Numayama-Tsuruta Keiko, Arai Yoko, Takahashi Masanori, Sasaki-Hoshino Makiko, Funatsu Nobuo, Nakamura Shun, Osumi Noriko
Division of Developmental Neuroscience, Center for Translational and Advanced Animal Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
BMC Dev Biol. 2010 Jan 18;10:6. doi: 10.1186/1471-213X-10-6.
The transcription factor Pax6 is essential for the development of the central nervous system and it exerts its multiple functions by regulating the expression of downstream target molecules. To screen for genes downstream of Pax6, we performed comprehensive transcriptome profiling analyses in the early hindbrain of Pax6 homozygous mutant and wild-type rats using microarrays.
Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up- or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally.
These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.
转录因子Pax6对中枢神经系统的发育至关重要，它通过调节下游靶分子的表达发挥多种功能。为了筛选Pax6的下游基因，我们使用微阵列对Pax6纯合突变体和野生型大鼠的早期后脑进行了全面的转录组谱分析。
使用两种计算方法对四重微阵列实验进行比较，使我们能够鉴定出具有相对较小倍数变化或低表达水平的差异表达基因。对差异表达分子的基因本体分析表明，Pax6参与了各种信号转导途径，在这些途径中它调节许多受体、信号分子、转运蛋白和转录因子的表达。这些基因的上调或下调通过定量RT-PCR进一步得到证实。Fabp7、Dbx1、Unc5h1和Cyp26b1 mRNA的原位染色显示，这些转录本的表达不仅在野生型和Pax6杂合突变体的后脑与Pax6的表达重叠，而且在Pax6纯合突变体的后脑明显降低。此外，Pax6纯合突变体后脑显示，Cyp26b1在菱脑节5和6的背侧和腹外侧区域缺乏表达，并且菱脑节5的大小在前后方向上扩大。
这些结果表明Unc5h1和Cyp26b1是被Pax6反式激活的靶基因的新候选者。此外，我们的结果提示了一个有趣的可能性，即Pax6通过激活Cyp26b1（一种代谢视黄酸的酶）来调节后脑的前后模式。
