Analysis of the Otd-dependent transcriptome supports the evolutionary conservation of CRX/OTX/OTD functions in flies and vertebrates

Abstract

Homeobox transcription factors of the vertebrate CRX/OTX family play critical roles in photoreceptor neurons, the rostral brain and circadian processes. In mouse, the three related proteins, CRX, OTX1, and OTX2, fulfill these functions. In Drosophila, the single founding member of this gene family, called orthodenticle (otd), is required during embryonic brain and photoreceptor neuron development. We have used global gene expression analysis in late pupal heads to better characterize the post-embryonic functions of Otd in Drosophila. We have identified 61 genes that are differentially expressed between wild type and a viable eye-specific otd mutant allele. Among them, about one-third represent potentially direct targets of Otd based on their association with evolutionarily conserved Otd-binding sequences. The spectrum of biological functions associated with these gene targets establishes Otd as a critical regulator of photoreceptor morphology and phototransduction, as well as suggests its involvement in circadian processes. Together with the well-documented role of otd in embryonic patterning, this evidence shows that vertebrate and fly genes contribute to analogous biological processes, notwithstanding the significant divergence of the underlying genetic pathways. Our findings underscore the common evolutionary history of photoperception-based functions in vertebrates and invertebrates and support the view that a complex nervous system was already present in the last common ancestor of all bilateria.

Figure 1. Differential gene expression in pupal heads of wild type and otd^UVI flies

Validation of differentially expressed genes identified by microarray. A) RT-PCR comparisons of expression levels in pupal heads of OR, CS, and one of two otd^UVI mutant lines used in our analysis; B) example of an RT-PCR test run with the internal control GAPDH1. Upper panel: gel of RT-PCR for GstE1 run with (lanes 1, 3, 5) or without (lanes 2, 4, 6) the GAPDH1 internal control. Lower panel: ImageJ plot showing change in GstE1 peaks relative to each GAPDH1 internal control; C) results of one test for β-Galactosidase activity (Rh4-lacZ) in adult heads of wild type versus otd^UVI mutant flies, negative control is wild type CS without Rh4-lacZ. Lower levels of β-Galactosidase activity in the otd^UVI mutant samples as compared to wild type were repeatedly observed in this and other tests using two detection methods (see Materials and Methods). In this experiment, n=6, error bars = ± 1 SE or standard error.

Figure 2. Otd is expressed in the Hofbauer-Buchner eyelet and a subset of the DN3 pacemaker neurons

Confocal microscope image of 10 micron cryostat sections from adult fly heads (wild type). Otd expression is found in the eyelet and in some of the DN3 circadian pacemaker neurons. Images were processed with Adobe Photoshop. (A): section stained with antibodies to visualize expression of Otd (red) and Rh6 (green). The eyelet (arrow) is located below the retina and expresses both Otd in the nucleus and Rh6 in the rhabdomeric membranes. Two of the 4 cells of the eyelet are visible in this section. In the retina (region marked by the vertical side bar), the Otd protein is found in the nucleus of multiple R-cells and Rh6 is found in the rhabdomere of a subset of the retinal R8 neurons. Additional neurons in the brain also express Otd (such as the three Otd-positive nuclei in the lower right end corner). (B): section stained with antibodies to visualize expression of Otd (red), the R32 enhancer-trap marker for all circadian pacemaker neurons (green), and the PDF marker for ventro-lateral pacemaker neurons and their projections (blue). Panel shows the projection of an A–Z series that spans part of the DN3 cluster (see inset for higher magnification). Otd is expressed in some but not all of the DN3 neurons (arrow). DN1 neurons are located nearby in a more dorso-medial position (arrowhead) but do not express Otd. The cell bodies of the PDF-positive ventro-lateral pacemaker neurons are outside the field shown in B and do not expressed Otd, but their projections help identify the DN3 and DN1 groups.