Nr2e1 regulates retinal lamination and the development of Müller glia, S-cones, and glycineric amacrine cells during retinogenesis
© Corso-Díaz and Simpson. 2015
Received: 1 March 2015
Accepted: 23 May 2015
Published: 20 June 2015
Nr2e1 is a nuclear receptor crucial for neural stem cell proliferation and maintenance. In the retina, lack of Nr2e1 results in premature neurogenesis, aberrant blood vessel formation and dystrophy. However, the specific role of Nr2e1 in the development of different retinal cell types and its cell-autonomous and non-cell autonomous function(s) during eye development are poorly understood.
Here, we studied the retinas of P7 and P21 Nr2e1 frc/frc mice and Nr2e1 +/+ ↔ Nr2e1 frc/frc chimeras. We hypothesized that Nr2e1 differentially regulates the development of various retinal cell types, and thus the cellular composition of Nr2e1 frc/frc retinas does not simply reflect an overrepresentation of cells born early and underrepresentation of cells born later as a consequence of premature neurogenesis. In agreement with our hypothesis, lack of Nr2e1 resulted in increased numbers of glycinergic amacrine cells with no apparent increase in other amacrine sub-types, normal numbers of Müller glia, the last cell-type to be generated, and increased numbers of Nr2e1 frc/frc S-cones in chimeras. Furthermore, Nr2e1 frc/frc Müller glia were mispositioned in the retina and misexpressed the ganglion cell-specific transcription factor Brn3a. Nr2e1 frc/frc retinas also displayed lamination defects including an ectopic neuropil forming an additional inner plexiform layer. In chimeric mice, retinal thickness was rescued by 34 % of wild-type cells and Nr2e1 frc/frc dystrophy-related phenotypes were no longer evident. However, the formation of an ectopic neuropil, misexpression of Brn3a in Müller glia, and abnormal cell numbers in the inner and outer nuclear layers at P7 were not rescued by wild-type cells.
Together, these results show that Nr2e1, in addition to having a role in preventing premature cell cycle exit, participates in several other developmental processes during retinogenesis including neurite organization in the inner retina and development of glycinergic amacrine cells, S-cones, and Müller glia. Nr2e1 also regulates various aspects of Müller glia differentiation cell-autonomously. However, Nr2e1 does not have a cell-autonomous role in preventing retinal dystrophy. Thus, Nr2e1 regulates processes involved in neurite development and terminal retinal cell differentiation.
KeywordsNr2e1 Amacrines S-cones Müller glia Brn3a Chimera Ectopic plexiform layer
During retinal development, six neuronal cell types and one type of glia are generated in a conserved histogenic yet overlapping order . Ganglion neurons are the first cells to be generated followed by amacrine, horizontal, cones, and rods during the embryonic period, and bipolar and Müller glia during the postnatal period . These cell types are organized into three nuclear layers and generate two plexiform layer neuropils where most of the synapses are confined. Retinal progenitor cell (RPC) proliferation and fate choice are regulated by intrinsic mechanisms [2–4] and extracellular signaling from differentiated cells [5–7].
During retinogenesis, the cell-cycle length increases over time in progenitor cells . In various vertebrates, including mice, premature cell cycle exit in the retina increases the number of cells that are born early and decreases the number of cells born towards the end of retinal histogenesis [9–12]. RPCs are heterogeneous in gene expression  and express transcription factors and cell cycle regulators that play dual roles in controlling RPC cell cycle and fate [12, 14, 15].
Nr2e1 is a conserved orphan nuclear receptor that regulates neural stem cell proliferation and maintenance . Nr2e1-null mice have smaller brains and retinas, are blind, and highly aggressive . Nr2e1 is mostly a repressor that operates in a cell-autonomous fashion, but it can also activate gene transcription [18, 19] and act non-cell-autonomously through the Wnt pathway to regulate neural stem cell proliferation and self-renewal . Lack of Nr2e1 results in premature cell cycle exit during corticogenesis and reduced thickness of superficial cortical layers due to a depletion of the neural stem cell pool . Lack of Nr2e1 in the retina results in precocious neurogenesis, impaired blood vessel development , and progressive dystrophy [21, 22]. This complex phenotype poses a challenge to understanding the role of Nr2e1 in specific retinal cell populations.
Chimeras provide valuable information regarding the autonomous and non-autonomous cellular consequences of gene mutations, the development of different cell-types and their interaction through cell-signaling, as well as the nature of tissue-tissue interactions in vivo . To better understand the role(s) of Nr2e1 in retinal development, we studied the cellular composition and morphology of Nr2e1 frc/frc , and Nr2e1 +/+ ↔Nr2e1 frc/frc chimeric mouse retinas. We found that dystrophy-related phenotypes in Nr2e1 frc/frc retinas are not generated cell-autonomously. In addition, we found that lack of Nr2e1 results in an ectopic plexiform layer in the inner retina, aberrant development of Müller glia and a bias towards the generation of glycinergic amacrine cells, S-cones and Müller glia.
To get insight into the cell autonomy of Nr2e1 during retinogenesis we used Nr2e1 frc/frc and chimeric mice comprised of both Nr2e1 frc/frc and wild-type cells. We studied abnormal phenotypes previously reported to be present in Nr2e1 null retinas, such as reduced retinal thickness and blood vessel numbers. We later focused on the role of Nr2e1 in cell type development by studying the numbers and localization of different cell types.
