Final Report Summary - COLOURHODOPSIN (Maintenance of functional identity of Drosophila colour photoreceptor neurons)
How small changes in our genomes cause dramatic changes in sensation, neuronal processing and behavior is poorly understood. Though genome-wide association studies have identified numerous potential genetic variants causing neurological disease or normal phenotypes, further studies to understand how these genetic changes affect phenotypes at the molecular level are often not feasible. Model organisms, including fruit flies, provide an excellent way to understand the variation of neurological development and behavior, the genetic causes, and the affected molecular mechanisms. In the COLOURHODOPSIN project, we investigated natural variation in the fruit fly Drosophila melanogaster visual system to address how natural variation affects the mechanisms that control color photoreceptor cell fate specification and maintenance.
We primarily focused on the mutually exclusive expression of two light receptor proteins, blue-sensitive Rhodospin5 (Rh5) and green-sensitive Rh6, which define two subtypes of R8 photoreceptor neurons (retinal neurons that are sensitive to light). Initiation and maintenance of expression of these two Rhodopsins are the last steps in the retinal photoreceptor differentiation program which arguably is among best-understood for a sensory system. Thus, any perturbations in this expression pattern would be indicative of a problem in a number of possible stages of retinal development. Our main experimental approach was to immunohistochemically visualize Rh5- and Rh6-expressing cells, i.e. to use antibody reagents to attach different fluorescent labels to Rh5 and Rh6 proteins in dissected fly retinas and then to image them under a fluorescence microscope. We did this either with flies captured in the wild or their descendants. And, we found a surprisingly high level of natural variation in adult retina photoreceptors.
We first examined a previously-established collection of approximately 200 inbred fly lines that were derived from wild-caught flies. A major advantage of this collection is that the genomes of each line have been sequenced, making identification of genetic variations among them much easier. Among these lines we identified at least nine distinct phenotypes (major differences from lab fly strains). For example, while in the normal lab strains the ratio of Rh5- to Rh6- expressing cells is approximately 30%:70%, in these fly lines it ranges from 0% to 80% of Rh5-expressing cells. In some lines, mechanisms responsible for mutually exclusive Rh5/Rh6 expression failed, resulting in cells with both Rhodopsin. Seven lines showed no Rh5 expression; instead, many photoreceptor cells contained neither Rhodopsin. We also observed completely novel phenotypes such as in one line where Rh5 is normal if flies are kept in light but disappears if the same flies are kept in the dark. Finally, a number of non-Rhodopsin phenotypes such as premature retinal degeneration were also found.
We next set out to identify some of the genes which are affected in these lines. 1) In collaboration with Robert Johnston’s group (Johns Hopkins University) we found that increase in proportion of Rh5-cells was often associated with alterations of the spineless gene and we specifically identified one such alteration that is present relatively broadly in wild fly populations. This alteration affects the way spineless gene is regulated and led Johnston group to identify the regulator protein, transcription factor klumpfus, whose binding to the spineless gene sequence is affected by the alteration. 2) On the other end of the spectrum, we found that the fly line with all R8 photoreceptors expressing Rh6 has a short deletion in the melted gene. This deletion removes a part of the sequence that is essential for the activation of this gene. And, melted needs to be active to produce Rh5-expressing photoreceptors. In collaboration with Jens Rister’s group (University of Massachusetts Boston) we are pursuing this genetic variant in order to extend our understanding of melted regulation during photoreceptor differentiation. 3) In the seven lines with no detectable Rh5, but with many “empty” photoreceptor cells, we identified two different mutations which affect Rh5 gene itself, such that it does not encode a full length Rh5 protein. We tested to what extent the two mutations affect Rh5 function, using drosophila larvae who need Rh5 to have a normal light-avoiding behaviour, and found that one mutation completely destroys Rh5 function, while the other one still allows larvae to avoid light. For a number of other phenotypes we are in process of identifying the affected genes: for some of them, we determined which chromosome carry the genetic changes, and for one we have narrowed down the possibilities to 10 genes.
While the above inbred-line approach has proved to be very efficient at identifying natural genetic variants, one can criticize it because of the possibility that at least some of the phenotypes would be due to inbreeding and would be encountered rarely in the wild. Therefore, we wanted to know whether we could apply the techniques we developed with inbred lines to identify natural variants in the flies that we catch ourselves, without generation of inbred lines. The aproach is to capture a number of flies from different locations in France (and later in Europe), breed them individually and when the progeny is secured, to stain them to reveal Rh5/Rh6 expression. Then, for the flies which show interesting phenotypes try to recover the same phenotypes in their progeny (showing inheritance of the phenotype) and identify first the affected gene and then the actual genetic change that causes the phenotype. We are well on the way developing this programme and have to date obtained two fly lines with interesting phenotypes. For one of them we already identified sevenless as the gene that carries the causative mutation.
In human species, there is a surprising level of natural variation affecting the visual system. The ratios of different colour photoreceptors differ vastly among the population. Colorblindness caused by mutations in the opsin genes are relatively common, affecting ~10% of male population. And, vast number of rare mutations affecting a long list of genes cause devastating retinal degeneration pathologies. Our work so far suggests that similar situation exists in the Drosophila natural populations: the ratios of photoreceptor types vary extensively, mutations in Rhodopsin genes occur with moderate frequency, and many of the retina developmental genes can carry rare strong-impact mutations. The questions we are left with are: 1) to understand deeper the structure of the natural variation affecting the visual system in Drosophila, 2) to ask whether there is something special about the natural variants affecting the eye development and function or if other aspects of the nervous system can be similarly affected and 3) what we can learn about retina development and function from the natural variants that we identify? The scientific program and the approaches that we have developed with the COLOURHODOPSIN project have set the solid foundation to address these questions and have already led not only to new insights in the eye development but also to an already mature collaboration to investigate the natural variation affecting the circadian rhythm entrainment in wild flies.
