Dynamic expression of the p53 family members p63 and p73 in the mouse and human telencephalon during development and in adulthood

Abstract

p63 and p73, family members of the tumor suppressor p53, are critically involved in the life and death of mammalian cells. They display high homology and may act in concert. The p73 gene is relevant for brain development, and p73-deficient mice display important malformations of the telencephalon. In turn, p63 is essential for the development of stratified epithelia and may also play a part in neuronal survival and aging. We show here that p63 and p73 are dynamically expressed in the embryonic and adult mouse and human telencephalon. During embryonic stages, Cajal-Retzius cells derived from the cortical hem co-express p73 and p63. Comparison of the brain phenotypes of p63- and p73- deficient mice shows that only the loss of p73 function leads to the loss of Cajal-Retzius cells, whereas p63 is apparently not essential for brain development and Cajal-Retzius cell formation. In postnatal mice, p53, p63, and p73 are present in cells of the subventricular zone (SVZ) of the lateral ventricle, a site of continued neurogenesis. The neurogenetic niche is reduced in size in p73-deficient mice, and the numbers of young neurons near the ventricular wall, marked with doublecortin, Tbr1 and calretinin, are dramatically decreased, suggesting that p73 is important for SVZ proliferation. In contrast to their restricted expression during brain development, p73 and p63 are widely detected in pyramidal neurons of the adult human cortex and hippocampus at protein and mRNA levels, pointing to a role of both genes in neuronal maintenance in adulthood.

Introduction

The transcription factor family p53 has three members, p53, p63, and p73. Each has a distinct expression pattern and plays distinct roles, although the three proteins may work in concert (Bourdon, 2007, Levrero et al., 2000, McKeon and Melino, 2007). p53 is the classical tumor suppressor, mutated in more than 50% of human cancers (Murray-Zmijewski et al., 2006). Like p53, p63 and p73 have tumor-suppressor activities (Flores et al., 2002), but in addition, they are necessary for the development of specific tissues and organs. p63 is essential for the development and maintenance of stratified epithelial tissues; p63^−/− mice are born without skin and limbs and die soon after birth (Mills et al., 1999, Yang et al., 1998). Mutations of the human TP63 gene are associated with a variety of syndromes, such as ectodermal dysplasia, cleft lip/palate syndrome; split-hand/foot malformation (SHFM); limb–mammary syndrome; Rap-Hodgkin syndrome (RHS), and orofacial cleft (Cabiling et al., 2007, Chan et al., 2004, Chan et al., 2005, Huang et al., 2005, Julapalli et al., 2009, Rinne et al., 2007, van Bokhoven et al., 2001). In turn, the inactivation of the p73 gene in mice gives rise to immunological, neurological, and pheromonal defects (Yang et al., 2000).

The p53 family members have a similar domain structure, displaying an N-terminal transactivation (TA) domain, a DNA-binding domain (DBD), and an oligomerization domain (OD). p73 and p63 share a sterile alpha motif domain (SAM) and an inhibitory domain (ID) at their C-termini and have a higher sequence homology as compared with p53. p53 family members have common target genes, e.g., p21 (Jost et al., 1997, Kaghad et al., 1997), as well as specific targets, such as CaN19 for p63 (Kirschner et al., 2008). The three proteins form a complex network, which may be of significance in cancer formation (Flores et al., 2002, Muller et al., 2006, Schilling et al., 2010). p63 and p73 use multiple promoters to generate an array of isoforms, including full-length TA isoforms, and amino-terminally truncated (∆N) isoforms, which lack the TA domain. The TA isoforms of p63 and p73 can activate downstream target genes and induce apoptosis, whereas the ∆N isoforms act as dominant inhibitors of the full-length forms of p53, p63, and p73, thereby inhibiting transactivation of target genes and induction of apoptosis (Flores et al., 2002, Grob et al., 2001, Melino et al., 2004, Muller et al., 2005, Wu et al., 2003, Wu et al., 2005). Further complexity derives from differential splicing of the last four exons of p63 and p73. For p73, the combination between N-terminal (TA or ∆Np73) isoforms and C-terminal splice variants (p73α-η) can produce up to 14 p73 proteins (Murray-Zmijewski et al., 2006).

p73 is important for the development of the central nervous system (CNS), in particular, for hippocampal development and cortical regionalization (Meyer et al., 2004). A remarkable aspect of p73-deficient mice is their lack of Cajal-Retzius (CR) cells (Meyer et al., 2002, Yang et al., 2000), which in wild-type mice represent the most important source of Reelin, a secreted glycoprotein that controls radial migration into the cortex (D'Arcangelo et al., 1995). The most severe brain phenotype is obtained by complete inactivation of the p73 gene (Yang et al., 2000), whereas selective inactivations of TAp73 (Tomasini et al., 2008) and ∆Np73 (Ravni et al., 2010, Tissir et al., 2009, Wilhelm et al., 2010) give rise to much milder brain defects. In the adult human cerebral cortex, both the TAp73 and ∆Np73 isoforms are widely expressed (Cabrera-Socorro et al., 2006). Several studies have linked ∆Np73 with neuronal survival pathways in the central and peripheral nervous system (Lee et al., 2004, Pozniak et al., 2002, Pozniak et al., 2000, Tissir et al., 2009), and there is increasing evidence involving p73 in neurodegeneration, Alzheimer 's disease, and cortical ischemia (Bui et al., 2009, Wetzel et al., 2008, Wilson et al., 2004). On the other hand, also p63 may play a role in neuronal survival (Dugani et al., 2009, Jacobs et al., 2005). As yet, the contributions of p63 and p53 to brain development is not well known, although a subset of p53-deficient mouse embryos exhibit severe brain malformations such as exencephaly (Armstrong et al., 1995, Sah et al., 1995).

In order to better understand the possible activities of the p53 family in the nervous system, we analyze here the normal expression patterns of p73 and p63 in the telencephalon during development and in adulthood. We find that prenatally they are co-expressed in CR cells of the cerebral cortex and hippocampus. At early postnatal stages, we study their expression in the neurogenetic niche of the subventricular zone of the lateral ventricle. We also examine the brains of p73 and p63 knockout (ko) mice. In addition, by RT-PCR and immunohistochemistry using pan-p63 antibodies, we demonstrate the presence of p63 in the adult human cortex, where it is widely expressed, and colocalized with TAp73.