Expression of EGFP and β-galactosidase in mouse chimeras
To better understand the cell-autonomous and non-cell autonomous roles of Nr2e1 during retinogenesis, we made chimeric mice comprised of Nr2e1 +/+ and Nr2e1 frc/frc cells, herein referred as Wt↔frc chimeras. Experimental and control chimeric mice were made by blastocyst injection of Nr2e1 +/+ or Nr2e1 frc/frc embryonic stem cells (ESCs) harboring a ubiquitous-expressing EGFP transgene (Additional file 1: Figure S1A and B). In contrast, host blastocyst contained the lacZ gene under the control of ROSA26 promoter (R26 lacZ ) thus expressing β-galactosidase (β-gal) (Additional file 1: Figure S1B). In this way ESC-derived cells could be identified by the green epifluorescence of EGFP and blastocyst-derived cells by the enzymatic product of β-gal.
We used two different embryonic stem cell lines per genotype to control for possible cell-line specific traits that could affect the phenotype of the mice. Four Wt↔Wt and four Wt↔frc chimeras were studied at P7. Nine Wt↔Wt and ten Wt↔frc chimeras were studied at P21. Eyes from these chimeras were subjected to funduscopy and collected for cryosectioning. First, we determined that the EGFP and β-gal markers were expressed appropriately in the chimeras. We assessed the expression of β-gal by its enzymatic activity and could clearly observe the blue precipitate formed by the hydrolysis of X-gal in perinuclear regions (Additional file 1: Figure S1C). Importantly, this enzymatic reaction did not interfere with the EGFP epifluorescence and both markers were expressed in mutually exclusive regions of the chimeric retinas (Additional file 1: Figure S1D). We assessed the percentage of chimerism by measuring the area displaying EGFP epifluorescence in the ONL plus INL of each retina and comparing it to the total ONL plus INL area. We excluded the IPL and GCL to decrease the interfering signal recovered from neural processes. Thus, we were able to use these two markers reliably as indicators of the origin of the different cell types; ESC or host blastocyst.
Nr2e1 frc/frc reduced retinal thickness and blood vessel numbers were rescued in Wt↔frc chimeras
Characteristics of the chimeras
Blood vessel #
Retinal thickness (μM)
213.63 ± 9.36
234.90 ± 13.84
205.67 ± 10.09
376.15 ± 38.77
183.67 ± 2.33
197.67 ± 5.61
146.33 ± 5.78
160.00 ± 0.00
170.00 ± 7.64
160.00 ± 5.77
149.33 ± 4.70
127.50 ± 7.50
108.33 ± 4.81
204.43 ± 18.80
259.95 ± 42.37
196.42 ± 4.99
180.5 ± 8.59
185.00 ± 4.93
190.33 ± 14.90
159.33 ± 6.33
178.00 ± 8.00
193.67 ± 5.84
172.67 ± 11.85
190 ± 0.00
204.33 ± 7.17
We measured the retinal thickness in a central section containing the optic nerve and two other adjacent sections 60 μm away in both directions. We found that the percentage of chimerism did not affect the retinal thickness of Wt↔Wt chimeras (Fig. 1b) but it affected the thickness of Wt↔frc chimeras (R2 = 0.67). In this case, a 66 % chimeric retina had a retinal thickness comparable to wild type (146.33 ± 5.78 μm, Z-score greater than −3) suggesting that ≥34 % wild-type cells were needed to rescue it (Fig. 1b). Only the two highest percentage chimeric retinas (67 % and 86 %) were not rescued by wild-type cells having thinner than wild-type retinas (127.5 ± 7.5 and 108.33 ± 4.81 μm, respectively, Z-score lower than −3).
In summary, a high contribution of wild-type cells (≥55 %) was needed to achieve rescue of blood vessels whereas retinal thickness was restored by fewer wild-type cells (≥34 %). The rescue of retinal thickness suggests that cell loss is not regulated cell autonomously by Nr2e1. We then studied whether other retinal abnormalities occurring in Nr2e1 null mice including gliosis and gross structural defects could also be rescued by wild-type cells in chimeric retinas.
Nr2e1 frc/frc Müller glia misexpression of GFAP and retinal structural defects were rescued in Wt↔frc chimeras
We found that none of the chimeric retinas, even those with a very high contribution of Nr2e1 frc/frc cells, express GFAP in central Müller glia, suggesting that wild-type cells rescue this defect (Fig. 2c). We also observed that the retina of high percentage chimeras was still very thin but did not have the structural defects seen in Nr2e1 frc/frc retinas (Fig. 2c).
In summary, misexpression of GFAP and gross structural defects do not emerge cell-autonomously in Nr2e1 frc/frc retinas and are likely a consequence of other defects such as abnormal cell type development. We then focused on studying the numbers of each retinal cell type in P7 Nr2e1 frc/frc and chimeric retinas.
Lack of Nr2e1 resulted in increased numbers of glycinergic amacrine cells and S-cones, and normal numbers of Müller glia
Although the role of Nr2e1 in regulating retinal thickness has been assessed [21, 22], the numbers of different retinal cell types in Nr2e1-null retinas has not yet been studied. Similarly, the presence of each retinal cell type has previously only been assessed in adult eyes after apoptosis has already severely altered the structure and composition of the retina [21, 22]. To gain better insight into the cell numbers generated in Nr2e1 frc/frc mouse retinas, we studied the retina at P7. At this time point most cells have already been differentiated and less than 5 % of bipolar and Müller glia are still being generated in the rat retina . At P7, cells have not been exposed to daily visual activity and are less prone to apoptosis in Nr2e1 null retinas . To quantitatively assess differences in cell numbers, we manually counted all cell types in the retinas of wild-type and Nr2e1 frc/frc mice at P7. Five sections throughout the eyes were analyzed.
To better understand the interplay between wild-type and Nr2e1 frc/frc cells during the development of chimeric retinas, and to better assess clonal composition, we looked at the density of different retinal cell types belonging to each genotype in Wt↔frc chimeras at P7. The number of each EGFP positive or EGFP negative cell type was divided over the EGFP-positive or EGFP-negative area, respectively.
