BrdU Labeling Pattern and Proliferation Kinetics
Our goal was to use incorporation of an S-phase marker, BrdU, to identify cells of the embryonic rod lineage as distinct from other retinal cell types. In general, rod production is delayed as compared to cones (Blaxter and Staines,1970; Carter-Dawson and LaVail,1979; Young,1985; Knight and Raymond,1990), although in the zebrafish, the first rodopsin+ rods are visible by 48 hr post-fertilization (hpf) (Raymond et al.,1995), and the wave of cone production is not complete until approximately 54 hpf (Hu and Easter,1999; Ochocinska and Hitchcock,2007), predicting some overlap in embryonic rod and cone neurogenesis. Therefore, we tested several cell labeling schemes to determine if selective labeling of the rod lineage was possible. Embryos were injected with 0.5 or 1.5 nL of 10 mM BrdU at 55 or 60 hpf, and were fixed at 72, 75, 77, 80, or 101 hpf for BrdU indirect immunofluorescence. Sections through the eye typically revealed BrdU labeling in regions corresponding to the newly-formed cgz, as well as scattered cells in the onl and in the inl (Fig. 1A). In some cases, radial arrays of BrdU+ cells spanning the inl and onl were revealed (Fig. 1A). We counted the average numbers of BrdU+ cells in each retinal layer, excluding the cgz (as identified as the terminus of the inner plexiform layer at each margin), to establish kinetic parameters of the various labeling schemes. Higher doses of BrdU resulted in more extensive labeling (Fig. 2A), and this labeling was detectable up to 24 hr after the injection, suggesting cumulative availability of BrdU from the injection site in the yolk. In addition, the high doses resulted in significantly higher (P < 0.01) numbers of BrdU+ cells in the onl at later survival times (Fig. 2A), consistent with movement of rod lineage cells from the inl to the onl (Julian et al.,1998). This shift in labeling was not seen following injection of low doses, suggesting possible dilution of label over the time of the experiment (Fig. 2A) and therefore a pulse-label rather than a cumulative label.
Figure 1. BrdU incorporation within the rod lineage in embryonic zebrafish. Embryos were injected with BrdU at 60 hpf and fixed at 75 hpf. A: Retinal cryosection showing distribution of BrdU in the circumferential germinal zone (cgz) and in radial arrays of cells (arrowhead) spanning the inner (inl) and outer nuclear layers (onl) in central retina; le, lens; rpe, retinal pigmented epithelium; gc, ganglion cell layer; v, ventral; d, dorsal. B,C: BrdU and rod opsin; Nomarski optics (B) merged with epifluorescent image (C); arrow indicates colabeled cell. D: BrdU and gnat2 colabeling showing BrdU-labeled cones in retinal periphery (arrow) and cells in the central onl labeled only for BrdU (arrowheads). Regions to the periphery of the thin white lines (located approximately 6 onl cell diameters from the unlaminated cgz) were excluded from subsequent analyses related to the rod lineage. E–G: BrdU and anti-GS; arrow indicates colabeled cell. H–J: BrdU and islet1; no colabeling was observed (arrowhead indicates singly-labeled cell). K–M: Section doubly-labeled for BrdU and PKC; no colabeling was observed (arrowhead indicates singly-labeled cell). Scale bar in A (applies to A, D) = 40 μm, in B (applies to B,C,E-M) = 10 μm.
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Figure 2. BrdU labeling kinetics, proliferation kinetics, and rod and cone colabeling in zebrafish embryos. A: Embryos were injected with BrdU and fixed at the indicated times, with low or high doses of BrdU, and sections stained for BrdU indirect immunofluorescence were analyzed for numbers of BrdU+ cells in the inner (inl) and outer nuclear layers (onl). *Significant difference (ANOVA + Fisher's post-hoc; P < 0.01). B: Sections were stained for PH3 indirect immunofluorescence, and numbers of PH3+ cells in the inl and onl were counted. *Significant difference (ANOVA + Fisher's post-hoc; P < 0.01). C: Embryos were injected with BrdU at the indicated times, with low or high doses of BrdU, and sections stained for BrdU in combination with in situ hybridization for rod opsin were analyzed for proportion of rod opsin+ cells colabeled with BrdU. *Significant difference (Kolmogorov-Smirnov; P < 0.01). D: Embryos were injected with BrdU and fixed at the indicated times, with low or high doses of BrdU, and sections were stained for BrdU and zpr-1 indirect immunofluorescence, or for BrdU in combination with in situ hybridization for gnat2, then analyzed for numbers of colabeled cones in dorsal and ventral retina.
