The Role of Combinational Coding by Homeodomain and Bhlh Transcription Factors in Retinal Cell Fate Specification

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Developmental Biology 285 (2005) 101 – 115 www.elsevier.com/locate/ydbio

The role of combinational coding by homeodomain and bHLH transcription factors in retinal cell fate specification

J.C.-C. Wang, W.A. Harris*

Department of Anatomy, Downing Site, Cambridge University, CB2 3DY, UK

Received for publication 17 March 2005, revised 26 May 2005, accepted 31 May 2005

Abstract

Two major families of transcription factors (TFs), basic helix–loop–helix (bHLH) and homeodomain (HD), are known to be involved in cell fate identity. Some recent findings suggest that these TFs are used combinatorially to code for cellular determination in the retina. However, neither the extent nor the efficiency of such a combinatorial coding mechanism has been tested. To look systematically for interactions between these two TF types that would address these questions, we used a matrix analysis. We co-expressed each of six retinally expressed bHLH TFs (XNeuroD; XNgnr-1; Xath3; Xath5; Xash1; Xash3) with each of eight retinally expressed HD TFs (XRx1; XOptx2; XSix3; XPax6; XOtx2; XOtx5b; XBH; XChx10) in retinal progenitors of Xenopus laevis using targeted lipofection. The effects of each of these combinations were assayed on the six major cell types in the retina: Retinal ganglion cells (GCs), Amacrines (ACs), Bipolars (BCs), Horizontals (HCs), Photoreceptors (PRs), and Muller cells (MCs), creating 288 result categories. Multiple-way ANOVA indicated that in 14 categories, there were interactions between the two TFs that produced significantly more or less of a particular cell type than either of the components alone. However, even the most effective combinations were incapable of generating more than 65% of any particular cell type. We therefore used the same techniques to misexpress selected combinations of three TFs in retinal progenitors, but found no further enhancements of particular cell fates, indicating that other factors are probably involved in cell type specification. To test whether particular combinations were essential for horizontal fates, we made VP16 and EnR fusion constructs of some of the factors to provide dominant negative transcriptional activities. Our results confirmed that normal activities of certain combinations were sufficient, and that individually these activities were important for this fate. D 2005 Elsevier Inc. All rights reserved.

Keywords: Retina; Cell determination; bHLH; Homeodomain; Combinatorial coding; Xenopus; Development; Transcription factor; Matrix analysis

Introduction combinatorial action of these factors is involved in this process. Before we develop the rationale for such a The vertebrate retina, being composed of few distinct cell combinatorial study, it is worth briefly considering what types organized in layers, is an excellent system to study the these factors have been shown to do individually. question of cell fate acquisition by multipotent progenitors. Pax6 is one of the best known of the HD transcription Two main sets of transcription factors (TFs) are known to factors in the retina. Lipofection studies reveal that Pax6 affect retinal cell fate: homeodomain (HD) and basic helix– does not influence cell fate when overexpressed in the loop–helix (bHLH) proteins, and have been the focus of Xenopus retinal progenitors (Hirsch and Harris, 1997a,b); particular attention (Cepko, 1999; Perron and Harris, however, a conditional knock-out of Pax6 in the peripheral 2000a,b; Vetter and Brown, 2001; Marquardt and Gruss, mammalian retina results in retinal tissue with only non- 2002; Dyer, 2003; Hatakeyama and Kageyama, 2004). The glycinergic amacrine cells, suggesting that Pax6 may have a present study is designed to test the idea that the role in maintaining multipotency of retinal progenitors (Ashery-Padan and Gruss, 2001). Another HD TF, Rx1, * Corresponding author. also appears necessary for the multipotency of retinal E-mail address: [email protected] (W.A. Harris). precursors (Mathers and Jamrich, 2000; Casarosa et al.,

