Inappropriate activation of the p53 transcription factor is thought to contribute to the developmental phenotypes in a range of genetic syndromes. Whether p53 activation drives these developmental phenotypes by triggering apoptosis, cell cycle arrest, or other p53 cellular responses, however, has remained elusive. As p53 hyperactivation in embryonic neural crest cells (NCCs) drives a number of phenotypes, including abnormal craniofacial and neuronal development, we investigate the basis for p53 action in this context. We show that p53-driven developmental defects are associated with the induction of a robust pro-apoptotic transcriptional signature. Intriguingly, however, deleting Puma or Caspase9, which encode key components of the intrinsic apoptotic pathway, does not rescue craniofacial, neuronal or pigmentation defects triggered by p53 hyperactivation in NCCs. Immunostaining analyses for two key apoptosis markers confirm that deleting Puma or Caspase9 does indeed impair p53-hyperactivation-induced apoptosis in NCCs. Furthermore, we demonstrate that p53 hyperactivation does not trigger a compensatory dampening of cell cycle progression in NCCs upon inactivation of apoptotic pathways. Together, our results indicate that p53-driven craniofacial, neuronal and pigmentation defects can arise in the absence of apoptosis and cell cycle arrest, suggesting that p53 hyperactivation can act via alternative pathways to trigger developmental phenotypes.
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Bowen ME, Attardi LD. The role of p53 in developmental syndromes. J Mol Cell Biol. 2019;11:200–11.
Vousden KH, Prives C. Blinded by the light: the growing complexity of p53. Cell. 2009;137:413–31.
Bronner ME, Simões-Costa M. The neural crest migrating into the twenty-first century. Curr Top Dev Biol. 2016;116:115–34.
Bowen ME, McClendon J, Long HK, Sorayya A, Van Nostrand JL, Wysocka J, et al. The spatiotemporal pattern and intensity of p53 activation dictates phenotypic diversity in p53-driven developmental syndromes. Dev Cell. 2019;50:212–28.e6.
Fischer M. Census and evaluation of p53 target genes. Oncogene. 2017;36:3943–56.
Kaiser AM, Attardi LD. Deconstructing networks of p53-mediated tumor suppression in vivo. Cell Death Differ. 2018;25:93–103.
Valente LJ, Tarangelo A, Li AM, Naciri M, Raj N, Boutelle AM, et al. p53 deficiency triggers dysregulation of diverse cellular processes in physiological oxygen. J Cell Biol. 2020;219:e201908212.
Hamard P-J, Barthelery N, Hogstad B, Mungamuri SK, Tonnessen CA, Carvajal LA, et al. The C terminus of p53 regulates gene expression by multiple mechanisms in a target- and tissue-specific manner in vivo. Genes Dev. 2013;27:1868–85.
Liu G, Terzian T, Xiong S, Van Pelt C, Audiffred A, Box N, et al. The p53-Mdm2 network in progenitor cell expansion during mouse postnatal development. J Pathol. 2007;213:360–8.
Terzian T, Wang Y, Van Pelt CS, Box NF, Travis EL, Lozano G. Haploinsufficiency of Mdm2 and Mdm4 in tumorigenesis and development. Mol Cell Biol. 2007;27:5479–85.
Van Nostrand JL, Brady CA, Jung H, Fuentes DR, Kozak MM, Johnson TM, et al. Inappropriate p53 activation during development induces features of CHARGE syndrome. Nature. 2014;514:228–32.
Zhang Q, He X, Chen L, Zhang C, Gao X, Yang Z, et al. Synergistic regulation of p53 by Mdm2 and Mdm4 is critical in cardiac endocardial cushion morphogenesis during heart development. J Pathol. 2012;228:416–28.
Pant V, Xiong S, Chau G, Tsai K, Shetty G, Lozano G. Distinct downstream targets manifest p53-dependent pathologies in mice. Oncogene. 2016;35:5713–21.
Delbridge ARD, Kueh AJ, Ke F, Zamudio NM, El-Saafin F, Jansz N, et al. Loss of p53 causes stochastic aberrant X-chromosome inactivation and female-specific neural tube defects. Cell Rep. 2019;27:442–54.e5.
Jones NC, Lynn ML, Gaudenz K, Sakai D, Aoto K, Rey J-P, et al. Prevention of the neurocristopathy Treacher Collins syndrome through inhibition of p53 function. Nat Med. 2008;14:125–33.
Johnson TM, Hammond EM, Giaccia A, Attardi LD. The p53QS transactivation-deficient mutant shows stress-specific apoptotic activity and induces embryonic lethality. Nat Genet. 2005;37:145–52.
Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol CB. 1998;8:1323–6.
Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2010;13:133–40.
Grier JD, Yan W, Lozano G. Conditional allele of mdm2 which encodes a p53 inhibitor. Genesis. 2002;32:145–7.