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The proliferation kinetics observed by BrdU incorporation over a 60- to 75-hpf time-course were verified by indirect immunofluorescence detection of phosphorylated histone 3 (PH3), a marker of M-phase. At 60, 72, and 75 hpf, PH3+ cells were rarely found in the inl and this frequency did not change significantly over time, suggesting a very low rate of proliferation in this layer (Fig. 2B). In embryos injected with BrdU at 60 hpf and fixed at 75 hpf (low or high doses), no BrdU+/PH3+ were observed in the inl (data not shown; 49 sections derived from 18 embryos). Mitotic cells were more common in the onl, with a significantly larger number found in embryos fixed at 72 hpf than at 60 or 75 hpf (Fig. 2B; P < 0.01). In BrdU-injected embryos, all of the PH3+ cells of the onl were also BrdU+ (data not shown). These data demonstrate that BrdU incorporation faithfully reports the spatiotemporal features of proliferative activity in the embryonic zebrafish retina.
Cell-Specificity of BrdU Labeling
We next confirmed that our labeling schemes could identify BrdU+, rod opsin+ cells. Sections from several of the different labeling schemes were processed for rod opsin in situ hybridization followed by BrdU indirect immunofluorescence; in nearly all cases, we could identify colabeled cells in the onl (Fig. 1B,C). Some BrdU+ cells in the onl were rod opsin-negative, consistent with an identity as either rod precursors or newly-generated cones (see below), and some rod opsin-positive cells in the onl were BrdU-negative, consistent with a birth date prior to the BrdU injection time (Fig. 2C). In embryos injected at 55 hpf, and in one embryo injected at 60 hpf, there were clusters of doubly-labeled cells near the optic nerve head (image not shown), a region of high rod abundance in embryonic retina (Raymond et al.,1995), and therefore a large proportion of rod opsin+ cells were also BrdU+ (Fig. 2C) in these sections. In the remaining embryos injected at 60 hpf, low doses of BrdU labeled fewer rods than the high doses of BrdU (Fig. 2C), consistent with our conclusions from the analysis of proliferation kinetics (Fig. 2A). Parallel experiments performed with a cRNA probe for rod transducin alpha subunit (gnat1) gave similar results (data not shown).
To determine to what extent our various labeling schemes identified newly-generated cones rather than rods, sections were processed for indirect immunofluorescence using the cone marker zpr-1 (Larison and Bremiller,1990), or for in situ hybridization with a probe for cone transducin alpha subunit (gnat2), in combination with BrdU indirect immunofluorescence. Statistically indistinguishable numbers of BrdU+ cells were labeled by either zpr-1 or gnat2, suggesting that the blue and UV cone populations represent a minor fraction of total cones being generated at this developmental time (see Raymond et al.,1995). The BrdU+ cones were restricted to regions near the cgz (Fig. 1D). High numbers of BrdU+ cones were found in embryos injected at 55 hpf (Fig. 2D), accounting for a major fraction of the BrdU+ cells in the onl (Fig 2A,D), and these colabeled cones were not restricted to the region near the cgz. Therefore, although the use of BrdU incorporation at 55 hpf resulted in higher rates of rod labeling (Fig. 2C), it also resulted in low confidence that BrdU+ cells in the onl were of the rod lineage. The proportion of BrdU+ cones (zpr-1+ or gnat2+) was substantially lower following BrdU injection at 60 hpf, using either the “pulse” (low dose) or “cumulative” (high dose) labeling scheme (Fig. 2D). For example, cumulative labeling beginning at 60 hpf resulted in a range of two to four colabeled cells near each margin (Fig. 2D), accounting for approximately half of the BrdU+ cells in the onl. Therefore, by injecting BrdU at 60 hpf rather than 55 hpf, and by eliminating from further analysis the retinal region six cell diameters from the border of the cgz (as shown in Fig. 1E, following cumulative labeling), or within three cell diameters in the case of pulse labeling, we could be confident that the BrdU+ cells in the onl were indeed related to the production of rods and not cones. Accordingly, all subsequent analyses, unless otherwise indicated, are restricted to these parameters.
