytomas, p53 deficiency caused by PAX-5- dependent transcriptional inhibition may contribute to Genotype PFT Normal NTD tumor development. Similarly, the PAX-5 paralog PAX-8 Sp/Sp −0 4may inhibit p53 gene expression in differentiated thyroid Sp/Sp +6 5carcinomas (Puglisi et al. 2000). However, unlike PAX-5 Sp/+ −3 0and its paralogs, PAX-2 and PAX-8, there are no identi- Sp/+ +3 0fiable binding sites for Pax-3 within a 530-bp upstream +/+ −3 0region of the murine p53 gene (Bienz-Tadmor et al. +/+ +2 01985). On the other hand, regulation of p53-dependent apoptosis is primarily posttranslational, involving pro- Embryos were obtained from Sp/+ matings and scored for NTD tein modifications such as phosphorylation or acetyla- as described for Table 1. Pregnant mice were injected with pifi- tion that regulate its stability (Appella and Anderson thrin-␣ (PFT) or saline on days 8.5 and 9.5. Yolk sac DNA was 2001). Therefore, an effect of Pax-3 on p53 protein levels used for genotype determination at the Pax-3 locus. The effect could also be possible. of PFT on Sp/Sp embryos was statistically significant (P < 0.05). To test whether Pax-3 might regulate either p53

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twofold difference in p53 protein levels in the whole embryo may under- estimate the magnitude by which Pax-3 affects p53 levels in individual Pax-3-expressing cells. Further inves- tigation will be necessary to fully un- derstand how Pax-3 regulates p53, as well as p53-dependent transcription and apoptosis. In addition, it will be important to determine whether inhibition of p53-dependent apoptosis is required for development of other Figure 1. Embryonic day 10.5 (E10.5) embryos following TUNEL assay to visualize apoptotic Pax-3-dependent tissues such as neu- cells. (Left) Sp/Sp p53+/+ embryo (TUNEL score = 8). Exencephaly and spina bifida, with large ral crest and somitic mesoderm de- numbers of apoptotic cells, indicated by arrows. (Inset) TUNEL-positive cells from the region rivatives. of the spina bifida. (Center) Normal Sp/Sp p53−/− embryo (TUNEL score = 2). Apoptotic cells can be visualized along the apical ectodermal ridge of the limb bud (indicated by arrow), as expected for this stage of development, but not along fused neural tube. (Right) Wild-type (+/+ Implications for Pax-3 p53+/+) embryo (TUNEL score = 2). Apoptotic cells are also visible on limb bud (indicated by in tumorigenesis arrow), but not along neural tube. (Brown staining along dorsal surface of head of wild-type (w.t.) embryo is artefactual owing to tissue compression and translucency.) The inhibition of p53-dependent apoptosis by Pax-3 has implications mRNA or protein levels, E10.5 embryos from Sp/+ mat- for tumorigenesis, as well as development. Expression of ings were assayed for p53 mRNA by semiquantitative a PAX-3/FKHR or PAX-7/FKHR fusion protein appears RT–PCR, or for p53 protein by Western blot analysis. to be a key step in the development of pediatric rhabdo- There was no difference in p53 mRNA in embryos of myosarcoma (Bernasconi et al. 1996; Fredericks et al. different Splotch genotypes (Fig. 3A,B). However, p53 2000). In addition, transcriptionally active PAX-3 is ex- protein levels were increased almost twofold in Sp/Sp pressed in human melanomas, but not in surrounding and Sp/+ embryos compared with wild-type embryos normal tissue (Barr et al. 1999; Galibert et al. 1999; (comparing the amount of p53 protein from each geno- Vachtenheim and Novotna 1999; Scholl et al. 2001). In type at intermediate dilution and the absence of p53 pro- the mouse, p53 inactivation has been shown to cooper- tein in wild-type embryos at greatest dilution), although ate with activated RAS to cause melanoma (Bardeesy et only the differences between wild-type and Sp/Sp levels al. 2001; Yang et al. 2001). Although loss of function of were statistically significant (Fig. 3C,D). It should be the p53 gene is associated with many malignancies, the noted that, on E10.5, only the neuroepithelium, neural evidence presented here indicates that an alternative crest, and dermomyotome express Pax-3. Therefore, a way to cause functional p53 deficiency is by reactivating or ectopically expressing Pax-3. Therefore, in human melanoma or rhabdomyosarcoma, PAX-3 inhibition of p53-dependent apoptosis may be critical to tumor estab- lishment. It should be noted that several Pax proteins (Pax-1, Pax-2, Pax-3, Pax-6, and Pax-8), which differ in their paired domain sequences and the presence of a complete or partial homeodomain, have the capacity to transform fibroblasts and induce tumors (Maulbecker and Gruss 1993). Furthermore, as noted above, PAX-5 inhibits p53 transcription, a process that may lead to astrocytoma (Stuart et al. 1995). Hence, induction of p53 loss of function by different mechanisms may be common to all Pax proteins during development, and may be an integral process leading to tumorigenesis when Pax proteins are inappropriately expressed.