2003; Andreazzoli et al., 2003) and when overexpressed only rods and Chx10 by itself is required for the generation promotes Muller glial fate (Mathers et al., 1997; Kimura et of INL cells. Similarly, Inoue et al. (2002) showed that mice al., 2000). Otx2 is involved in the specification of retinal mutant for both Math3 and NeuroD lack amacrine cells pigment epithelium (Bovolenta et al., 1997; Martinez- though overexpression of either Math3 or NeuroD alone Morales et al., 2001) and is able to promote bipolar cell promoted only rod genesis. However, when either Pax6 or fate in Xenopus (Viczian et al., 2003) as well as rods in Six3 was co-expressed with either Math3 or NeuroD, there rodents (Bobola et al., 1998; Baas et al., 2000; Nishida et was a significant increase in amacrine cells. Such work al., 2003). Otx5b, like Crx, has a role in photoreceptor shows that combinations of bHLH and HD factors are identity and differentiation (Furukawa et al., 1997, 2002; involved in the specification of correct retinal cell type and Chau et al., 2000; Yanagi et al., 2000; Vignali et al., 2000; favor the notion of combinatorial coding. Viczian et al., 2003). Bipolar cell determination is promoted It therefore seemed useful to explore the possibility of by Chx10 (Belecky-Adams et al., 1997; Hatakeyama et al., combinatorial coding of retinal cell specification more 2001; Vetter and Brown, 2001; Toy et al., 2002; De Melo et comprehensively. Thus, we took advantage of the Xenopus al., 2003), as is the determination of horizontal cells by lipofection system (Holt et al., 1990) where it is easy to co- Prox1 (Belecky-Adams et al., 1997; Dyer et al., 2003), and transfect multiple genes into single retinal precursors in an ganglion cells by Bar and Brn3 (Hirsch and Harris, 1997a,b; otherwise normal or wild-type environment. We used this Xiang, 1998; Hutcheson and Vetter, 2001; Liu et al., 2001; strategy to establish a comprehensive matrix analysis of six De Melo et al., 2003). retinally expressed bHLHs by 8 retinally expressed HD For the bHLH transcription factors used in this study, genes. The cell types resulting from each combination was Ash1 seems to favor rod fates (Ahmad, 1995; Tomita et al., compared to the results from same dose of single gene by 1996; Kageyama et al., 1997; Perron and Harris, 2000a,b; itself and to GFP controls using a multiple ANOVA with Hatakeyama et al., 2001). Neurogenin and NeuroD also post hoc significance tests to see whether there was an promote photoreceptor differentiation at the expense of later interaction between the genes being tested. Each of the 48 born cell types (Kageyama et al., 1997; Perron et al., 1999; combinations was analyzed with respect to each of the six Ahmad et al., 1998; Yan and Wang, 1998; Inoue et al., 2002). major cell types in the retina, giving 288 categories to Ath5 acts as a potent determinant for the generation of evaluate. In addition, we re-examined the expression ganglion cells (Brown et al., 1998; 2001; Perron et al., 1998; domains of each transcription factor by in situ hybridization Liu et al., 2001; Schneider et al., 2001; Wang et al., 2001) and to find out if the interactions seen in the matrix analysis had Ath3 seems to be capable of specifying both photoreceptors potential validity in vivo, i.e., whether it is the case that and ganglion cells (Kageyama et al., 1997; Perron et al., transcription factors that interact in these assays are 1998, 1999; Hatakeyama et al., 2001; Inoue et al., 2002). expressed in the same cells during normal development. The idea that these factors may work combinatorially in To try to enhance the effects of combinations that promoted retinal cell fate specification comes from several observa- specific cell types, we misexpressed eight combinations of tions. First, in situ studies indicate that many of these three transcription factors at once. To test the necessity of transcription factors are expressed in more than one retinal some double combinations, we made VP16-activator and cell type and that most are expressed in many, if not all, Engrailed-Repressor fusions of selected transcription fac- multipotent retinal precursors at some stage of development tors. These were used in 8 sets of combinations. Through (Perron et al., 1998, 1999). Second, the overexpression or this analysis, we aimed to shed some light on the strength, knock-out of single transcription factors often affects more extent, sufficiency, and necessity of intrinsic combinatorial than one cell type and changes ratios of cell types rather coding in retinal cell fate specification. than driving all the transfected cells down one particular fate pathway. Moreover, in many cases, it is known that the action of these factors on retinal fates is context-dependent. Materials and methods For instance, Vetter and Brown (2001) showed that the function of Xath5 varied depending upon the time it is Xenopus embryos overexpressed. While early misexpression favors ganglion cell fates, later misexpression promotes the differentiation of The Xenopus laevis embryos used in this study were later-born cell types. Similarly, NeuroD is able to induce from a colony maintained in the Department of Anatomy in amacrine cells or photoreceptors based on when it is University of Cambridge. All embryos were the products of expressed (Morrow et al., 1999). Some recent evidence in vitro fertilizations from wild-type frogs. directly supports the combinatorial specification in the retina. Hatakeyama et al. (2001) observed that Chx10 and In situ hybridization Math3/Mash1 are co-expressed in the inner nuclear layer (INL) of the retina and that expression of both transcription Digoxigenin (DIG)-labeled antisense RNA probes were factors is involved in generating bipolar cells. This is generated from full-length cDNA templates of XNeuroD, particularly interesting as Math3 or Mash1 alone induces XNgnr-1, Xath3, Xath5, Xash1, Xash3, XRx1, XPax6, J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115 103

