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Biol Open
2012 Jan 15;11:30-6. doi: 10.1242/bio.2011016.
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Generation of a genetically encoded marker of rod photoreceptor outer segment growth and renewal.
Willoughby JJ
,
Jensen AM
.
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Vertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability.
Fig. 1.
Methods to measure rates of rod outer segment growth.
(A) The original method to measure outer segment growth used injection of radioactive amino acids, which are incorporated into newly synthesized proteins. The displacement of predominantly H3-labelled Rhodopsin was measured over time. (B) A new method to measure outer segment growth using heat-shock induction to transiently express a red fluorescent protein that is incorporated into newly synthesized outer segment discs. The displacement of a stripe of red fluorescent protein can be followed over time. (C) A diagram of the construct used to generate a stable transgenic line to express heat-shock inducible red fluorescent protein. The hsp70 promoter was placed upstream of an expression construct where a signal peptide (SP) is fused to the hemagglutinin (HA) peptide tag followed by a transmembrane domain (TM) and mCherry fluorescent protein.
Fig. 2.
Expression of HA-mCherryTM in the eye of a 69.5 hpf larva at 1.5 hours hpHS (heat shocked at 68 hpf).
(A, C, E, G) Single confocal z-section images of an eye labeled with anti-HA antibodies. (B, D, F, H) Single confocal z-section image of an eye labeled with anti-HA antibodies merged with DIC-like image. (A, B) Heat-shocked Tg(hsp70:HA-mCherryTM) larva shows ubiquitous expression of HA-mCherryTM in retinal cells. (C, D) Heat-shocked wild-type eye shows only weak autofluorescence. (E, F) In the absence of heat-shock, an eye of a Tg(hsp70:HA-mCherryTM) larva shows only weak expression of HA-mCherryTM in a small number of amacrine cells (arrows). (G, H) An eye of a wild-type larva shows weak autofluorescence in the absence of heat-shock. Scale bar: A, 50 .
Fig. 3. Expression of HA-mCherryTM in photoreceptors after heat-shock at 3 dpf.
(A) At 5 hpHS, a single confocal z-section of a Tg(hsp70:HA-mCherryTM) photoreceptor layer labeled with anti-HA antibody (red), anti-Rhodopsin antibody (blue) and GFP-expressing rods (green) shows membrane expression of HA-mCherryTM in rods and neighboring cones, and a stripe of HA-mCherryTM at the base of rod outer segments (arrows, A, B). (D) At 1 dpHS, a single confocal z-section of a Tg(hsp70:HA-mCherryTM) photoreceptor layer labeled with anti-HA antibody (red), anti-Rhodopsin antibody (blue) and GFP-expressing rods (green) shows that membranous HA-mCherryTM labeling in the cell body and inner segment has largely disappeared and a stripe of HA-mCherryTM in a rod outer segment has been displaced distally (double arrows, D). Continuous HA-mCherryTM labeling largely fills cone outer segments (arrowheads, D). Scale bar: A, 5 .
Fig. 4. Expression of HA-mCherryTM in photoreceptors after heat-shock at 5 dpf.
Confocal z-projections of Tg(hsp70:HA-mCherryTM) photoreceptor layers labeled with anti-HA antibody (red), anti-Rhodopsin antibody (blue) and with GFP-expressing rods (green). (A) At 6 dpf, 1 dpHS, HA-mCherryTM in rod outer segments is seen as a stripe, an oval or a circle, depending on the orientation of the outer segment to the plane of section. (C) A magnification of the boxed area in (A) shows the displacement of the HA-mCherryTM stripe from the base of the outer segment in 2-dimensions. (B) At 8 dpf, 3 dpHS, HA-mCherryTM in rod outer segments is seen as a stripe, an oval or a circle, depending on the orientation of the outer segment to the plane of section and HA-mCherryTM has moved distally compared to that seen at 1 dpHS. (D) A magnification of the boxed area in (B) shows the displacement of the HA-mCherryTM stripe from the base of the outer segment in 2-dimensions. Scale bars: 5 , A, B; 5 , C, D.
Fig 5. Expression of HA-mCherryTM in photoreceptors after heat-shock in adult fish.
Confocal z-projections of adult Tg(hsp70:HA-mCherryTM) photoreceptor layers labeled with anti-HA antibody (red), anti-Rhodopsin antibody (blue) and with GFP-expressing rods (green) at 1 dpHS (A), 4 dpHS (B), 7 dpHS (C), 9 dpHS (D), 15 pHS (E), 16 dpHS (F). Over the time-course, HA-mCherryTM moves distally toward the RPE, and has largely disappeared from rod outer segments by 16 dpHS (F). Scale bar: 20 , A.
Fig 6. Methods using hsp70:HA-mCherryTM to identify molecular mechanisms of rod outer segment growth.
(A) The role of candidate gene x function in outer segment growth can be analyzed in mosaic animals where some photoreceptors express gene x driven by the Xenopus laevis rod opsin promoter (Xop). This method uses injection of a Xop:tag-gene x pTol DNA construct into one-cell Tg(Xop:EGFP); Tg(hsp70:HA-mCherryTM) embryos. Injected individuals are heat-shocked to induce HA-mCherryTM expression and since only a subset of rod photoreceptors will have incorporated the transgene Xop:tag-gene x, the distance (D) of HA-mCherryTM from the base of the outer segment in Xop:tag-gene x-expressing rods (DTG) can be compared to non-expressing rods (DWT). (B) A stable transgenic line can be generated, Tg(Xop:tag-gene x);Tg(Xop:EGFP); Tg(hsp70:HA-mCherryTM), where all rods express tag-gene x. Following heat-shock, the distance of HA-mCherryTM from the base of the outer segment in Tg(Xop:tag-gene x);Tg(Xop:EGFP); Tg(hsp70:HA-mCherryTM) rods (DTG) can be compared to Tg(Xop:EGFP); Tg(hsp70:HA-mCherryTM) rods (DWT).
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