FIG. 8.

Neuronal differentiation of embryonic stem cells induces the expression of both Zac1 and p73. (A) Light microscopy of mES cells and qRT-PCR analysis of embryonic stem cell markers. Top panels, mES cells differentiate progressively into neurons following Lif withdrawal for the indicated number of days (d). Bottom panels, the expression of the pluripotency marker Oct4 is rapidly downregulated in mES cells upon Lif withdrawal. In contrast, the expression of the early neuroectoderm marker Sox1 and the progenitor marker Nestin were increased and followed by the increased expression of the late neuronal marker Tuj. (B to D) qRT-PCR analysis. The expression of Zac1 (B), entire p73, TAp73, or ΔNp73 (C), and p53 (D) was measured following Lif withdrawal for the indicated number of days. Zac1 and TAp73 are coinduced by Lif withdrawal, while p53 is downregulated. (E) IB analysis. WCE from mES cells grown for the indicated number of days in the absence of Lif were immunoblotted and tested with the indicated antibodies. Isoforms of p73 (α, β, and γ) are indicated. The coinduction of TAp73 and Zac1 mRNAs translates into their increased protein expression and the upregulation of the cyclin kinase inhibitor proteins p21^Cip1 and p57^Kip2. (F) qRT-PCR analysis. Expression of the Cip/Kip (p21^Cip1, p27^Kip1, and p57^Kip2) and INK4 (p16^Ink4a, p15^Ink4b, p18^Ink4c, and p19^Ink4d) families following Lif withdrawal for the indicated number of days. The coinduction of TAp73 and Zac1 coincides with the upregulation of the direct p73 target p21^Cip1 and p57^Kip2 genes.