XSix3, XOptx2, XOtx2, and XOtx5b, using T3 or T7 RNA Lipofection was performed by injecting the DNA mix- polymerase (Promega, USA). In situ hybridization was then tures into the presumptive eye fields of de-jellied Xenopus performed as described (Perron et al., 1998) on 12-Am retinal embryos at stages 16–18. At stage 41, the embryos with clear sections of stage 32 and 41 Xenopus eyes. After staining with GFP signals in their eyes were fixed with 4% PFA (Agar BMPurple (Roche, USA), images were visualized on a Scientific Ltd., USA) at 4-C for 1.5 h, cryoprotected in 30% Nikon microscope and captured by Improvision software. sucrose solution at 4-C for 30 min to an hour, mounted with O.C.T. (Sakura, Netherlands) at À20-C and then sectioned at DNA constructs 10 Am on a cryostat (Leica, Germany). The sections were then rehydrated in 1Â PBS for 5 min, incubated with The basic helix–loop–helix transcription factors 1:20,000 DAPI for 10 min for nuclei staining, and mounted in XNeuroD (Lee et al., 1995), XNgnr-1 (Ma et al., 1996), FluorSave (CalBioChem, Germany) after washing twice for Xath3 (Takebayashi et al., 1997), Xath5 (Kanekar et al., 5 min in 1Â PBS. The sections were then stored at 4-C in the 1997), Xash1 (Ferreiro et al., 1993), and Xash3 (Zimmer- dark until viewed with a Nikon microscope. man et al., 1993), and the homeodomain transcription factors XRx1 (Casarosa et al., 1997), XPax6 (Hirsch and Cell counting Harris, 1997a,b), XSix3 (Zhou et al., 2000), XOptx2 (Zuber et al., 1999), XOtx2 (Viczian et al., 2003), XOtx5b (Vignali Cells misexpressing GFP were counted using fluores- et al., 2000), XBH1 (Poggi et al., 2004), and XChx10 cence microscopy. The identification of different cell types (Viczian AS, unpublished data) were ligated into specific was based upon their laminar positions and morphology sites of pCS2R or pCS2+ so that they could be expressed in (Dorsky et al., 1997). The retinal images were taken using vivo under the CMV promoter. Constructs were amplified in de-convolution in Open Lab software. E. coli and purified with a Qiagen Midi Prep Kit (UK). VP16 and Engrailed-Repressor (EnR) versions of Xash3, Statistical analysis XPax6, and XOptx2 were made. cDNAs for each genes were produced by PCR using specific designed pairs of Statistical analysis was performed using multiple-way primers (Genosys, UK). After running the PCR products on ANOVAwith post hoc tests (SPSS). For each set of sections 1% agarose gel (Duchefa, Netherlands), the cDNA bands injected with the same lipofection mixture, the total number were collected under UV lights and extracted using Gel of GFP-positive cells for every cell type in each retina was Extraction Kit (Qiagen, UK). They were then ligated into counted, and the corresponding mean and standard error of opened VP16-pCS2 or Engrailed-Repressor pCS2 plasmids. mean (SEM) were calculated from the counts of GFP- The positive insertions were amplified in E. coli and positive cells. Comparisons were then made between GFP purified with Qiagen Midi Prep Kit (UK). and each individual transcription factors as well as between each combination of factors, individual factors, and GFP In vivo lipofection alone. P values of <0.05 suggest significant interactions between the combined factors. One needs to be cautious that The pCS2-DNAs of interest were mixed by vortexing the comparisons were performed between lipofection sets prior to use and then mixed with pCS2-GFP, Green from the same experiment to avoid possible variances such Fluorescent Protein, for cell labeling and DOTAP (Roche, as the different sensitivity of different clutches of Xenopus USA). The ratio of DOTAP:total plasmid-DNA is 3 Alto1 embryos. At least 3 different sets of experiments were carried Ag as described (Holt et al., 1990). For single transcription out for each combinational matrix and each experiment factor lipofection, the ratio between GFP and single tran- consisted of no less than 35 Xenopus retinas. For each scription factor was 2 to 1. Every 9 Al of DOTAP would lipofection, the number of GFP-positive cells per retina contain 2 Ag of pCS2-GFP and 1 Ag of transcription factor ranged from 20 in the least transfected retinas to up to more inside. For double combinatorial experiments (GFP, tran- than 300 in heavily transfected retinas. For the VP16 and scription factor 1, transcription factor 2), the DNA ratio was EnR fusion experiments, we used an unpaired t test to test 1:1:1, which made 1 Ag of each factor (3 Ag of total DNA) each of the particular combinations against GFP control data. in 9 Al of DOTAP. Therefore, same amount of each transcription factor was lipofected into the presumptive eye field to provide a reliable comparison between what the Results factor does by itself versus what it does in combination with another factor. Further triple combinatorial experiments The expression of bHLH and homeodomain transcription (GFP:transcription factor 1:transcription factor 2:transcrip- factors in the Xenopus retina is compatible with tion 3) formed a new set of experiments and a DNA ratio of combinatorial coding 1:1:1:1 was used by diluting out GFP further, which implied 1 Ag each type of DNA in 12 Al of DOTAP to maintain 1 Ag The retinal expression patterns of the eight homeodomain total DNA per 3 Al DOTAP ratio. (XRx1; XOptx2; XSix3; XPax6; XOtx2; XOtx5b; XChx10; 104 J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115