There is evidence that the pax6+ stem cells at the apex of the rod lineage in post-embryonic teleosts (Hitchcock et al.,1996) are Müller glia (Bernardos et al.,2007). Therefore, we performed dual immunolabeling experiments using the Müller glial marker anti-glutamine synthetase (GS; Linser and Moscona,1979) in combination with BrdU in embryonic zebrafish. We identified multiple cases of colabeling in embryos injected at 55 hpf as well as at 60 hpf; these cells were located within the proximal (vitreal) half of the inl (Fig. 1E–G). In embryos injected at 55 hpf, over half (56%) of the BrdU+ cells in this location were GS+, but this ratio declined in embryos injected at 60 hpf (3–12%), even when the cumulative labeling scheme was utilized. Therefore, the embryonic Müller cell population may slow or cease proliferation over the 55–75 hpf time-course. In support of the latter interpretation, we have never observed the presence of the M-phase marker PH3 in GS+ Müller glia in embryos fixed at 60, 72, or 75 hpf (data not shown; 36 sections derived from 8–12 embryos at each time point). The use of other Müller glia markers, anti-GFAP and zrf1, gave the same result (data not shown). These findings are consistent with a lineage relationship between Müller glia and rod photoreceptors at the developmental time when production of both cell classes commences; however, it is unclear whether this embryonic glial population participates in the production of new rods.
To establish that the BrdU+ cells in the inl were exclusively related to the rod lineage (and/or to Müller glia), but did not label other, late-born neurons, we performed additional colabeling experiments with the retinal neuronal markers anti-protein kinase C (PKC), which labels rod bipolar neurons (Koulen et al.,1997), and anti-islet1, which labels ganglion cells and amacrine cells (Masland and Tauchi,1986), using embryos injected at both 55 hpf and at 60 hpf. In none of our labeling schemes were we able to locate doubly-labeled cells (Fig. 1H–M; 2 embryos and 14 sections for anti-islet 1; 3 embryos and 17 sections for anti-PKC), allowing us to be confident that, following any of our labeling schemes, the BrdU+ cells of the inl corresponded to cells of the rod/Müller glial lineage(s) rather than to postmitotic retinal neurons.
Transcription Factor Expression in the Rod Lineage
Several transcription factors have already been associated with the rod lineage of teleost fish, most notably pax6, NeuroD/nrd, Nr2e3, and crx, primarily in postembryonic retinas. We wished to determine the relative expression domains of these transcription factors in the rod lineage of embryonic retina, and to establish whether the rx1 and nrl genes were also in this lineage.
Pax6 is expressed by retinal progenitors, ganglion cells, and amacrine cells, and in adult goldfish and juvenile zebrafish, pax6 has been established as a putative marker for the apex of the rod lineage (Hitchcock et al.,1996; Otteson et al.,2001). To determine whether this is also the case in the embryonic zebrafish, we performed pax6 in situ hybridizations followed by BrdU indirect immunofluorescence on sections derived from BrdU-injected embryos. We regularly (1 cell per section on average; 12 sections) observed colabeled cells in the inl (Fig. 3A,B). These cells were typically located just within the proximal half of the inl, near the outer limit of the pax6 expression domain. These findings suggest that pax6 is expressed in a small population of cells of the rod lineage in the embryonic zebrafish.
