Developmental Biology, Institute Biology I, Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany.
Biological Services, Weizmann Institute of Science, Rehovot, Israel.
PLoS One. 2014 Mar 18;9(3):e92356. doi: 10.1371/journal.pone.0092356. eCollection 2014.
Myc proteins control cell proliferation, cell cycle progression, and apoptosis, and play important roles in cancer as well in establishment of pluripotency. Here we investigated the control of myc gene expression by the Pou5f1/Oct4 pluripotency factor in the early zebrafish embryo. We analyzed the expression of all known zebrafish Myc family members, myca, mycb, mych, mycl1a, mycl1b, and mycn, by whole mount in situ hybridization during blastula and gastrula stages in wildtype and maternal plus zygotic pou5f1 mutant (MZspg) embryos, as well as by quantitative PCR and in time series microarray data. We found that the broad blastula and gastrula stage mych expression, as well as late gastrula stage mycl1b expression, both depend on Pou5f1 activity. We analyzed ChIP-Seq data and found that both Pou5f1 and Sox2 bind to mych and mycl1b control regions. The regulation of mych by Pou5f1 appears to be direct transcriptional activation, as overexpression of a Pou5f1 activator fusion protein in MZspg embryos induced strong mych expression even when translation of zygotically expressed mRNAs was suppressed. We further showed that MZspg embryos develop enhanced apoptosis already during early gastrula stages, when apoptosis was not be detected in wildtype embryos. However, Mych knockdown alone did not induce early apoptosis, suggesting potentially redundant action of several early expressed myc genes, or combination of several pathways affected in MZspg. Experimental mych overexpression in MZspg embryos did significantly, but not completely suppress the apoptosis phenotype. Similarly, p53 knockdown only partially suppressed apoptosis in MZspg gastrula embryos. However, combined knockdown of p53 and overexpression of Mych completely rescued the MZspg apoptosis phenotype. These results reveal that Mych has anti-apoptotic activity in the early zebrafish embryo, and that p53-dependent and Myc pathways are likely to act in parallel to control apoptosis at these stages.
Myc 蛋白控制细胞增殖、细胞周期进程和细胞凋亡，并在癌症以及多能性的建立中发挥重要作用。在这里，我们研究了 Pou5f1/Oct4 多能性因子在早期斑马鱼胚胎中对 myc 基因表达的控制。我们通过全胚胎原位杂交技术，在野生型和母体加合子 pou5f1 突变型（MZspg）胚胎的囊胚和原肠胚阶段分析了所有已知的斑马鱼 Myc 家族成员（myca、mycb、mych、mycl1a、mycl1b 和 mycn）的表达情况，同时还通过定量 PCR 和时间序列微阵列数据进行了分析。我们发现，广泛的囊胚和原肠胚阶段 mych 表达，以及晚期原肠胚阶段 mycl1b 表达，都依赖于 Pou5f1 活性。我们分析了 ChIP-Seq 数据，发现 Pou5f1 和 Sox2 都结合在 mych 和 mycl1b 的调控区域。Pou5f1 对 mych 的调控似乎是直接的转录激活，因为在 MZspg 胚胎中过表达 Pou5f1 激活融合蛋白，即使抑制合子表达的 mRNA 的翻译，也能诱导强烈的 mych 表达。我们进一步表明，MZspg 胚胎在早期原肠胚阶段就出现了增强的细胞凋亡，而在野生型胚胎中则没有检测到细胞凋亡。然而，单独敲低 Mych 并不能诱导早期凋亡，这表明几种早期表达的 myc 基因可能具有冗余作用，或者 MZspg 中影响几种途径的组合。在 MZspg 胚胎中过表达实验性 mych 确实显著，但不完全抑制细胞凋亡表型。同样，p53 敲低仅部分抑制 MZspg 原肠胚胚胎的凋亡。然而，p53 和 Mych 的联合敲低完全挽救了 MZspg 凋亡表型。这些结果表明，Mych 在早期斑马鱼胚胎中具有抗凋亡活性，并且 p53 依赖性和 Myc 途径可能平行作用于这些阶段的细胞凋亡控制。
