Downstream genes of Pax6 revealed by comprehensive transcriptome profiling in the developing rat hindbrain

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.Pax6 is a highly conserved transcription factor that contains two DNA-binding domains, i.e., a paired domain (PD) and a homeodomain. Pax6 has been identified as an essential regulator for the development of the central nervous system (CNS), eyes, nose, pancreas and pituitary gland, mostly through study of the phenotypes of several mouse and rat lines that possess either a spontaneous or an artificial mutation in their Pax6 gene. Although Pax6 heterozygous mutant mice/rats with a semi-dominant mutation are known as Small eye (Sey) mutants and can be bred, their homozygous mutant embryos die soon after birth and exhibit severe phenotypes such as a lack