Abstract
Purpose: :
Research on the development of visual systems has revealed remarkable conservation of eye-associated genes across the animal kingdom. Among such genes are those involved in eye specification, including pax6 (paired box gene 6), eya (eyes absent), six (sine oculis), and dac (dachshund), which function in concert during and are critical for eye formation. The bobtail squid, Euprymna scolopes, has evolved at least two independent sets of tissues that interact with light, a complex eye and a ‘light organ’ that houses the luminous bacterial symbiont Vibrio fischeri. Recent studies of the system demonstrated remarkable biochemical similarities between these two organs, including the presence in the light organ of all proteins critical for visual transduction. Such findings prompted the question: are the two organs under the same developmental specification? The goal of the study reported here was to determine whether the eye-specification genes are expressed in the light organ and, if so, how their expression compares to that of the eye.
Methods: :
We used RACE-PCR to obtain full-length light-organ sequences of each gene and standard PCR to determine whether the same gene was expressed in the eye. We are also localizing gene transcripts and encoded proteins with in situ hybridization (ISH) and immunocytochemistry (ICC), respectively, throughout the trajectory of development, comparing the patterns of expression in the two organs.
Results: :
Phylogenetic comparisons of the full-length sequences confirmed the conserved homeodomains typical of these genes. At least one isoform of each gene is expressed in both the eye and light organ of embryonic, early postembryonic, and adult squid. Thus far, ISH and ICC results localized the pax6, six, and dac genes, and the Six protein to the pores of the light organ, where V. fischeri enters, and to the tissues surrounding the lens of the eye.
Conclusions: :
We are continuing to examine the effect of light production on these patterns by manipulating exposure of: 1) the eye to environmental light; and 2) the light organ to endogenous light, using both wild-type luminous strains of V. fischeri and those defective in light production. The results of these analyses promise to provide evidence that the eye-specification genes are critical for development of light-interacting tissues, independent of their embryonic origin, and are not solely associated with sensory structures, such as the eye.
Keywords: development • fluorescent in situ hybridization • comparative anatomy