Expression of transcription factors divides retinal ganglion cells into distinct classes

2.1 Mouse lines

Most experiments were performed on Isl2-GFP transgenic reporter mice, genotyped by polymerase chain reaction (PCR) for GFP (Triplett et al., 2014) or using GFP-exciting flashlight and detecting filter glasses to visualize expression in newborn pups (P0–P4) (BlueStar model Bls2, Lexington, MA). C57Bl/6 mice (Charles River, Wilmington, MA) were used for experiments that did not require visualization of Isl2. CB2-GFP, DRD4-GFP, and Cdh3-GFP mice were genotyped as described previously (Huberman et al., 2008, 2009; Osterhout et al., 2011).

Both male and female mice were used for these experiments, with no differences noted between sexes. Approximately 50 Isl2-GFP, 10 C57 Bl/6, and 20 mice from GFP-labeled lines were used in these experiments. All procedures were performed in accordance with the UCSC Institutional Animal Care and Use Committee.

2.2 EdU-based cellular birth dating

5-ethynyl-2'deoxyuridine (EdU) is a thymidine analog that integrates into the DNA of dividing cells during replication (Salic & Mitchison, 2008). Pregnant dams were injected intraperitoneally (IP) with EdU (Invitrogen/Thermo Fisher, Waltham, Ma) dissolved in phosphate buffered saline (PBS) (136.9 mM NaCl, 2.7 mM KCl, 10.1 mM Na₂HPO₄, 1.4 mM K₂PO₄) at 50 mg/kg bodyweight. To detect EdU incorporation, we first performed click-chemistry aided staining (Invitrogen/Thermo Fisher, Waltham, Ma) before immunohistochemical detection of other antigens.

2.3 Immunohistochemistry

Postnatal mice were sacrificed and intracardially perfused with ice-cold PBS followed by ice-cold paraformaldehyde (PFA) (pH 7.4, 4% in PBS). Eyes were dissected out and fixed in 4% PFA in PBS for either 30 min at room temperature or overnight at 4 °C. The retinas were isolated, placed in 30% sucrose in PBS overnight, embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA), cryosectioned at 12–25 µm on a CM1520 Cryostat (Leica Microsystems, Buffalo Grove, IL) maintained at −20 to −25 °C, and collected on Superfrost Plus glass slides (Thermo Fisher, Waltham, Ma). In some cases, we first performed click-chemistry aided EdU staining before immunohistochemical detection of antigens (Invitrogen/Thermo Fisher). All immunostained sections were blocked for 1 hr in 5% heat inactivated serum (either horse, goat, or donkey) in PBS + 0.1% Triton-X100. Primary and secondary antibodies were diluted in this blocking medium, and multiple washes were done with PBS. 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO, D8417) was included in a penultimate PBS rinse at a dilution of 1:2,000 to visualize nuclei. The following primary antibodies were used:

2.4 Antibody characterization

2.5 Microscopy

Sections were imaged using a Leica DM5500 B Widefield Microscope, with either a 10×/0.3 or 20×/0.5 objective and Leica filter cubes optimized for DAPI, GFP, Cy3, and Cy5, or on an Olympus BX51 epifluorescent microscope (Olympus, Center Valley, PA) equipped with QImaging Retiga EXi digital camera (QImaging, Surrey, BC, Canada).

2.6 Data analysis

Cells were manually counted with ImageJ. DAPI and lamination patterns were used to restrict analysis to the RGC layer where applicable. GraphPad's Prism software program was used to calculate and graph means, standard deviations and standard errors, following cell counts. For some figures, brightness and contrast were changed in ImageJ to standardize the appearance of cells and enhance figure legibility.