Previous studies have shown a reduced outer nuclear layer (ONL) but have conflicting results regarding the thickness of the inner nuclear layer (INL) in P4-P7 Nr2e1 mutant retinas [21, 22]. Miyawaki et al., reported a thicker INL  whereas Zhang et al., reported a thinner INL . We observed, in agreement with a reduced ONL, a 33 % reduction in rods in Nr2e1 frc/frc retinas when compared to wild type (Additional file 2: Figure S2A and C). We found no difference in INL thickness between wild-type and Nr2e1 frc/frc P7 central retinas measured in sections taken through the optic nerve (Wt = 85.9 ± 0.17; frc = 85.86 ± 3.27; n = 3 for Wt; n = 3 for frc).
We also found a 27 % reduction in bipolar cells in Nr2e1 frc/frc retinas when compared to wild type (Additional file 2: Figure S2B and C). A reduction in the numbers of rods and bipolar cells is expected given that they differentiate at later time-points in the mouse retina . However, since the thickness of the Nr2e1 frc/frc INL is not overall reduced at P7, other cell types must be overrepresented in these retinas.
In Wt↔frc chimeras, Nr2e1 frc/frc rods (Additional file 2: Figure S2D and F) and bipolar cells (Additional file 2: Figure S2E and G) were 50 % and 53 % less abundant, respectively, compared to wild type. This suggests that wild-type cells cannot rescue the numbers of Nr2e1 frc/frc rods and bipolar cells that fail to differentiate.
In summary, P7 Nr2e1 frc/frc retinas have a marked reduction of ganglion, bipolar, and rod cells, increased numbers of glycinergic amacrine cells, and normal numbers of cones, horizontal, and Müller glial cells. In Wt↔frc P7 chimeric retinas, an increase in Nr2e1 frc/frc S-cone numbers was also evident. In the same chimeras, wild-type cells did not rescue Nr2e1 frc/frc abnormal cell numbers with the exception of ganglion and amacrine cells in the GCL. We further studied whether these cellular defects were accompanied by abnormalities in retinal lamination as explained below.
Nr2e1 frc/frc retinas displayed an ectopic plexiform layer and a disorganized inner plexiform layer, which were not rescued by wild-type cells in Wt↔frc retinas
To evaluate whether bipolar cells that normally establish synaptic connections with retinal ganglion cells (RGCs) and amacrine cells in the inner plexiform layer (IPL)  also had terminals in the EPL, we stained retinas for PKC-alpha expressed in ON bipolars. We found that PKC-alpha positive bipolar cells had mislocalized axon branchings in the EPL (Fig. 8c and d, solid arrow).
We then asked if the lamination defects generated by Nr2e1 mutant cells could be rescued by extracellular signals from wild-type cells in Wt↔frc chimeras. At P7 the EPL was present in all four chimeras, and regions of wild-type cells failed to form this layer, showing that wild-type cells cannot rescue this lamination defect or be influenced by mutant cells to form this ectopic layer (Fig. 8e).
Furthermore, the IPL of Wt↔frc chimeras was thicker throughout the whole retina and less defined than that of Wt↔Wt chimeras suggesting that the development of the IPL is also affected by the lack of Nr2e1 (Fig. 8e, brackets).
To better characterize the EPL, we stained Nr2e1 frc/frc P7 retinas with antibodies that normally recognize the inner plexiform layer (IPL) and found that unlike syntaxin-1A and GlyT1, calbindin and mGluR1 were occasionally but not always found in this ectopic layer (Additional file 4: Figure S4A-D).
Moreover, some calbindin and mGluR1 positive cells were observed in proximity to the EPL of Nr2e1 frc/frc retinas (Additional file 4: Figure S4B and D, asterisks) and Pax-6-positive cells were found between mGluR1 positive sublaminae (Additional file 4: Figure S4D, arrowhead) revealing lamination defects.
Taken together, these results suggested a role of Nr2e1 in constraining the neurites of INL neurons to an organized IPL. P21 chimeric retinas also revealed a rescue of amacrine cell numbers by wild-type cells that we further studied.
Nr2e1 frc/frc amacrine cell loss is rescued by wild-type cells in Wt↔frc retinas
We observed few syntaxin-1A and GlyT1 positive cells in P21 Nr2e1 frc/frc retinas suggesting extensive cell loss of amacrine cells as expected from dystrophy (Fig. 9a-d). However, in P21 Wt↔frc chimeras the numbers of Nr2e1 frc/frc GlyT1 positive amacrine cells appeared normal (Fig. 9h). Quantification of syntaxin-1A cell numbers in the INL of chimeras revealed normal numbers of Nr2e1 frc/frc amacrine cells (Additional file 5: Figure S5), suggesting that wild-type cells prevented the excessive loss of mutant amacrine cells during the postnatal period and that amacrine cell death is not a primary consequence of Nr2e1 loss.
The primary roles of Nr2e1 in the retina are then the regulation of RPC cell cycle, cell type numbersand inner neurite development. Nr2e1 remains to be expressed in the adult Müller glia and thus it may also have specific primary roles in this cell type. Therefore, we further characterized Müller glia with specific markers as shown below.