Parant J, Chavez-Reyes A, Little NA, Yan W, Reinke V, Jochemsen AG, et al. Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat Genet. 2001;29:92–5.
Villunger A, Michalak EM, Coultas L, Müllauer F, Böck G, Ausserlechner MJ, et al. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science. 2003;302:1036–8.
Simon DJ, Weimer RM, McLaughlin T, Kallop D, Stanger K, Yang J, et al. A caspase cascade regulating developmental axon degeneration. J Neurosci. 2012;32:17540–53.
Brady CA, Jiang D, Mello SS, Johnson TM, Jarvis LA, Kozak MM, et al. Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression. Cell. 2011;145:571–83.
Aubrey BJ, Kelly GL, Janic A, Herold MJ, Strasser A. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell Death Differ. 2018;25:104–13.
Green DR, Llambi F. Cell death signaling. Cold Spring Harb Perspect Biol. 2015;7:a006080.
Sinha SK, Chaudhary PM. Induction of apoptosis by X-linked ectodermal dysplasia receptor via a Caspase 8-dependent mechanism. J Biol Chem. 2004;279:41873–81.
Tinel A, Tschopp J. The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science. 2004;304:843–6.
Resch U, Schichl YM, Winsauer G, Gudi R, Prasad K, de Martin R. Siva1 is a XIAP-interacting protein that balances NFkappaB and JNK signalling to promote apoptosis. J Cell Sci. 2009;122:2651–61.
Kawase T, Ichikawa H, Ohta T, Nozaki N, Tashiro F, Ohki R, et al. p53 target gene AEN is a nuclear exonuclease required for p53-dependent apoptosis. Oncogene. 2008;27:3797–810.
Singh P, Ravanan P, Talwar P. Death associated protein kinase 1 (DAPK1): a regulator of apoptosis and autophagy. Front Mol Neurosci. 2016;9:46.
Ohki R, Saito K, Chen Y, Kawase T, Hiraoka N, Saigawa R, et al. PHLDA3 is a novel tumor suppressor of pancreatic neuroendocrine tumors. Proc Natl Acad Sci USA. 2014;111:E2404–13.
Mork CN, Faller DV, Spanjaard RA. Loss of putative tumor suppressor EI24/PIG8 confers resistance to etoposide. FEBS Lett. 2007;581:5440–4.
Li Z, Zhao J, Tikhanovich I, Kuravi S, Helzberg J, Dorko K, et al. Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression. Cell Death Differ. 2016;23:583–95.
Ishii K, Ishiai M, Morimoto H, Kanatsu-Shinohara M, Niwa O, Takata M, et al. The Trp53 - Trp53inp1 - Tnfrsf10b pathway regulates the radiation response of mouse spermatogonial stem cells. Stem Cell Rep. 2014;3:676–89.
Tan Y, Huang N, Zhang X, Hu J, Cheng S, Pi L, et al. KIAA0247 suppresses the proliferation, angiogenesis and promote apoptosis of human glioma through inactivation of the AKT and Stat3 signaling pathway. Oncotarget. 2016;7:87100–13.
Ham J, Eilers A, Whitfield J, Neame SJ, Shah B. c-Jun and the transcriptional control of neuronal apoptosis. Biochem Pharm. 2000;60:1015–21.
Li B, Chen R, Chen L, Qiu P, Ai X, Huang E, et al. Effects of DDIT4 in methamphetamine-induced autophagy and apoptosis in dopaminergic neurons. Mol Neurobiol. 2017;54:1642–60.
Jeffers JR, Parganas E, Lee Y, Yang C, Wang J, Brennan J, et al. Puma is an essential mediator of p53-dependent and -independent apoptotic pathways. Cancer Cell. 2003;4:321–8.
Michalak EM, Villunger A, Adams JM, Strasser A. In several cell types tumour suppressor p53 induces apoptosis largely via Puma but Noxa can contribute. Cell Death Differ. 2008;15:1019–29.
Hakem R, Hakem A, Duncan GS, Henderson JT, Woo M, Soengas MS, et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell. 1998;94:339–52.
Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C, Karasuyama H, et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell. 1998;94:325–37.
Marsden VS, O’Connor L, O’Reilly LA, Silke J, Metcalf D, Ekert PG, et al. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature. 2002;419:634–7.
Gown AM, Willingham MC. Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved Caspase 3. J Histochem Cytochem. 2002;50:449–54.
Loo DT. In situ detection of apoptosis by the TUNEL assay: an overview of techniques. In: Didenko VV, editor. DNA damage detect situ ex vivo vivo methods protoc [Internet]. Totowa, NJ: Humana Press; 2011. p. 3–13. https://doi.org/10.1007/978-1-60327-409-8_1.