Figure 3. Gene expression in cells of the BrdU+ rod lineage. All embryos were injected with BrdU at 60 hpf and fixed at 75 or 80 hpf. A,B: BrdU and pax6; Nomarski optics (A) merged with epifluorescence image (B); arrow indicates colabeled cell. C,D: BrdU and NeuroD; Nomarski optics (C), merged with epifluorescence image (D); two colabeled cells are indicated by arrows. E,F: BrdU and crx; Nomarski optics (E), merged with epifluorescence image (F); arrow indicates colabeled cell. G–J: BrdU and Nr2e3; Nomarski optics (G,I), merged with epifluorescence images (H,J); colabeled cells are indicated by arrows. K: Retinal expression pattern of nrl; both rod (arrow) and cone progenitors (arrowhead) are labeled, as well as cells of the lens (*). L,M: BrdU and nrl; Nomarski optics (L), merged with epifluorescence image (M); arrow indicates colabeled cell. N,O: BrdU and rx1; Nomarski optics (N), merged with epifluorescence image (O); colabeled cells are indicated by arrows. rpe, retinal pigmented epithelium; onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer; le, lens. Scale bar in A (applies to all except K) = 10 μm, in K = 40 μm.
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NeuroD is expressed by cells of the rod lineage in the inl and onl, and by newly-generated cone photoreceptors (Hitchcock et al.,2004; Ochocinska and Hitchcock,2007). However, the BrdU labeling scheme used (exposure at 55 hpf) did not exclusively detect rods as the BrdU+ cells of the onl (as noted by the authors; and see Fig. 2), and so we performed colabeling experiments using embryos injected at 60 hpf. In these experiments, we frequently (2–3 cells per section on average; 32 sections) observed colabeled cells in the inl (Fig. 3C, D), consistent with Ochocinska and Hitchcock (2007). The location of these cells was variable. A large number of NeuroD+ cells in the inl were not BrdU+, and likely correspond to amacrine cells (Ochocinska and Hitchcock,2007). We also observed colabeled cells in the onl, in regions that we had confirmed did not contain BrdU+ cones (Fig. 3C, D). These findings extend the results of Ochocinska and Hitchcock, indicating that NeuroD is expressed by cells of the rod lineage in the onl as well as in the inl. All cells of the onl that were BrdU+ were also NeuroD+. Because our labeling scheme provided sufficient BrdU to label the entire rod lineage, up through rod opsin expression, this NeuroD labeling pattern indicates that rod precursors and maturing rods are NeuroD+.
The cone-rod homeobox (crx) gene is expressed by differentiating cones in the zebrafish (Liu et al.,2001) embryo, and is co-expressed with NeuroD in this cell population (Ochocinska and Hitchcock,2007). The crx gene is also expressed in the rod lineage of juvenile zebrafish (Bernardos et al.,2007). We wished to determine the expression pattern of crx in the embryonic rod lineage. In embryos injected with BrdU at 55 or 60 hpf, we frequently observed crx+/BrdU+ cells in the onl and in the middle region of the inl (Fig. 3E,F). In fact, all of the BrdU+ cells of the onl were also crx+ and most of the BrdU+ cells of the inl were also crx+ (3–4 cells per section on average; 9 sections). This raises the possibility that crx and NeuroD cooperate during the migration and maturation of cells of the rod lineage. We note that Ochocinska and Hitchcock (2007) were unable to colocalize NeuroD with crx in the inl. This issue is addressed in the Relationship of rx1 to Other Transcription Factors of the Rod Lineage section, in the Results section. A large number of crx+ cells in the inl were not BrdU+, however, indicating, that, like pax6 and NeuroD, crx is not an exclusive marker of the rod lineage.
