May 1, 2009;
DeltaNp63 antagonizes p53 to regulate mesoderm induction in Xenopus laevis.
, a homolog of the tumor suppressor p53
, is critical for the development and maintenance of complex epithelia. The developmentally regulated p63
isoform, DeltaNp63, can act as a transcriptional repressor, but the link between the transcriptional functions of p63
and its biological roles is unclear. Based on our initial finding that the mesoderm
-inducing factor activin A is suppressed by DeltaNp63 in human keratinocytes
, we investigated the role of DeltaNp63 in regulating mesoderm
induction during early Xenopus laevis development. We find that down-regulation of DeltaNp63 by morpholino injection in the early Xenopus embryo
formation whereas ectopic expression of DeltaNp63 inhibits mesoderm
formation. Furthermore, we show that mesodermal induction after down-regulation of DeltaNp63 is dependent on p53
. We propose that a key function for p63
in defining a squamous epithelial phenotype is to actively suppress mesodermal cell fates during early development. Collectively, we show that there is a distinct requirement for different p53
family members during the development of both mesodermal and ectodermal tissues. These findings have implications for the role of p63
in both development and tumorigenesis of human epithelia.
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References [+] :
Fig. 2. ?Np63 loss and gain of function results in anterior patterning defects in Xenopus laevis. (A) Four-cell Xenopus embryos were injected dorsally with ?Np63alpha or ?Np63alpha-R304W mRNA (2 ng each) and scored at the tailbud stage. (B) Four-cell Xenopus embryos were injected dorsally with p63MO-A (200 ng) in the presence or absence of ?Np63alpha mRNA (2 ng), and scored at the tailbud stage. (C) Table summarizing results from embryo injections in (A) and (B). (D) Animal caps injected with control or p63MO, or ?Np63-encoding RNA were fixed, sectioned, and stained for p63 (4A4 antibody). (E) Whole embryo RT-PCR shows that the pan-mesodermal marker Xbra and the anterior mesodermal marker Gsc are down-regulated in ?Np63alpha-injected but not ?Np63alpha-R304W-injected embryos. (F) In situ hybridization analysis shows that ?Np63 is expressed in the animal pole of late blastula (stage 9) embryos and in most of the epidermis of mid-neurula (stage 18) embryos (anterior is on the left).
Fig. 3. ?Np63 suppresses activin-mediated mesoderm induction. (A) ?Np63alpha suppresses activin-mediated explant elongation. Animal caps from embryos injected with ?Np63alpha mRNA (2 ng), ?Np63alpha-R304W mRNA (2 ng), or p63MO-A (200 ng) were dissected at stage 9 and cultured overnight in the presence or absence of activin A (10 ng/ml). Embryos were then fixed and photographed at sibling stage 18. (B) Activin treatment of animal caps results in down-regulation of ?Np63 expression. Caps were treated with activin (+ Act, 10 ng/ml) for 2 h or left untreated, fixed, sectioned, and stained for p63 (4A4 antibody). Scale bar is 50 ?m. (C) Animal caps injected with p63MO-A and B (100 ng each) were cultured in the presence of the indicated concentrations of activin, fixed, and photographed at sibling stage 18. Length-to-width (LWR) ratios for these explants were determined as previously described (Tahinci et al., 2007). Numbers in parentheses indicate numbers of explants scored. Asterisks indicate significant difference between p63MO injected and the corresponding uninjected explants (p <0.01). (D) Animal caps injected with p63MO-A or p63-MO-B (200 ng) or a morpholino against ?-globin (control MO, 200 ng) were cultured in the presence or absence of activin (10 ng/ml). RNA was isolated and levels of the mesodermal marker brachyury (Xbra), the ectodermal marker epidermal keratin, and ODC (control for RNA extraction) were assayed by RT-PCR.
Fig. 4. Mesoderm induction due to loss of p63 is p53-dependent. (A) Embryos were injected with δNp63 mRNA (2 ng), p63MO-A/B (200 ng), p53 mRNA (100 pg), p53MO (30 ng), p53 mRNA + p63MO-A/B, or p53MO + p63MO-A/B. The relative extent of the Xbra expression domain between the injected and uninjected sides of each embryo was quantified as shown (two lower right image panels) and plotted on a bar graph. β-galactosidase mRNA (β-gal, 250 pg) was co-injected to mark the injection site. (B) RT-PCR analysis in animal caps injected with p63MO, p53MO, or both morpholinos confirms that down-regulation of p63 can induce mesoderm (as assessed by the pan-mesodermal marker Xbra) only in the presence of p53. (C) Embryos were injected as in (A), fixed at stage 26, stained for β-galactosidase activity (X-gal, blue color), and processed for whole-mount immunohistochemistry with an antibody specific for somitic mesoderm (12/101). The relative extent of the somitic mesoderm between the injected and uninjected sides of each embryo was measured as shown (two lower right image panels) and plotted on a bar graph. (D) Embryos were injected with δNp63 mRNA (2 ng), p53 mRNA (100 pg), or both, and β-galactosidase mRNA (β-gal, 250 pg) was co-injected to mark the injection site. The relative extent of the Xbra expression domain between the injected and uninjected sides of each embryo was quantified as in (A) and plotted on a bar graph. For (A) and (D), injected embryos were fixed at stage 10.5, stained for β-galactosidase activity (Red-gal, red color) and processed for in situ hybridization with probes for the pan-mesodermal marker brachyury (Xbra). Analysis of the extent of mesoderm tissue formation for (A), (C), and (D) is described in the Materials and methods section. , : single asterisks indicate significant changes from control embryos; double asterisks indicate significant changes between p63 MO and p63MO + p53 injected embryos (Student's t-test, p < 0.05). Numbers in parentheses indicate numbers of embryos scored.
Fig. 5. Loss and gain of δNp63 function affects dorsal but not ventral mesoderm. (A) In situ hybridization of early gastrulae with a chordin (dorsal mesoderm) probe. Dorsal sides of representative embryos for each experimental condition are shown. (B). In situ hybridization of early gastrulae with a Xwnt8 probe (ventral mesoderm). Representative embryos are shown with their vegetal poles oriented so that the dorsal side is facing down.
Supplemental Fig. 1. Modulation of p53/δNp63 regulates the expression of markers of cell fate. (A) In situ hybridization analysis of embryos injected with δNp63 mRNA and morpholino show that expression of the ectodermal marker Dlx3 is regulated by δNp63, indicating that the prospective ectoderm is competent to respond to δNp63 signaling at an early developmental stage. Animal poles of embryos are shown. (B) Xenopus animal caps were injected with morpholino oligonucleotides targeting δNp63 and p53 singly, or in combination, or with a control morpholino targeting β-globin. RNA was isolated and the levels of the mesodermal marker, Mix.2, was measured using quantitative real-time PCR and normalized to the expression of ornithine decarboxylase (ODC). Data is representative of at lease three individual experiments
Supplemental Fig. 2. Endogenous p63/p53 in situ analysis. In situ hybridization shows that p53 is expressed in both the prospective ectoderm and mesoderm of the developing gastrula [as previously described, Hoever et al, Oncogene, 9, 10920 (1994)], while p63 is primarily expressed in the ectoderm. This suggests that regulation of p63 expression can be endogenously controlled in a dynamic fashion between mesodermal and ectodermal tissues and that p53 and p63 interact endogenously in the prospective ectoderm. The limits of p63 expression in the equatorial region determine the extent of ectodermal (vs. mesodermal) formation.
Endodermal Nodal-related signals and mesoderm induction in Xenopus.