3.1 Postnatal RGCs express either Tbr2, Isl2, and one or both of Satb1 and Satb2 with very little overlap

To identify transcriptional regulators involved in RGC type specification, we performed an immunohistochemistry screen to search for transcription factors that are expressed in subsets of RGCs in the developing or adult mouse retina. We conducted this screen in an Isl2-GFP BAC transgenic line; we have previously characterized and demonstrated that this line faithfully reports endogenous Isl2 expression in RGCs (Triplett et al., 2014). We initially found that the transcription factor Satb2 is expressed in a subset of non-Isl2-GFP RGCs that also coexpress the transcription factor Brn3a in the adult retina (Figure 1a). Brn3a is a transcription factor important for RGC development and is expressed in approximately 80% of RGCs (Badea, Cahill, Ecker, Hattar, & Nathans, 2009; Quina et al., 2005; Sweeney et al., 2014). Remarkably, when immunostaining with an antibody that recognizes Satb2 and its homologue Satb1, we found that Satb1/2-Satb1- and Satb2 expressing RGCs have very little overlap with Isl2-expressing or Tbr2-expressing RGCs in the mature retina (Figure 1b, Table 1). This non-overlapping pattern of expression for Isl2, Satb2, and Tbr2 is already present at P0 (Figure 1c).

Lacking a pan-RGC marker, we were only indirectly able to ask whether each RGC expresses at least one of the three transcription factors. Brn3a is expressed in 80% of adult RGCs but is not expressed in RGCs that express Tbr2, which comprise about 20% of total RGCs (Badea et al., 2009; Quina et al., 2005; Sweeney et al., 2014). In the adult retina, we find that 98% of RGCs that express Brn3a also express either Isl2 or Satb1/2 (Figure 1d; n = 360 Brn3a-expressing cells), and very few Isl2-expressing or Satb1/2-expressing RGCs are Brn3a negative. Therefore, we can divide RGCs into three categories—Brn3a/Satb1/2-expressing, Brn3a/Isl2-expressing, and Tbr2-expressing—and together these categories comprise nearly all RGCs; however, it is possible that a small percentage of RGCs do not express any of these transcription factors.

3.2 Satb1 and Satb2 are expressed in overlapping subsets of ON-OFF DS RGCs

Based on the percentage of RGCs that express Satb1 or Satb2, we hypothesized that these were a collection of types that share common features rather than a single RGC type. To test this idea, we determined the degree of overlap between Satb1 and Satb2 expression and markers of defined RGC types. We find that 79% of Satb1- and Satb2-expressing RGCs also express the ON-OFF DS marker CART, although CART is not a perfect marker of DS RGCs (Kay et al., 2011) and have very little coexpression with non-direction selective RGCs labeled with Smi32 and melanopsin (OPN4, Figure 1e,f).

Consistent with the hypothesis that Satb1 and Satb2 are expressed in ON-OFF DS RGCs, we find that Satb1 and Satb2 are almost always expressed in the GFP-labeled posterior DS RGCs labeled by the DRD4-GFP (>90%) and TRHR-GFP (>90%) transgenic mouse lines (Huberman et al., 2009; Rivlin-Etzion et al., 2011; Figure 2a–d). Satb1 (>90%) is also expressed in the HB9-GFP line that labels upward motion DS RGCs (Trenholm, Johnson, Li, Smith, & Awatramani, 2011) while Satb2 is expressed in only 3% of HB9-GFP DS RGCs (Figure 2e,f). We also find that neither Satb1 or Satb2 are expressed in OFF-transient, CB2-GFP labeled RGCs (Huberman et al., 2008) (<5%), nor in the non-image-forming, Cdh3-GFP RGCs (<5%, Figure 2g–j; Osterhout et al. 2011).

3.3 Isl2, Tbr2, and Satb2 are all expressed in postmitotic RGCs but differ in their onset of expression

Within the retina, the different cell types—RGCs, amacrine cells, bipolar cells, horizontal cells, photoreceptors, and Müller glia—are generated from retinal progenitor cells (RPCs) in successive yet overlapping temporal waves (Cepko, Austin, Yang, Alexiades, & Ezzeddine, 1996). RGCs are among the earliest born; they begin to appear at embryonic day 8 (E8) and are completely generated by E17, with birth rate peaking at E11 (Farah & Easter, 2005; Voinescu, Kay, & Sanes, 2009). To determine the onset of expression of these transcription factors, we examined expression of Isl2, Satb2, and Tbr2 in the mouse retina on different days of retinal development (E12, E14, E16, P0, and P10). In agreement with previous reports (Pak, Hindges, Lim, Pfaff, & O'Leary, 2004), we detected widespread Isl2 in the ganglion cell layer of the retina at the earliest embryonic time points examined (Figures 3aA1–A5), and saw 97% overlap with Brn3a-expressing RGCs at E14 Figure 3b. Isl2 gradually refined into its mature pattern by P0. This refinement correlates with the onset of expression of Satb2 (Figure 3aA6–A10) and Tbr2 (Figure 3aA11–A15), consistent with Mao et al. (2008). Even at E16, Isl2, and Tbr2 mark separate RGC populations (Figure 3c).