The expression of crx mRNA was rather diffuse, and so we confirmed these findings through antibody staining using a crx-specific antibody in combination with anti-BrdU. Colabeled cells were found in both the inl and onl (Fig. 4A–C). Expression of crx/crx was extensive within the inl (Shen and Raymond,2004), raising the possibility that many of the crx+ cells were postmitotic neurons. We verified that at least some crx+ cells are actively proliferative through colabeling studies using the crx antibody in combination with a monoclonal anti-phosphoprotein antibody, MPM-2. In embryos examined at 72 hpf, we found examples of crx+/MPM-2+ cells in the inl (Fig. 4D–F), and in the onl (not shown). This is an important finding as is the first demonstration (in undamaged retina) that mitotic cells, likely part of the rod photoreceptor lineage, can express a “photoreceptor-specific” transcription factor.
Figure 4. Crx protein expression in mitotic cells of the BrdU+ rod lineage. A–C: Embryo was injected with BrdU at 60 hpf and fixed at 75 hpf, and processed for indirect immunofluorescence with anti-crx (A) and anti-BrdU (B). C shows merged image and colabeled cells (arrows). D–F: Embryo fixed at 72 hpf and processed for indirect immunofluorescence with anti-crx (D) and MPM-2 (mitotic cells; E). F shows merged image and colabeled cells (arrow). Scale bar in A (applies to all) = 10 μm.
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Photoreceptor cell-specific nuclear receptor (pnr), also known as Nr2e3, is an orphan nuclear receptor known to activate rod-specific genes and repress cone-specific genes in mouse (Chen et al.,2005). In the zebrafish, Nr2e3 is expressed throughout the onl (Kitambi and Hauptmann,2007), but then becomes restricted to rod photoreceptors (Chen et al.,2005), and is upregulated in a zebrafish model for rod degeneration and replacement (Morris et al.,2008). Our preliminary experiments revealed Nr2e3 expression in scattered cells in the inl (similar to that seen by Morris et al. (2008) in a zebrafish model for rod pathology and regeneration), and so we pursued colabeling experiments with BrdU. In embryos injected with BrdU at 60 hpf, we regularly observed Nr2e3+/BrdU+ cells in the onl (Fig. 3G,H), consistent with the identity of these cells as new rods, but also occasionally observed Nr2e3+/BrdU+ cells in the outermost or innermost regions of the inl (Fig. 3I,J). However, some Nr2e3+ cells in the inl were not colabeled, indicating that Nr2e3 expression in the inl may not be exclusive to the rod lineage in wild-type embryonic retina.
The neural retina leucine zipper (nrl) gene is required for rod photoreceptor differentiation in mammals (Mears et al.,2001; Daniele et al.,2005; Akimoto et al.,2006); the Nrl protein interacts synergistically with Crx at the rod opsin promoter (Mitton et al.,2000; Liu et al.,2001). A putative, and phylogenetically distant, ortholog of nrl has been identified in zebrafish, and this gene is expressed in the developing lens and the adult onl (Coolen et al.,2005). However, a screen for expressed sequence tags from adult zebrafish retina failed to find this nrl ortholog (Vihtelic et al.,2005), raising questions as to the true expression pattern and function of zebrafish nrl. In embryonic and early larval (80 hpf) retina, this nrl gene was expressed in cells of the lens, and also in cells of the onl in a pattern reminiscent of that of Nr2e3 (Fig. 3K, and see Chen et al.,2005). We observed weak expression of nrl in a scattered population of cells of the onl, consistent with their identity as rods or possibly rod precursors (Fig. 3K). However, nrl was also strongly expressed in the cells of the onl adjacent to the cgz, having a likely identity of cone progenitors (Fig. 3K). In BrdU-injected embryos, we regularly observed co-expression of nrl in some of the BrdU+ cells of the onl sufficiently distant from the cgz to be identified as rods or rod precursors (Fig. 3L,M).