We primarily focused on the mutually exclusive expression of two light receptor proteins, blue-sensitive Rhodospin5 (Rh5) and green-sensitive Rh6, which define two subtypes of R8 photoreceptor neurons (retinal neurons that are sensitive to light). Initiation and maintenance of expression of these two Rhodopsins are the last steps in the retinal photoreceptor differentiation program which arguably is among best-understood for a sensory system. Thus, any perturbations in this expression pattern would be indicative of a problem in a number of possible stages of retinal development. Our main experimental approach was to immunohistochemically visualize Rh5- and Rh6-expressing cells, i.e. to use antibody reagents to attach different fluorescent labels to Rh5 and Rh6 proteins in dissected fly retinas and then to image them under a fluorescence microscope. We did this either with flies captured in the wild or their descendants. And, we found a surprisingly high level of natural variation in adult retina photoreceptors.
We first examined a previously-established collection of approximately 200 inbred fly lines that were derived from wild-caught flies. A major advantage of this collection is that the genomes of each line have been sequenced, making identification of genetic variations among them much easier. Among these lines we identified at least nine distinct phenotypes (major differences from lab fly strains). For example, while in the normal lab strains the ratio of Rh5- to Rh6- expressing cells is approximately 30%:70%, in these fly lines it ranges from 0% to 80% of Rh5-expressing cells. In some lines, mechanisms responsible for mutually exclusive Rh5/Rh6 expression failed, resulting in cells with both Rhodopsin. Seven lines showed no Rh5 expression; instead, many photoreceptor cells contained neither Rhodopsin. We also observed completely novel phenotypes such as in one line where Rh5 is normal if flies are kept in light but disappears if the same flies are kept in the dark. Finally, a number of non-Rhodopsin phenotypes such as premature retinal degeneration were also found.
We next set out to identify some of the genes which are affected in these lines. 1) In collaboration with Robert Johnston’s group (Johns Hopkins University) we found that increase in proportion of Rh5-cells was often associated with alterations of the spineless gene and we specifically identified one such alteration that is present relatively broadly in wild fly populations. This alteration affects the way spineless gene is regulated and led Johnston group to identify the regulator protein, transcription factor klumpfus, whose binding to the spineless gene sequence is affected by the alteration. 2) On the other end of the spectrum, we found that the fly line with all R8 photoreceptors expressing Rh6 has a short deletion in the melted gene. This deletion removes a part of the sequence that is essential for the activation of this gene. And, melted needs to be active to produce Rh5-expressing photoreceptors. In collaboration with Jens Rister’s group (University of Massachusetts Boston) we are pursuing this genetic variant in order to extend our understanding of melted regulation during photoreceptor differentiation. 3) In the seven lines with no detectable Rh5, but with many “empty” photoreceptor cells, we identified two different mutations which affect Rh5 gene itself, such that it does not encode a full length Rh5 protein. We tested to what extent the two mutations affect Rh5 function, using drosophila larvae who need Rh5 to have a normal light-avoiding behaviour, and found that one mutation completely destroys Rh5 function, while the other one still allows larvae to avoid light. For a number of other phenotypes we are in process of identifying the affected genes: for some of them, we determined which chromosome carry the genetic changes, and for one we have narrowed down the possibilities to 10 genes.
While the above inbred-line approach has proved to be very efficient at identifying natural genetic variants, one can criticize it because of the possibility that at least some of the phenotypes would be due to inbreeding and would be encountered rarely in the wild. Therefore, we wanted to know whether we could apply the techniques we developed with inbred lines to identify natural variants in the flies that we catch ourselves, without generation of inbred lines. The aproach is to capture a number of flies from different locations in France (and later in Europe), breed them individually and when the progeny is secured, to stain them to reveal Rh5/Rh6 expression. Then, for the flies which show interesting phenotypes try to recover the same phenotypes in their progeny (showing inheritance of the phenotype) and identify first the affected gene and then the actual genetic change that causes the phenotype. We are well on the way developing this programme and have to date obtained two fly lines with interesting phenotypes. For one of them we already identified sevenless as the gene that carries the causative mutation.
In human species, there is a surprising level of natural variation affecting the visual system. The ratios of different colour photoreceptors differ vastly among the population. Colorblindness caused by mutations in the opsin genes are relatively common, affecting ~10% of male population. And, vast number of rare mutations affecting a long list of genes cause devastating retinal degeneration pathologies. Our work so far suggests that similar situation exists in the Drosophila natural populations: the ratios of photoreceptor types vary extensively, mutations in Rhodopsin genes occur with moderate frequency, and many of the retina developmental genes can carry rare strong-impact mutations. The questions we are left with are: 1) to understand deeper the structure of the natural variation affecting the visual system in Drosophila, 2) to ask whether there is something special about the natural variants affecting the eye development and function or if other aspects of the nervous system can be similarly affected and 3) what we can learn about retina development and function from the natural variants that we identify? The scientific program and the approaches that we have developed with the COLOURHODOPSIN project have set the solid foundation to address these questions and have already led not only to new insights in the eye development but also to an already mature collaboration to investigate the natural variation affecting the circadian rhythm entrainment in wild flies.