In some developmental systems, distinct types of progenitor cells give rise to progeny that share their marker expression profiles. A striking example of this is that of the Cdh6-expressing RPCs, which give rise mainly to Cdh6-expressing RGCs (and other neuronal cell types) in the retina (De la Huerta, Kim, Voinescu, & Sanes, 2012). We wanted to determine whether Isl2, Tbr2, or Satb2 expression is restricted to postmitotic RGCs or if these transcription factors can also be found in proliferative RPCs, which might give rise to a limited repertoire of RGCs. To do this, we labeled dividing cells with a pulse of EdU introduced by IP injection of timed-pregnant dams or neonatal mice, sacrificed the animals 4 hr after EdU injection, and stained the retinas to visualize EdU incorporation and transcription factor expression in retinal sections. Consistent with previous work that showed Isl2 and Tbr2 are not expressed in progenitors (Mao et al., 2008; Pak et al., 2004), we found no time point at which any of the three transcription factors were expressed in EdU labeled cells in the retina (even outside of the ganglion cell layer), or within the neuroblast layer where the RPCs reside. This demonstrates that Isl2, Satb2, and Tbr2 are only expressed postmitotically in RGCs (Figure 4a–c).

3.4 There is no specific temporal order in generating the Isl2/Satb2/Tbr2 RGC classes

Different neuronal cell types are born in successive temporal waves in the mammalian cerebral cortex, spinal cord, and retina (Berry, Rogers, & Eayrs, 1964; Soula et al., 2001) (Livesey & Cepko, 2001). Recently, it was shown that this is also true for some RGC types (Osterhout, El-Danaf, Nguyen, & Huberman, 2014). To test if the RGC classes defined by Isl2, Satb2, and Tbr2 are born with a temporal order, we used EdU to label dividing cells at several embryonic time points from E9 to E15, spanning the window of RGC genesis. We then euthanized the pups at P10 and calculated the percentage of mature RGCs that express Isl2, Satb2, or Tbr2 and terminally differentiated on a particular day (EdU labeled). We found that RGCs born at each time point examined could express each transcription factor (Figure 5a,b). Therefore, despite the fact they are first detectable at different developmental points (Figure 3), we find no evidence that Isl2-, Satb2-, or Tbr2-expressing RGCs are biased to be born on a particular day of RGC genesis. However, we note that at each time point we detected only low percentages of Tbr2-expressing RGCs being born. Since we normalized by the total number of Tbr2-expressing RGCs present in the retina at P10, this result is surprising and may mean that there is a sharp peak of Tbr2-expressing RGC birth that was missed in our time course.

4.1 Isl2, Satb1/2, and Tbr2 subdivide RGCs into three molecular types

Here we have shown that nearly all mature RGCs express one of the transcription factors: Isl2, Tbr2, and Satb1 and/or Satb2. This hints that a precise transcriptional network mediates RGC type identity. Isl2 has been shown to play a role in specifying ipsilateral versus contralateral axonal projections of ventral-temporal RGCs early in development (Pak et al., 2004). In the mature retina, Isl2 is restricted to a population of RGCs with characteristic dendritic stratification in layer S3 of the inner plexiform layer, suggesting that Isl2-expressing RGCs share common functional properties (Triplett et al., 2014). Expression of Isl2 in known types of transient-OFF and OFF-alpha RGCs (marked by CB2-GFP and SMI-32, respectively) combined with lack of expression in posterior DS RGCs (marked by DRD4-GFP) provide evidence that Isl2-expressing RGCs are primarily contrast-sensitive and non-DS (Triplett et al., 2014). Tbr2 is expressed in several types of Brn3a-negative non-image-forming RGCs, including ipRGCs. In Tbr2-null mice, the Tbr2 cells either die or fail to develop; furthermore, the pupillary light reflex, mediated by non-image-forming RGCs, is greatly reduced (Mao et al., 2014; Sweeney et al., 2014).