Nr2e1 frc/frc Müller glia were aberrantly positioned in the inner nuclear layer and cell-autonomously misexpressed Brn3a
Nr2e1 is expressed in the Müller glia of adult mice . To better characterize the expression of Nr2e1 in Müller glia during postnatal development, we stained P3, P7, P14, and P21 retinas with antibodies against β-gal and SOX-2 in mice expressing β-gal under the control of the human NR2E1 promoter (NR2E1-lacZ) . We made use of this mouse strain due to the lack of a reliable commercial antibody for Nr2e1. Previous work by others has shown that SOX-2 is expressed in progenitors and a subpopulation of amacrine cells starting at P0. By P7, SOX-2 expression becomes mainly restricted to the inner nuclear layer in a subpopulation of amacrine cells and in Müller glia. This expression in amacrine cells and Müller glia continues through adulthood . We observed co-localization of SOX-2 and β-gal at all time-points in cells that extend apical and basal processes indicative of Müller glia (Additional file 6: Figure S6A). This suggests sustained expression of Nr2e1 in Müller glia throughout postnatal development. Furthermore, we observed SOX-2 positive cells in Nr2e1 frc/frc retinas at all time-points (Additional file 6: Figure S6B), further showing that Nr2e1 frc/frc Müller glia numbers are comparable to wild type.
Overall, these results suggested that Nr2e1 regulates the maturaion of Müller glia cell-autonomously.
The phenotype of the Nr2e1-null eye is very complex involving various cell types and tissues, thus making the identification of the primary function of Nr2e1 during eye development challenging. In this study we used chimeras to clarify the cell-autonomous roles of Nr2e1 during retinogenesis, and described novel phenotypes of the Nr2e1 frc/frc mice in the P7 retina before extensive dystrophy has occurred. Our results suggest that Nr2e1 regulates the development of specific retinal cell-types and the organization of inner retinal neurites.
Nr2e1 does not prevent retinal dystrophy cell-autonomously
The thickness of the P21-P28 Nr2e1 mutant retina is highly reduced compared to wild type due to progressive postnatal cellular loss [21, 22]. Our findings suggest that wild-type cells can rescue cell loss in Wt↔frc chimeras. First, we showed that in P21 Wt↔frc chimeras, the reduced Nr2e1 frc/frc retinal thickness could be rescued by ≥34 % wild-type cells. Second, we observed that postnatal amacrine cell loss is rescued in P21 Wt↔frc chimeric retinas. These retinas appeared to have normal instead of reduced numbers of Nr2e1 frc/frc glycinergic amacrines and had normal numbers of Nr2e1 frc/frc total amacrine cells. Third, chimeras still had low numbers of mutant rods and bipolar cells that do not account for the observed rescue of retinal thickness. Fourth, we showed that low numbers of ganglion cells were rescued in P7 Wt↔frc chimeras. According to a previous study, increased apoptosis in the ganglion cell layer occurs in Nr2e1 mutant retinas as early as P0 . Thus, excessive cell-death may be the cause of ganglion cell reduction in P7 Nr2e1 frc/frc retinas that should be otherwise overrepresented as a consequence of premature neurogenesis. Therefore, the rescue of mutant ganglion cell numbers that we observed in chimeras is likely due to a restoration of ganglion cell survival. In conclusion, Nr2e1 does not have a cell-autonomous role in preventing postnatal cell death as wild-type cells are able to rescue cell loss.
Moreover, structural and gliosis-related phenotypes were rescued in P21 Wt↔frc chimeras. In Nr2e1 frc/frc retinas, some cells in the INL protrude into the ONL, and cells in the ONL migrate into the subretinal space . In Wt↔frc chimeras, we do not see overlapping of INL and ONL cells or any other structural defect even in a retina with 86 % Nr2e1 frc/frc cells, suggesting that these defects are not regulated cell-autonomously by Nr2e1. Interestingly, expression of GFAP in Müller glia, which is typical during gliosis but also in Nr2e1 frc/frc retinas , was absent in Nr2e1 frc/frc Müller glia of chimeric retinas in spite of GFAP being a direct target of Nr2e1 . This further supports the suggestion that dystrophy-related phenotypes are not regulated cell-autonomously by Nr2e1.
Furthermore, we observed that blood vessel numbers do not influence retinal thickness. Nr2e1 frc/frc reduced blood vessel number was rescued by ≥55 % wild-type cells. The high number of cells needed to rescue the blood vessel numbers indicates that this defect is not corrected by rescuing the mutant cells and is likely cell-autonomous. Nr2e1 is expressed in proangiogenic astrocytes  and thus may act cell-autonomously in these cells to regulate retinal blood vessel development. In addition, the contribution of mutant cells to the astrocyte lineage could have been different than that to the retinal lineage, as astrocytes and retinal progenitors come from different cell populations. Regardless of the mechanism, it appears that the blood vessel and retinal thickness phenotypes of Nr2e1 frc/frc retinas are not influenced by each other and thus retinal dystrophy may not be influenced by abnormal blood vessel development. In addition, Nr2e1 frc/frc defects such as abnormal cellular proportions and lamination were never observed in wild-type regions of chimeric retinas suggesting that vascular abnormalities are not the primary cause of retinal defects in the mutant retinas.
These results taken together suggest that cell death, abnormal retinal structure, and gliosis, all related to dystrophy in Nr2e1 mutant retinas, are not regulated cell-autonomously and can be rescued by wild-type cells. However, blood vessel abnormalities may not be a crucial factor in influencing dystrophy in Nr2e1 mutant retinas. What are then the primary roles of Nr2e1 during retinogenesis? We found that in addition to the previously described role in cell-cycle regulation, Nr2e1 regulates the development of specific retinal cell types as discussed below.
Nr2e1 regulates the development of specific retinal cell types
In the retina, Nr2e1 is thought to regulate cell numbers through shortening cell-cycle length and thus delaying neurogenesis . We found that at P7, the density of some cell-types in Nr2e1 frc/frc retinas does not correspond to the proportions expected from premature cell-cycle exit. Importantly, the same cellular proportions were seen in Nr2e1 frc/frc regions of chimeras suggesting they emerge intrinsically from each clone of RPCs and are not secondary to eye volume differences.
