Identification of differentially expressed genes in mouse Pax6 heterozygous lenses.

The critical regulatory gene Pax6 is located on human chromosome 11p13 and mouse chromosome 2. It is expressed in many developing ocular tissues, brain, and pancreas.1 The Pax6 gene encodes a specific DNA-binding transcription factor capable of initiating ectopic lens2 and eye3 development. Heterozygous mutations in human PAX6 induce a spectrum of ocular diseases including aniridia, Peters anomaly, autosomal dominant keratitis, foveal hypoplasia, and earlyonset cataracts.1 In addition, more recent studies showed that PAX6 haploinsufficiency in humans leads to cerebral malformations and olfactory dysfunction.4 Homozygous PAX6 mutations cause anophthalmia, brain malformation, and neonatal lethality.5 Two major forms of the protein, Pax6 and Pax6(5a), result from alternate splicing of Pax6 mRNA.6 The function of Pax6(5a) has not been studied as extensively as Pax6; however, overexpression of PAX6(5a) relative to PAX6 was detected in human congenital cataracts.5 Ectopic expression of Pax6(5a) in lens fiber cells of transgenic mice results in an abnormal lens phenotype, associated with changes in the levels of cell adhesion proteins.7 The expression pattern of Pax6 in the developing mouse embryo and other vertebrate systems reveals a dynamic behavior of Pax6 from the onset of expression (mouse embryonic day [E]8.0) up to the end of organ morphogenesis.8 Pax6 is also expressed in many adult tissues.9-11 Despite extensive studies indicating roles for Pax6 in biological processes, as diverse as cellular proliferation, differentiation, cell migration, cell-to-cell adhesion, and signal transduction pathways, the genes directly regulated by Pax6 are largely unidentified. Studies on the transcriptional regulation of crystallin genes in vertebrate lenses implicate Pax6 as a critical regulatory factor influencing, at least, αA-, αB-, δ1-, βB1-, and ζ-crystallins.12,13 In addition, two genes expressed in the cornea, keratin K12 and gelatinase B, are known to be transcriptionally regulated by Pax6.14-15 Pax6 has also been implicated in transcriptional control of a small set of genes expressed in the developing lens, encoding diverse transcription factors, including Eya-1 and -2, and c-Maf.16,17 In the optic cup and stalk, Pax6 has been shown to control expression of Pax2.18 Finally, in nonocular tissues, the genes directly regulated by Pax6 in the brain and pancreas are L1 CAM and insulin, glucagon and somatostatin, respectively.19,20 In all instances, except for Eya-1 and -2, Pax6 has been shown to directly bind regulatory elements of the genes just listed.12-15,17-20 In addition, analysis of mouse embryonic Pax6-null brains has shown that cadherin 6, Lim-1, Gsh1, Islet-2, and Wnt-7b are also possibly directly activated by Pax6.21-23 High-throughput technologies based on expression analysis of mRNA involving cDNA microarrays24 and differential display RT-PCR (RT-PCR-DD)25 offer rapid detection of novel candidate target genes for developmental regulatory factors. Pax6 haploinsufficient lenses can serve as an advantageous system to identify genes regulated by Pax6, because Pax6 homozygous embryos are completely without lenses. In the current study, we used RT-PCR-DD and a candidate-gene approach to identify target genes of Pax6. These methods were chosen over others, because they can be reliably conducted with relatively small amounts of RNA,26 and, in contrast to cDNA microarrays, can detect low-level transcripts. These strategies yielded eight genes showing reduced expression in the Pax6 haploinsufficient lenses. The functional roles of these genes agree with established roles of Pax6 in lens biology. Collectively, the present data provide the molecular basis for understanding the role of Pax6 and other critical transcription factors required for lens development and maintenance.