Vydianathan Ravi1,¶, Shipra Bhatia2,¶, Phillippe Gautier2, Felix Loosli3, Boon-Hui Tay1, Alice Tay1, Emma Murdoch2, Pedro Coutinho2, Veronica van Heyningen2, Sydney Brenner1, Byrappa Venkatesh1,* and Dirk A. Kleinjan2,*
Institute of Molecular and Cell Biology, Agency for Science Technology and Research (A*STAR), Biopolis, Singapore 138673, Singapore.
MRC Human Genetics Unit at the MRC IGMM at the University of Edinburgh, EdinburghEH4 2XU, United Kingdom.
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann von Helmholtz Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany.
¶ - joint first-authors
* - Corresponding authors
Published in PLoS Genetics on 24 January 2013.
To read paper, please click here.
Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii), and found two distinct Pax6 loci in its genome. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships we identify homologs of this novel paired-less Pax6.2 gene in lizard, frog, as well as in zebrafish and other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event, and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a 'small eye' phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and independent, lineage-specific gene losses.
Functional assay of PAX6 conserved noncoding element (CNE) -200 in transgenic zebrafish and mouse. Upper panel: VISTA plot of PAX6 locus from human, mouse, chicken, zebrafish (Pax6a and b copies) and elephant shark. The human sequence (x-axis) was used as base and a cut-off of ≥70% identity and >100 bp window size was used for identifying CNEs. The y-axis represents percent identity and shaded peaks represent CNEs (pink) and conserved exons (blue). CNE-200 is present in human, mouse, chicken, zebrafish-a, and elephant shark PAX6 loci but lost in zebrafish-b. Middle panel: Transgenic assay of human CNE-200 in zebrafish (A-C). Lateral view of an E-200-gata2-GFP reporter line showing expression in olfactory bulbs (OB) and hindbrain (HB) in a 48 hpf of zebrafish embryo (A); ventral (B) and dorsal (C) views of the embryo head region showing expression in the OB and HB, respectively. Lower panel: Transgenic assay of human CNE-200 in mouse (H-J). LacZ reporter mouse transgenics using the human CNE-200 element (H-J). Expression in the neural tube is intrinsic to the Hsp68-LacZ cassette (H); expression starts to appear from E12.5 in the olfactory tracts at the base of the cortex (I); at E15.5 (J) expression is seen in OB, lateral olfactory tracts (LOT) and a thin band in the dorsal cerebellum (CB).
For more information on Byrappa VENKATESH's laboratory, please click here.