We found that Nr2e1 frc/frc retinas are biased to generate Müller glia. The reduction in rods and bipolar cell numbers in Nr2e1 frc/frc retinascould be explained by underproduction of late-born cell types due to premature neurogenesis. Müller glia is the last retinal cell type to be generated  and thus premature neurogenesis would also predict reduced numbers. The fact that their numbers are not reduced in Nr2e1 frc/frc mice suggests a bias towards Müller glia differentiation in the absence of Nr2e1 regulation. In adult rat hippocampal-progenitors, Nr2e1 represses glial differentiation and activates the neuronal lineage . Nr2e1 also directly binds to the promoter of glial-specific genes . All of this taken together suggests a role for Nr2e1 in repressing the Müller glial lineage during development.
We also found an expanded population of glycinergic amacrine cells in the INL of Nr2e1 frc/frc retinas and increased numbers of Nr2e1 mutant amacrine cells compared to wild type in chimeras. Amacrine cells are mainly subdivided in two groups: GABAergic and glycinergic . Amacrine cell overproduction in Nr2e1 frc/frc retinas could be a consequence of precocious neurogenesis. However, a bias towards a particular subtype was not expected, especially when glycinergic amacrines are generated after the GABAergic subtype  making it more likely that GABAergic amacrines would be overrepresented. This suggests that Nr2e1 may have a role in regulating amacrine subtype development during mouse retinogenesis and that a switch in cell fate may have occurred in favor of glycinergic amacrine cells at the expense of other cell types that are reduced in number such as bipolar cells and rods.
We also observed that Nr2e1 regulates most retinal cell numbers cell-autonomously. To assess how Nr2e1 regulates extrinsic and intrinsic signals that affect RPC proliferation and differentiation in vivo, we evaluated the interplay between wild-type and Nr2e1 frc/frc cells in Wt↔frc chimeras. Intriguingly, the cellular composition of the GCL, but not the INL or ONL, was rescued by extra-cellular signals from wild-type cells. The GCL is comprised of ganglion and amacrine cells. The rescue of ganglion cell numbers in this layer is likely due to prevention of cell-death, as explained above. The fact that Nr2e1 frc/frc amacrine cell numbers were normal instead of overabundant in the GCL of chimeras suggests a role of extracellular signals in regulating their migration or survival in this layer rather than in preventing their generation because amacrines in the INL were still overabundant. Therefore, we think that wild-type cells did not affect the proportions of cells generated from RPCs. This suggests that RPC proliferation and differentiation does not depend on non-cell-autonomous roles of Nr2e1 as is the case of adult neural stem cell proliferation . In other words, the role of Nr2e1 in differentiation and cell-cycle regulation is likely cell-autonomous in the retina.
Furthermore, chimeras revealed a role of Nr2e1 in S-cone development. In Nr2e1 frc/frc retinas, total cones and S-cones numbers were normal but in Wt↔frc chimeras Nr2e1 frc/frc S-cones were more abundant than wild-type S-cones. The late stage of development of S-cones, which includes S-Opsin expression, can be regulated by extracellular signaling such as the one mediated by thyroid hormone . It is then possible that Nr2e1 regulates extrinsic signals necessary for the development and/or survival of S-cones. However, we cannot rule out that a bigger sample size would yield a significant difference in S-cones between wild-type and mutant retinas given that the quantified difference has a p-value of only 0.1. In agreement with this, a previous study reported increased S opsin transcript levels in P14 Nr2e1 mutant retinas . However, without information regarding protein levels and cell numbers, we cannot know with certainty that the numbers of S-cones were higher in those mutant retinas. Finally, the fact that Nr2e3, the closest relative of Nr2e1, regulates photoreceptor development and is mutated in enhanced S-cone syndrome in humans  further supports the hypothesis that Nr2e1 has a role in S-cone development.
Interestingly, in Nr2e1 frc/frc retinas, the densities of horizontal cells, cones, and Müller glia remained comparable to wild type from P7 through P21 when retinogenesis has long been completed. This indicates that these cells are spared from the excessive cell-death seen in Nr2e1 frc/frc retinas during the postnatal period. This also suggests that the majority of cell death observed between P7 and P21 in Nr2e1 frc/frc retinas may be due to the loss of amacrine cells that make most of the INL at P7.
Nr2e1 then has an important role in the development of certain cell-types during retinogenesis perhaps through the regulation of genetic networks that are also involved in cell-fate. During retinogenesis, transcription factors such as Prox1 , P57(Kip2)  and Math5  are reused to perform multiple functions including cell proliferation and cell fate. Interestingly, Nr2e1 remains to be expressed in Müller glia but the specific roles of Nr2e1 in this cell type remains elusive. We further studied the expression of Nr2e1 during Müller glia development and the localization of Müller glia within the retina of Nr2e1 frc/frc and chimeric retinas as discussed below.
Nr2e1 regulates Müller glia differentiation cell-autonomously
We observed that Nr2e1 is expressed in Müller glia throughout postnatal development and that Nr2e1 frc/frc Müller glia misexpress Brn3a and position their soma ectopically in the upper INL. Brn3a is a transcription factor normally expressed only in ganglion cell precursors and mature ganglion cells in the retina . Brn3a ectopic expression was likely specific in Müller glia as 1) it was present only in the ventral retina; 2) it co-localized with Müller glial markers; and 3) it was present only in Nr2e1 frc/frc cells of chimeras. Thus, Nr2e1 may be required to maintain a differentiated state in Müller glia by directly or indirectly repressing Brn3a. Interestingly, Brn3a misexpression in Müller glia was also cytoplasmic and not exclusively nuclear. However, this is not surprising since various studies have reported cytoplasmic localization of other transcription factors including Crx , Hes , Pax-6, SOX-2 and Chx10  in cultured Müller glia. This suggests that nuclear trafficking mechanisms of various transcription factors may work differently in Müller glia under atypical conditions. Importantly, Brn3a misexpression in the ventral retina suggests that Nr2e1 may have a role during dorso-ventral patterning in the retina as it does in the brain .
