Natural antisense transcripts (NATs) are a class of genes whose role in controlling gene expression is becoming more and more relevant. We describe the identification of eight novel mouse NATs associated with transcription factors (Pax6, Pax2, Six3, Six6, Otx2, Crx, Rax and Vax2) that play an important role in eye development and function. These newly identified NATs overlap with the mature processed mRNAs or with the primary unprocessed transcript of their corresponding sense genes, are predicted to represent either protein coding or non-coding RNAs and undergo extensive alternative splicing. Expression studies, by both RT-PCR and RNA in situ hybridization, demonstrate that most of these NATs, similarly to their sense counterparts, display a specific or predominant expression in the retina, particularly at postnatal stages. We found a significant reduction of the expression levels of one of these NATs, Vax2OS (Vax2 opposite strand) in a mouse mutant carrying the inactivation of Vax2, the corresponding sense gene. In addition, we overexpressed another NAT, CrxOS, in mouse adult retina using adeno-associated viral vectors and we observed a significant decrease in the expression levels of the corresponding sense gene, Crx. These results suggest that these transcripts are functionally related to their sense counterparts and may play an important role in regulating the molecular mechanisms that underlie eye development and function in both physiological and pathological conditions.

SUMMARYVax2 is an eye-specific homeobox gene, the inactivation of which in mouse leads to alterations in the establishment of a proper dorsoventral eye axis during embryonic development. To dissect the molecular pathways in which Vax2 is involved, we performed a transcriptome analysis of Vax2 -/-mice throughout the main stages of eye development. We found that some of the enzymes involved in retinoic acid (RA) metabolism in the eye show significant variations of their expression levels in mutant mice. In particular, we detected an expansion of the expression domains of the RA-catabolizing enzymes Cyp26a1 and Cyp26c1, and a downregulation of the RA-synthesizing enzyme Raldh3. These changes determine a significant expansion of the RA-free zone towards the ventral part of the eye. At postnatal stages of eye development, Vax2 inactivation led to alterations of the regional expression of the cone photoreceptor genes Opn1sw (S-Opsin) and Opn1mw (M-Opsin), which were significantly rescued after RA administration. We confirmed the above described alterations of gene expression in the Oryzias latipes (medaka fish) model system using both Vax2 gain-and loss-of-function assays. Finally, a detailed morphological and functional analysis of the adult retina in mutant mice revealed that Vax2 is necessary for intraretinal pathfinding of retinal ganglion cells in mammals. These data demonstrate for the first time that Vax2 is both necessary and sufficient for the control of intraretinal RA metabolism, which in turn contributes to the appropriate expression of cone opsins in the vertebrate eye.