Many of the molecular mechanisms underlying photoreceptor signal transduction, synaptic transmission, and ciliogenesis have been identified, but yet very little is known about how photoreceptors maintain their outer segments through the combined processes of outer segment growth and shedding and what molecular mechanisms underlie those processes. The renewal of rod photoreceptor outer segments was described over forty years ago by following the fate of radioactively labeled proteins (largely Rhodopsin) in the outer segment over time in several different vertebrate species (Young, 1967
; Young and Bok, 1969
; Bok and Young, 1972
; LaVail, 1973
). The role of light in the process of renewal was studied and the rate of outer segment growth in Xenopus laevis
rods was found to be the same in those maintained in constant light and those maintained to cyclic light, although growth was reduced significantly in those maintained in constant dark (Hollyfield and Rayborn, 1979
). While outer segment growth was greatly reduced under constant dark conditions, this reduction could be due to a general reduction in protein synthesis, including reduced Rhodopsin levels (Hollyfield and Anderson, 1982
). Light exposure, however, may play a role in outer segment growth, as experiments examining Xenopus laevis
rods indicated that exposure to light accelerates outer segment growth during the first 8 hours of a 12
12 hour light-dark cycle and growth was undetectable during the last 8 hours of darkness (Besharse et al., 1977a
, Besharse et al., 1977b
). Light also plays a vital role in the shedding process; rods initiate outer segment shedding at the onset of light (Basinger et al., 1976
In many mouse models of human retinal degeneration diseases where it has been examined, photoreceptor outer segments progressively shorten before photoreceptors die (Heckenlively et al., 1995
; Chen et al., 1999
; Hawes et al., 2000
; Hong et al., 2000
; Gao et al., 2002
; Collin et al., 2005
; Pang et al., 2005
; Vasireddy et al., 2006
). This observation raises the interesting question of whether there is a causal link between outer segment size and photoreceptor viability. We consider two possibilities. One, disease-associated mutations physically compromise photoreceptors and as they sicken they no longer are able to sustain the enormous metabolic load required to maintain their outer segments. Thus, as the cell sickens, the outer segment shortens secondarily and concomitantly, and finally the cell is so sick it undergoes apoptosis. Two, the cell undergoes apoptosis because its primary functional organ, the outer segment, is no longer functional. Thus, the loss of outer segment function induces photoreceptor apoptosis. If the latter possibility is true, then the question becomes could stimulating outer segment growth during photoreceptor degeneration disease prolong photoreceptor function and viability? In order to test this possibility, the underlying cellular and molecular mechanisms of outer segment growth must first be identified.
Given the fascinating and largely unaddressed question of how photoreceptors maintain their outer segments through the continuous processes of growth and shedding and the likely importance of these processes to retinal health, we developed a new method that allows us to rapidly measure growth rates of rod outer segments. Now that we easily measure rod outer segment growth, we can next ask the question what genes and molecular mechanisms contribute to outer segment growth. We can approach this question using two methods. One method to study gene function in outer segment growth uses mosaic analysis. This method uses the injection of a DNA construct where the Xenopus rod opsin promoter drives expression of a gain-of-function or loss-of-function candidate gene (Xop:gene X) in a subset of rods and then the rates of outer segment growth in these rods are compared to neighboring non-transgene-expressing rods (). The other method is to generate stable transgenic lines, Tg(Xop:gene X), that express gain-of-function or loss-of-function candidate genes driven by Xenopus rod opsin promoter. The rate of rod outer segment growth in the transgenic line Tg(Xop:gene X);Tg(Xop:EGFP);Tg(hsp70:HA-mCherryTM) is compared to the rate of growth in the Tg(Xop:EGFP);Tg(hsp70:HA-mCherryTM) line. The advantage to the mosaic analysis is that it is rapid. The advantage to the stable transgenic line analysis is that transgene copy number can be determined and growth rates in individual transgenic rods should be more consistent.
Methods using hsp70:HA-mCherryTM to identify molecular mechanisms of rod outer segment growth.
Given that we know virtually nothing about the cellular and molecular mechanisms of outer segment growth, what kind of genes and mechanisms might contribute to this process? We consider two different mechanisms that might control outer segment growth– cilia size control mechanisms and cell size control mechanisms. Although the ciliary axoneme appears to extend to the tip of cone outer segments, it does not in rods and as yet, there is no data supporting the renewal of the axoneme in rods (Roof et al., 1991
; Eckmiller, 1996
). There are, however, a number of molecular pathways identified that modulate ciliary and flagellar length that could be examined (see review by Ishikawa and Marshall, 2011
). Photoreceptor outer segments are unlike typical cilia: they are longer and have a much greater volume, which is filled largely with disc membrane. Using data from LaVail (1973)
, we roughly calculate that a mouse rod makes about 230 µm2
of membrane daily to replace that which was shed. The growth process clearly requires a great deal of membrane and protein synthesis and thus, pathways like the mTor pathway (for review see Zoncu et al. 2011
) that are involved in cell size control may be regulators of outer segment growth. In the case of photoreceptor, increased cell growth could be invested in the outer segment. The generation of the Tg(hsp70:HA-mCherryTM)
line should allow us to determine whether either of these two mechanisms– cilia size control or cell size control contribute to the growth of rod outer segments.