Both, the Brn3a misexpression and ectopic soma positioning of Müller glia were present only in Nr2e1 frc/frc cells of Wt↔frc chimeras. The fact that Nr2e1 is expressed in Müller glia and that wild-type Müller glia are unaffected by Nr2e1 frc/frc cells, suggests that Nr2e1 may have a cell-autonomous role in the maturation of Müller glia by regulating their transcriptional profile and localization within the retina. Furthermore, Müller glia mislocalization and misexpression of Brn3a are not a consequence of dystrophy since dystrophy is rescued by wild-type cells in chimeras but these Müller glia defects are not. Nr2e1 null Müller glia were shown by others to have thinner processes compared to wild-type . Our results suggest that this phenotype is likely cell-autonomous and not a consequence of other cellular defects in Nr2e1 mutant retinas. However, this was not determined in that study.
The role of Nr2e1 in regulating the function of Müller glia or any other cell-type was not assessed in this study and deserves further investigation. However, defects in neuropil development were observed in Nr2e1 mutant retinas, which suggest major defects in retinal connectivity and are discussed below.
Nr2e1 regulates retinal lamination by preventing disorganization of inner retinal neurites
Nr2e1 frc/frc retinas have a disorganized IPL, an ectopic plexiform layer (EPL) within the INL, and abundant interplexiform amacrine cells. Together these observations suggest that Nr2e1 is involved in a cellular mechanism that organizes and constrains the neurites of inner retinal neurons to the IPL. Interestingly, the EPL can attract normal pre-synaptic partners, as bipolar cells arborized within it.
In Wt↔frc chimeras, wild-type regions did not have an EPL, however an EPL was evident in mutant regions. This suggests that a long-range extracellular molecule is not involved in inducing the formation of the EPL. On the other hand, wild-type columns had a disorganized IPL, suggesting that Nr2e1 mutant cells can misdirect the neurites of wild-type cells. Wt↔frc chimeras also demonstrate that abnormal ganglion cell numbers per se are not the cause of IPL disorganization since they are restored in these chimeras.
The regulation of laminar targeting specificity in the retina depends on repulsive and attractive interactions mediated mostly by multiple short-range molecules [41–44]. A disrupted patterning of the IPL and disorganized amacrine branching is seen in Pten −/− and Dscam −/− mice [45, 46]. Pten −/− mouse retinas have an expanded IPL with scattered cell bodies of abnormal morphology and clusters of dendrites in the IPL [45, 47]. Since Pten is a direct target of Nr2e1, its abnormal levels in Nr2e1 frc/frc retina could contribute to aberrant neurite organization. Pten remains expressed in the INL, IPL and GCL at P7 . Whether Nr2e1 is expressed in immature amacrine cells during development deserves further investigation.
Interestingly, a clear single ectopic layer in the INLhas been shown only in mice lacking the transmembrane repellents Sema5A and Sema5B  and the atypical cadherin Fat3 . Fat3 −/− amacrine cells develop a second dendritic tree that projects away from the IPL and forms an additional synaptic layer in the INL . Thus, our work suggests the interesting hypothesis that the Sema5 or Fat3 pathways may be affected in Nr2e1 frc/frc retinas.
The mechanisms by which signaling molecules orchestrate the development of a normal neuropil in the nervous system are still vastly unknown. Understanding how the lack of a transcription factor, such as Nr2e1, allows the formation of an ectopic neuropil in the retina would help to shed light into the cellular and molecular mechanisms leading to this process.
In summary, we found that Nr2e1 has numerous roles beyond preventing premature cell cycle exit in RPCs during retinal development including neurite organization and terminal cell differentiation. Our results show that Nr2e1 regulates the organization of inner retina neurites to the IPL and the development of neuronal subtypes, such as S-cones and glycineric amacrines as well as Müller glia. In addition, Nr2e1 regulates Müller glia differentiation cell-autonomously. Moreover, we have shown that prevention of retinal dystrophy is not regulated cell-autonomously by Nr2e1. A model summarizing the defects in cell numbers and lamination of the P7 Nr2e1 frc/frc and chimeric retina is depicted in Fig. 12.
Materials and methods
Mouse strains, husbandry, and breeding
Nr2e1 frc mice harbor a spontaneous deletion of the entire coding region of Nr2e1 with no transcripts detected in the brain. These mice are highly aggressive and display hypoplastic cerebrum, olfactory lobes and retina . Mice for study were generated by crossing Nr2e1 frc/+ heterozygote females, which were congenic (>N30) on the C57BL/6 J (B6) (JAX stock # 000664) background, to Nr2e1 frc/+ heterozygote males, which were congenic (>N30) on the 129S1/SvImJ (129) (JAX stock # 002448) background, thus generating Nr2e1 +/+ control and Nr2e1 frc/frc mutant littermates on the hybrid B6129F1 background . Males harboring the NR2E1-lacZ reporter BAC (bEMS86) were used to study NR2E1 expression in the postnatal retina; strain B6.129P2(Cg)-Hprt tm73(Ple142-lacZ)Ems , abbreviated here as NR2E1-lacZ (MMRRC stock # 032962-JAX) . Males harboring a random insertion of multiple-linked copies of the enhanced green fluorescent protein (EGFP) under an ubiquitous promoter were used for embryonic stem cell (ESC) generation; strain B6-Tg(CAG-EGFP)1Osb/J, abbreviated here as B6-EGFP (N > 10; JAX Stock # 003291) . Mice containing the lacZ transgene at the ROSA26 locus were used as a source of host blastocysts; strain 129S-Gt(ROSA)26Sor/J (JAX Stock # 002292) , backcrossed to B6 (N2-3) and abbreviated here as B6-R26 lacZ . Eye studies were carried out on mice of either sex.