Chavez-Reyes A, Parant JM, Amelse LL, de Oca Luna RM, Korsmeyer SJ, Lozano G. Switching mechanisms of cell death in mdm2- and mdm4-null mice by deletion of p53 downstream targets. Cancer Res. 2003;63:8664–9.
Belle JI, Petrov JC, Langlais D, Robert F, Cencic R, Shen S, et al. Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance. Cell Death Differ. 2016;23:759–75.
Stadanlick JE, Zhang Z, Lee S-Y, Hemann M, Biery M, Carleton MO, et al. Developmental arrest of T cells in Rpl22-deficient mice is dependent upon multiple p53 effectors. J Immunol. 2011;187:664–75.
Little JN, Dwyer ND. p53 deletion rescues lethal microcephaly in a mouse model with neural stem cell abscission defects. Hum Mol Genet. 2019;28:434–47.
Williams SE, Garcia I, Crowther AJ, Li S, Stewart A, Liu H, et al. Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice. Development. 2015;142:3921–32.
Liu D, Ou L, Clemenson GD, Chao C, Lutske ME, Zambetti GP, et al. Puma is required for p53-induced depletion of adult stem cells. Nat Cell Biol. 2010;12:993–8.
Zheng TS, Hunot S, Kuida K, Momoi T, Srinivasan A, Nicholson DW, et al. Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nat Med. 2000;6:1241–7.
Abbas HA, Maccio DR, Coskun S, Jackson JG, Hazen AL, Sills TM, et al. Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity. Cell Stem Cell. 2010;7:606–17.
Barkić M, Crnomarković S, Grabusić K, Bogetić I, Panić L, Tamarut S, et al. The p53 tumor suppressor causes congenital malformations in Rpl24-deficient mice and promotes their survival. Mol Cell Biol. 2009;29:2489–504.
Ceccaldi R, Parmar K, Mouly E, Delord M, Kim JM, Regairaz M, et al. Bone marrow failure in fanconi anemia is triggered by an exacerbated p53/p21 DNA damage response that impairs hematopoietic stem and progenitor cells. Cell Stem Cell. 2012;11:36–49.
Ranjan A, Iwakuma T. Non-canonical cell death induced by p53. Int J Mol Sci. 2016;17:2068.
Kroemer G, Martin SJ. Caspase-independent cell death. Nat Med. 2005;11:725–30.
Chautan M, Chazal G, Cecconi F, Gruss P, Golstein P. Interdigital cell death can occur through a necrotic and caspase-independent pathway. Curr Biol. 1999;9:967–S1.
Carter BZ, Kornblau SM, Tsao T, Wang R-Y, Schober WD, Milella M, et al. Caspase-independent cell death in AML: caspase inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin does not affect cell survival or prognosis. Blood. 2003;102:4179–86.
Hirsch T, Marchetti P, Susin SA, Dallaporta B, Zamzami N, Marzo I, et al. The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene. 1997;15:1573–81.
McCarthy NJ, Whyte MK, Gilbert CS, Evan GI. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J Cell Biol. 1997;136:215–27.
Xiang J, Chao DT, Korsmeyer SJ. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci USA. 1996;93:14559–63.
Shan B, Pan H, Najafov A, Yuan J. Necroptosis in development and diseases. Genes Dev. 2018;32:327–40.
Nassour J, Radford R, Correia A, Fusté JM, Schoell B, Jauch A, et al. Autophagic cell death restricts chromosomal instability during replicative crisis. Nature. 2019;565:659–63.
Ke FFS, Vanyai HK, Cowan AD, Delbridge ARD, Whitehead L, Grabow S, et al. Embryogenesis and adult life in the absence of intrinsic apoptosis effectors BAX, BAK, and BOK. Cell. 2018;173:1217–30.e17.
Li T, Kon N, Jiang L, Tan M, Ludwig T, Zhao Y, et al. Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence. Cell. 2012;149:1269–83.
Valente LJ, Gray DHD, Michalak EM, Pinon-Hofbauer J, Egle A, Scott CL, et al. p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa. Cell Rep. 2013;3:1339–45.
We thank Dr. David Simon and Dr. Mark Tessier-Lavigne for insightful discussions and for providing mice carrying Casapse9-null and Puma-null alleles. This work was supported by a March of Dimes Foundation grant no. 6-FY15-189 (to LDA), R35 grant CA197591 (to LDA), and Jane Coffin Childs Fund Postdoctoral Fellowship (to MEB).
Conflict of interest
The authors declare that they have no conflict of interest.
All mouse work was approved and performed in compliance with the Stanford University Administrative Panel on Laboratory Animal Care (APLAC).
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Bowen, M.E., Mulligan, A.S., Sorayya, A. et al. Puma- and Caspase9-mediated apoptosis is dispensable for p53-driven neural crest-based developmental defects. Cell Death Differ (2021). https://doi.org/10.1038/s41418-021-00738-7