Materials and methods Mice Heterozygous Splotch (Sp/+) mice on a C57Bl/6J background, and heterozygous p53 knockout (p53+/−) mice on a FVB background were obtained from Jackson Laboratories. Embryos were recovered on E10.5 to assay p53 mRNA or protein, or apoptosis and NTD. Embryos to be used for Figure 2. Effect of p53 inhibition on apoptosis in Sp/Sp embryos. TUNEL assay were fixed in 4% paraformaldehyde, and embryos for RT– (A) Relative apoptosis scores (on a scale of 1–10) in Sp/Sp embryos as PCR or Western blot analysis were stored at −80°C. Pifithrin-␣ (Calbio- a function of p53 genotype and normal or abnormal morphology. (N) chem) was administered by intraperitoneal injection of 2.2 mg/kg dis- Normal neural tube; (NTD) exencephaly and/or spina bifida. (*) solved in PBS on E8.5 and E9.5. P < 0.05 versus p53+/+ or p53+/− with NTD. (B) Relative apoptosis scores in Sp/Sp embryos whose mothers had been administered sa- TUNEL assay line or pifithrin-␣ (PFT). Mean apoptosis scores for normal embryos Apoptosis was assayed by a whole-mount TUNEL procedure as described and embryos with exencephaly and/or spina bifida are shown sepa- (Phelan et al. 1997). Apoptosis specifically localized to the neural tube rately. (*) P < 0.05 versus PFT normal embryos. was scored by an individual who was blinded to embryo genotype. A

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Pax-3 and p53 during neural tube closure

determined using yolk sac DNA as described above; 500 ng of RNA was reverse transcribed as described (Phelan et al. 1997). PCR was performed using primers specific to p53 (Toda et al. 1998) or to 36B4 (Hill et al. 1998), which was used as a control, and fourfold serial dilutions of RT reaction products (6.25–0.39 ng for p53,24–1.5pgfor36B4) and cycling conditions as described (Hill et al. 1998; Toda et al. 1998). p53 cDNA was expressed relative to 36B4 cDNA by scanning densitometry of bands that were present in a linear range following autoradiography of PCR products.

Western blot analysis Immunoblot analysis of p53 protein was performed using twofold serial dilutions of protein (250–62.5 µg) from individual embryos and p53 antibodies (Ab-1 and Ab-3 from Calbiochem, both diluted 1:500) and goat- anti-mouse secondary antibody (Pierce, diluted 1:5000). p53 was normalized to ␤-tubulin, which was detected with a primary anti-tubulin antibody (Santa Cruz Biotechnology, diluted 1:1000) and goat-anti-rabbit secondary antibody (Pierce, diluted 1:2500). Secondary antibodies were detected by chemiluminescence (Pierce).

Statistical analysis Data were analyzed by 1-Way Analysis of Variance and Neuman Keuls post-hoc test, or ␹-square analysis, using Prism 3 software (GraphPad Software).

Acknowledgments

This work was supported by grants from the National Institutes of Health, the Juvenile Diabetes Foundation, and the March of Dimes Birth Defects Foundation to M.R.L. We are grateful to Peter Howley for critical comments on the manuscript, to Guo Jun Zhang for advice on p53 immunoblotting procedures, and to Rakhi Patel and Rebecca Clark for tech- nical assistance. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 USC section 1734 solely to indicate this fact.

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Rescue of neural tube defects in Pax-3-deficient embryos by p53 loss of function: implications for Pax-3- dependent development and tumorigenesis

Lydie Pani, Melissa Horal and Mary R. Loeken

Genes Dev. 2002, 16: Access the most recent version at doi:10.1101/gad.969302

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