Mice were kept in a pathogen-free animal facility at the Centre for Molecular Medicine and Therapeutics, University of British Columbia (Vancouver, BC, Canada) on a 6 am to 8 pm light cycle with, 20 ± 2 °C, 50 % ± 5 % relative humidity, and food and water ad libitum. All procedures involving animals were in accordance with the Canadian Council on Animal Care (CCAC) and UBC Animal Care Committee (ACC) (Protocol numbers A11-0370 and A11-0081).
Generation of embryonic stem cells (ESCs)
To obtain ESCs that contained both the Nr2e1 frc allele and the EGFP transgene as a marker, a two generation cross was performed. First, B6-Nr2e1 frc/+ mice were crossed to B6-EGFP/0 mice to obtain B6-Nr2e1 frc/+ EGFP/0 females. Then, these females were crossed to 129-Nr2e1 frc/+ males to obtain B6129F1-Nr2e1 frc/frc EGFP/0 blastocysts. ESC lines were derived as previously described . Briefly, blastocysts at 3.5 days post fertilization (dpf) were cultured in KSOM + AA media (Caisson Laboratories, North Logan, UT) under oil at 37 °C for 3–5 h. Then, each blastocyst was transferred to a single (1 x) 96-well containing mitomycin-C-inactivated mouse embryonic fibroblasts (MEFs) and cultured in KSR-ESC media (Knockout™ D-MEM with 2 mM l-glutamine, 0.1 mM MEM nonessential amino acid solution, 16 % Knockout™ Serum Replacement (all from Life Technologies Inc., Burlington, ON), and 1000 U/ml ESGRO® (LIF) (Millipore, Temecula, CA) . Once the blastocyst hatched and had a well-defined inner cell mass, it was trypsinized and transferred to 1 x 24-well containing inactivated MEFs. Cells were replated 1:1 if necessary. When cells reached confluence, they were split into 3 x 24-wells in 100 % ESC media. All 3 wells were combined at confluence and frozen in 3 separate vials until needed.
Generation of chimeras
Chimeras were derived by microinjection of ESC into host blastocysts as previously described . Either B6129F1-Nr2e1 +/+ EGFP/0 or B6129F1-Nr2e1 frc/frc EGFP/0 ESCs were microinjected into B6-R26 lacZ/+ host blastocysts. The generated chimeras were thus comprised of blastocyst-derived cells that were wild-type for Nr2e1 and harbored the R26 lacZ transgene and ESC-derived cells that were either wild-type or mutant for Nr2e1, and harbored the EGFP transgene. A chimera is defined by the genotype of the blastocyst-derived and ESC-derived cells (blastocyst↔ESC): (Nr2e1 +/+ , R26 lacZ/+ ) ↔ (Nr2e1 +/+ , EGFP/0) or (Nr2e1 +/+ , R26 lacZ/+ ) ↔ (Nr2e1 frc/frc , EGFP/0); and abbreviated as Wt↔+/+ or Wt↔frc/frc, respectively. Two Nr2e1 +/+ (mEMS4919 and mEMS4926) and two Nr2e1 frc/frc (mEMS4914 and mEMS4922) ESC lines were used to generate chimeras. After injection, blastocysts were implanted into the uterine horns of B6 2.5 day pseudopregnant females. Four Wt↔+/+ and four Wt↔frc/frc P7 chimeric eyes were included in the study. Nine Wt↔+/+ and ten Wt↔frc/frc P21 adult chimeric eyes were also included.
Assessment of chimerism
Chimerism was initially assessed by coat color. Then, in slides prepared of the retina, chimerism was assessed by measuring the area of EGFP epifluorescence signal in the inner nuclear layer (INL) plus outer nuclear layer (ONL) using the software ImageJ . The percentage of this fluorescence was assessed over the total INL plus ONL area.
Eyes were fixed by intracardial perfusions performed on avertin-anesthetized mice with 4 % paraformaldehyde (PFA) in phosphate buffered saline (PBS). Eyes were then post-fixed in 4 % PFA for 30 min prior to cryoprotection in 25 % sucrose-PBS overnight. Subsequently, eyes were embedded in optimal cutting temperature (OCT) medium, cryosectioned at 12 μm and mounted on SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA). For β-galactosidase (β-gal) immunohistochemistry, staining was performed using X-gal substrate (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside) for 18 h at 37 °C. To evaluate retinal thickness, retinal sections were subjected to hematoxylin and eosin staining. Briefly, tissue was incubated in hematoxylin for 5 min, washed in tap water and incubated in 1 % lithium carbonate solution for 30 sec. After washing in tap water again, the tissue was incubated in acid alcohol (1 %) for 5 s followed by another tap water wash and incubation in eosin Y solution for 5 min. After a final tap water wash, tissue was dehydrated in a gradient of ethanol, and xylene before mounting for microscopy. For immunofluorescence, at least three Nr2e1 +/+ and three Nr2e1 frc/frc retinal sections per epitope were incubated in blocking solution (5 % bovine serum albumin, 0.3 % Triton X-100 in PBS) for 1 h. Subsequently, sections were incubated in primary antibody in blocking solution at 4 °C overnight. After three washes of 10 min in PBS, sections were incubated in secondary antibody in blocking solution with the DNA dye Hoechst-33342 for 1 h. Sections were washed 3 times in PBS and mounted in ProLong® Gold Antifade Reagent (Life Technologies Inc., Carlsbad, CA, USA). At least three transverse retinal sections were immunostained for each antibody using at least three eyes per genotype for imaging analysis.