Expression of the retinal homeobox (rx) genes has been characterized in the zebrafish embryo (Chuang et al.,1999), but this characterization did not include a complete evaluation of rod versus cone expression, nor of expression in the rod lineage. We therefore processed BrdU-injected embryos for in situ hybridization using cRNAs corresponding to the zebrafish rx1 gene, together with BrdU immunofluorescence. All BrdU+ cells of the onl were also rx1+ (Fig. 3N,O), suggesting that rx1 is expressed by both rods and rod precursors. We also regularly observed rx1+/BrdU+ cells in variable locations within the inl (Fig. 3P,Q). However, not all BrdU+ cells were rx1-positive (Fig. 3N,O), and not all rx1-positive cells were BrdU+, even in the cumulative labeling experiments. This indicates that rx1, like NeuroD, crx, and Nr2e3, is not an exclusive marker of cells of the rod lineage. Weaker retinal expression of the rx2 and rx3 genes prevented us from performing interpretable dual labeling experiments; the color reaction from the in situ hybridization steps did not survive the BrdU labeling procedure. However, the expression patterns of rx1 and rx2 are virtually identical (Chuang et al.,1999, and data not shown), predicting a similar association with the rod lineage.
For each transcription factor evaluated, we counted the proportion of BrdU+ cells that were colabeled following pulse-labeling (60–75 hpf), within defined compartments of the inl (Fig. 5A), in order to establish a putative genetic hierarchy within the rod lineage. The proximal inl (p-inl) is defined as the region spanning the inner limit of the crx expression domain and the boundary of the inner plexiform layer (ipl), and is approximately three cell diameters thick. The distal inl (d-inl) is defined as a layer that is approximately two cell diameters thick and its outer border is the outer plexiform layer (opl). Finally, the medial inl (m-inl) lies between the inner border of the d-inl and the inner boundary of the crx expression domain and is approximately four cell diameters thick. Within the p-inl, BrdU coexpression with pax6 was more consistently observed than that with any of the other transcription factors tested (Fig. 5B). Coexpression with NeuroD was seen less frequently, though still more frequently than that with rx1 or Nr2e3; in these latter cases colabeling was seen only rarely. Within the m-inl, BrdU+ cells were always crx+, and approximately half of them were NeuroD+, as was the case in the p-inl. The proportion of BrdU+ cells that were also rx1+ remained low, although it was higher than the corresponding proportion in the p-inl. Within the d-inl, crx coexpression with BrdU remained at 100%, NeuroD coexpression at 50%, rx1 coexpression remained low but increased compared to that in the m-inl, and Nr2e3 coexpression was again rarely observed (Fig. 5B). Within the onl, all BrdU+ cells were also NeuroD+, rx1+, and crx+, and nearly all were also Nr2e3+. This quantitative evaluation reveals heterogeneity of transcription factor expression within the rod lineage in the inl, followed by remarkable homogeneity once the progenitors reach the onl. Only a fraction (39%) of the BrdU+ cells of the onl also expressed rod opsin (Fig. 5B), the remainder correspond to an identity as rod precursors or immature rods. An even smaller fraction (22%) of the BrdU+ cells of the onl expressed nrl (Fig. 5B), suggesting that this transcription factor is expressed only in a subpopulation of developing rods, or is expressed transiently.
Figure 5. Colabeling quantification by onl and inl compartments. A: Retinal cryosection from 72-hpf embryo showing subdivision of the inner nuclear layer (inl) into proximal (p-inl), medial (m-inl), and distal (d-inl) compartments; further explanation is provided in the Results section. B: BrdU colabeling with rod opsin, nrl, Nr2e3, crx, rx1, NeuroD, and pax6, by retinal lamina and defined inl compartments. C:Rx1 colabeling with rod opsin, crx, NeuroD, and pax6, by retinal lamina and defined compartments of the inl. rpe, retinal pigmented epithelium; onl, outer nuclear layer; gcl, ganglion cell layer.
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Relationship of rx1 to Other Transcription Factors of the Rod Lineage
We wished to establish the in vivo relationship of expression of the rx1 gene to that of other rod lineage markers, in order to better understand the genetic hierarchy of the rod lineage. BrdU+/rx1+ cells were occasionally observed in the proximal region of the inl, and so we tested whether these cells may correspond to the pax6+ stem cells of the rod lineage, by performing rx1/pax6 colabeling experiments. Using two alternative sets of visualization reagents, on a total of 126 sections from 37 embryos, we could not see convincing evidence of colabeling (Fig. 7A–D). However, we observed several instances of rx1 labeling in cells immediately adjacent to the region of pax6 expression. Rx1 and pax6 clearly segregated into discrete expression domains within the inl. Therefore, rx1 is not retained in the pax6+/Müller glia that serve as stem cells for the production of rods (Bernardos et al.,2007).