While Isl2- and Tbr2-expressing RGCs have been previously described, very little is known about the RGC types that express Satb1 and Satb2. One study showed that they are expressed in a subset of Foxp2-expressing RGCs (Rousso et al., 2016), but did not do extensive analysis of the RGC types that express Satb1 and Satb2. Here we show that Satb1 and Satb2 are expressed in the posterior ON-OFF DS RGCs labeled in the DRD4-GFP and THRH-GFP lines, and Satb1 is expressed in the upward ON-OFF DS RGCs labeled in the HB9-GFP mouse line. Satb1 and Satb2 also show a large overlap of expression (79%) in RGCs with the putative ON-OFF DS marker CART. Taken together, these results suggest that Satb1 and Satb2 are expressed in ON-OFF DS RGCs. However, we cannot definitively conclude that Satb1 and/or Satb2 are expressed in all ON-OFF DS RGCs because we do not have a marker of the anterior or dorsally tuned ON-OFF DS RGCs and because some ON-OFF DS RGCs do not express CART (Dhande et al., 2013). Future functional analysis of Satb1- and Satb2-expressing RGCs will need to be performed to confirm our hypothesis.

4.2 No evidence for RGC type-specific progenitors

Isl2, Satb2, and Tbr2 are only found in postmitotic cells (Figure 3), excluding a model in which RPCs express these transcription factors and give rise to class-restricted progeny as shown for Cdh6 expressing RPCs (De la Huerta et al., 2012). Birthdating experiments show that there is no strict temporal order of birth for these three RGC classes, thus discounting a temporal model for generating these three RGC classes. This is in agreement with previous work showing that three non-overlapping RGC types are born in a highly overlapped fashion (De la Huerta et al., 2012). However, another study showed that there are differences in birth timing for three other non-overlapping RGC types (Osterhout et al., 2014), including a comparatively early peak birth date for the RGC types labeled by the Cdh3-GFP mouse line, which make up ∼9% of the Tbr2-expressing RGCs (Sweeney et al., 2014). Thus, some RGC types within the classes that we propose may show a temporal bias in birth date that is absent at the class level.

Taken together, these data suggest that cell identity emerges gradually in postmitotic RGCs. It could be that expression levels of certain genes activate mutual inhibition and/or positive feedback-like transcriptional programs that solidify cell identity. The transcription factors described here—Isl2, Satb1, Satb2, and Tbr2—may be involved directly in such a process. Several pieces of evidence suggest that they play a role in generating RGC identity. First, their expression patterns are non-overlapping and together, they account for nearly all RGCs; this positions them as potential master regulators of RGC identity. Second, the internal division of these three classes into Brn3a-coexpressing (Satb1, Satb2-expressing, or Isl2-expressing) and Brn3a non-expressing (Tbr2-expressing) is suggestive of their potential to influence expression of other transcription factors. Alternatively, it may be that other genes upstream of these transcription factors are necessary for generating the RGC classes that we describe, and that Isl2, Tbr2, Satb1, and Satb2 are simply markers for these classes. In the future, gain and loss of function experiments will explore the role that these genes play in RGC development.

In glaucoma, a common neurodegenerative disease, patients become blind due to RGC death. Cell replacement therapy, that is, injection of RGCs or RPCs into the glaucoma patient's retina, is an attractive mode of treatment currently under study (Riazifar, Jia, Chen, Lynch, & Huang, 2014; Sluch et al., 2015). The success of this approach will depend on our collective ability to faithfully generate the endogenous RGC types, each with its own functional role. In other CNS circuits, reprogramming and transdifferentiation experiments have powerfully shown that transcription factor induction is sufficient to generate specific neuronal types (Pfisterer et al., 2011; Yang, Ng, Pang, Sudhof, & Wernig, 2011). The data presented here comprise one step toward the goal of faithful RGC recapitulation. Future work will examine which, if any, of Isl2, Tbr2, Satb1, and Satb2 are necessary and/or sufficient to drive RGC identity.