To label ganglion cells, we stained the retinas with antibodies to Brn3a, which is expressed in approximately 80 % of these cells . For amacrines we used antibodies to the pan-amacrine markers Pax-6, and syntaxin-1A, which label amacrine cells and horizontal cells in the inner nuclear layer (INL) and amacrine and ganglion cells in the ganglion cell layer (GCL) [53, 54]. To visualize horizontal cells we used antibodies to calbindin, which is expressed in horizontal cells and a subpopulation of amacrine cells . We used for: cones, cone-arrestin (arrestin-C) and S-Opsin antibodies that at P7 label the cell body, processes and outer segment of cones, which are located throughout the outer nuclear layer ; rods, rhodopsin antibody; bipolar cells, CHX10 antibody ; and Müller glia, Sox9 antibody . The following primary antibodies were used: mouse anti-Brn3a 14A6 from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); mouse anti-Pax-6, anti-ISL1/2; rabbit anti-Pax-6 from Coavance (Princeton, NJ, USA); mouse anti-calbindin and anti-syntaxin from Sigma-Aldrich (St. Louis, MO, USA); mouse anti-Rhodopsin (ID4) from Dr. R.S. Molday, University of British Columbia (Vancouver, BC, Canada); sheep anti-Chx10 from Exalpha Biologicals, Inc. (Shirley, MA, USA); rabbit anti-cone-arrestin, rabbit anti-Sox9, rabbit anti-S-opsin, rabbit anti-mGluR1, goat anti-calretinin, chicken anti-vimentin, and guinea pig anti-GABA from Millipore (Billerica, MA, USA); mouse anti-PKCalpha, chicken anti-β-galactosidase, and rabbit anti-SOX-2 and anti-GlyT1 from Abcam (Cambridge, England); and mouse anti-GFAP from New England Biolabs (Ipswich, MA, USA).
Imaging and cell counting
For dual visualization of X-gal blue precipitate and EGFP fluorescence in chimeras, images were taken with the Olympus BX61 motorized microscope using both DP Controller and In Vivo software (Olympus Corporation of the Americas, Center Valley, PA, USA). All remaining fluorescent images were taken with a Leica TCS SP5 II confocal microscope (Leica Microsystems, Wetzlar, Germany). To assess the numbers of each cell type in the retina, images were taken at 200 times magnification throughout a 12 μM retinal section and tiled. At least five equidistant transverse retinal sections throughout the entire eye were imaged: two images were close to the edge; two close to the middle half; and one central that included the optic nerve. One eye, from three different mice of each genotype, was included. Labeled cells were manually counted using the Cell Counter tool of ImageJ. Images were taken with a scan size of at least 1024 X 1024 pixels using 200 times magnification and visualized at 18 X 18 cms for accurate counting. The retinal marker channels and blue channel (Hoeschst stained-nuclei) images were superimposed followed by alternative switching between the channels to make sure individual cells were counted and to assess co-localization. Using the Cell Counter Tool a dot was placed on each cell counted to avoid double counting or miscounting of cells. Cell numbers were normalized to retinal length (μm). This length was assessed by drawing a line from edge to edge of retinal tissue along the nuclear layer where the cell-type to be counted was located. To count cells in chimeric retinas, marker-positive and EGFP-negative singled-labeled cells or marker-positive and EGFP-positive double-labeled cells were counted throughout a central retinal section. Cell numbers were normalized to retinal area. The EGFP-positive area was assessed by measuring the area of EGFP epifluorescence signal in the INL plus ONL using ImageJ. The EGFP-negative area was assessed by subtracting the EGFP positive area from the total INL plus ONL area. Cell density was expressed as the number of single-labeled cells over the EGFP-negative area or double-labeled cells over the EGFP-positive area. One eye, from three different chimeras for each ESC genotype, was included; totaling 6 eyes. Only chimeras with a contribution of >46 % mutant cells were counted. Quantification of retinal structural defects where INL and ONL cells overlap was performed in 3 eyes per genotype and 5 retinal sections as explained above for retina cell number quantification. A structural defect was counted when INL and ONL cells where in such proximity to each other that the OPL was no longer evident.
To assess the number of retinal blood vessels, funduscopy was performed as previously described [29, 58]. Briefly, eyes were dilated with 1 % atropine in PBS and photographed after 30 min. Animals were manually restrained without sedation.
Images were processed using ImageJ and Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA, USA). Brightness, contrast, and scaling adjustments were performed as necessary.
Statistical analyses were performed using Microsoft Excel (Microsoft Corporation, Redmond, Washington, USA) and XLSTAT (Addinsoft, New York, NY, USA). Student’s t-test was used to compare total cell numbers using raw data. In some cases, data was graphed as percentage of wild-type cells. Results were considered significance with a P-value ≤ 0.05. The standard error of the mean (SEM) was indicated with error bars. Z-scores were calculated as (retinal thickness or blood vessel number of Wt↔frc chimeras) minus ((mean of Wt↔Wt chimeras) divided by (standard deviation of Wt↔Wt chimeras)).
This work was supported by a Sharon Stewart Aniridia Research Award [grant number 20R64586] and Sharon Stewart Aniridia Trust [grant number 20R67715] to EMS, and a CFRI Partnership Graduate Studentship award to XCD. We are very grateful to: the Microinjectionists Sonia F. Black, Tom W. Johnson and Tess C. Lengyell for their role in making the chimeric mice as part of the Mouse Animal Production Service at the Centre for Molecular Medicine and Therapeutics, Vancouver, BC, Canada; Dr. Robert S. Molday, University of British Columbia, Vancouver, BC, Canada, for providing us with mouse anti-Rhodopsin (ID4) antibody; and Dr. Dan Goldowitz, University of British Columbia, Vancouver, BC, Canada, for his input during manuscript preparation.
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