Figure 7. Rx1 coexpression with rod lineage markers. A–C:rx1 (A) and pax6 (B) double in situ; merged image (C) showing no colabeled cells (arrowhead). D:rx1 (purple) and pax6 (pink) using alternative reaction products; arrowhead = rx1+ cell only. E–G:rx1 (E) and NeuroD (F); merged image (G) showing colabeling in the inl (arrow) and throughout the onl; arrowhead = rx1+ cell only. H:rx1 (purple) and NeuroD (pink) using alternative reaction products; doubly-labeled cell (arrow) and rx1+ cell only (arrowhead). I–K:rx1 (I) and crx (J); merged image (K) showing a colabeled cell in the inl (arrow) and throughout the onl; arrowhead = rx1+ cell only. L:rx1 (purple) and crx (pink) using alternative reaction products, showing a doubly-labeled cell (arrow) and an rx1+ cell only (arrowhead). M–O:NeuroD (M) and crx (N); merged image (O) showing a colabeled cell in the inl (arrow) and throughout the onl; arrowhead = NeuroD+ cell only. P:NeuroD (pink) and crx (purple) using alternative reaction products, showing doubly-labeled cell (arrow). onl, outer nuclear layer; inl, inner nuclear layer; scale bar in A (applies to A–C,E–G,I–K,M–O) = 20 μm; in D (applies to E,H,L,P) = 20 μm. *, areas enlarged in insets.
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Many of the rx1+/BrdU+ cells in the inl were observed in locations corresponding to locations of both NeuroD+/BrdU+ cells, and crx+/BrdU+ cells. In the interests of determining whether these three genes may cooperate in regulating the maturation of cells in the rod lineage, we performed rx1/NeuroD, rx1/crx, and crx/NeuroD colabeling experiments. For each combination, we used two alternative sets of visualization reagents, on an average of 60 sections from 16 embryos. In each case, colocalization of gene expression was observed throughout the onl (Fig. 7E–P), indicating that cones and rods, as well as rod precursors, co-express rx1, crx, and NeuroD (see also Bernardos et al.,2007; Ochocinska and Hitchcock,2007). In addition, colocalization of rx1 and NeuroD expression was seen regularly in cells residing in the inl (Fig. 7E–H; in 84 sections of 23 embryos). Expression of rx1 together with crx was also observed in a population of cells residing within the crx expression domain (Fig. 7I–L; in 72 sections of 19 embryos). These experiments, along with those presented earlier (Fig. 3C–F) predicted that NeuroD and crx may be co-expressed by cells in the middle and distal inl. Our colabeling experiments, using two alternative sets of visualization reagents, confirmed that this is the case (Fig. 7M–P). Colabeling experiments using Nr2e3, nrl, rx2, and rx3 in combination with rx1, were challenging and/or difficult to interpret because of weaker hybridization signals.
We then counted the proportion of rx1+ cells that were colabeled with pax6, NeuroD, or crx, to understand potential combinatorial relationships of these transcription factors, using the compartments of the inl defined previously (Fig. 5A). Within the p-inl, rx1 colabeled only with NeuroD, and in only 25% of the cases (Fig. 5B). Within the m-inl, rx1+ cells were also NeuroD+ in 50% of the cases observed, but were always also crx+ (Fig. 5B). Interestingly, the rx1+ cells of the d-inl were less likely to be colabeled with either of these transcription factors, but the rx1+ cells of the onl coexpressed NeuroD and crx in all cases. These results corroborate the different temporal patterns of expression within the inl of the developing retina, and suggest that a complete combinatorial relationship among the three retinal transcription factors is associated only with cells of the embryonic onl. Only 19% of the rx1+ cells of the onl were rod opsin+ (Fig. 5B), the remainder being cones.