XBH) and six bHLH (XNeuroD; XNgnr-1; Xath3; Xath5; these factors is also expressed in distinctive laminar patterns Xash1; Xash3) transcription factors in Xenopus embryos are of the stage 41 central retina. XSix3, XPax6, and XBH are shown in Figs. 1A and B, respectively. We examined stage all observed in GCL and INL, with XPax6 and XBH among 32, a time point when cells are being generated in the central these being restricted just to the inner part of the INL retina, and stage 41, when almost all cells in the central (Perron et al., 1998; Ghanbari et al., 2001; Poggi et al., retina are postmitotic. 2004). XOptx2 is also weakly expressed in the INL (Zuber All of the homeodomain genes, except XChx10, are et al., 1999). Both XOtx2 (Viczian et al., 2003) and XChx10 highly expressed in the neural retina of stage 32 Xenopus are expressed in outer part of the INL. Finally, XRx1 and embryos. At stage 41, all, including XChx10, are expressed XOtx5b were expressed in the ONL and outer INL (Perron in the CMZ where there are multipotent progenitors, with et al., 1998; Viczian et al., 2003). XRx1 and XSix3 expression extending to the most At stage 32, all six bHLH factors were expressed in the peripheral part of CMZ, the home of retinal stem cells neural retina with a central-to-peripheral gradient. As with (Perron et al., 1998). Interestingly for this study, each of the HD genes at stage 41, all of the bHLH transcription

Fig. 1. In situ hybridizations for HD and bHLH TFs at stage 32 (upper panel) and stage 41 (lower panel). (A) For homeodomain transcription factors, all are expressed in neural retina at stage 32 except XChx10. XSix3, XOptx2, and XPax6 are also expressed in the lens. By stage 41, all HD TFs are expressed in the CMZ but only XRx1 and XSix3 extend to the most peripheral region. Distinctive expression patterns are also observed in the retinal layers: ONL and outer INL for XRx1 and XOtx5b; outer INL for XOtx2 and XChx10; GCL and inner INL for XOptx2, XPax6, and XBH; GCL and INL for XSix3. (B) For bHLH transcription factors, all are expressed in the neural retina by stage 32 and localized in the CMZ at stage 41. XNeuroD is the only bHLH gene whose expression is maintained at stage 41. bHLH = basic helix–loop–helix; HD = homeodomain; ONL = outer nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer; TF = transcription factor. J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115 105 factors are expressed in the CMZ, but none is expressed in amacrine cells was found with XBH1 (Poggi et al., 2004). the most peripheral region (Perron et al., 1998; Poggi et al., XRx1 was observed to increase Muller cells as shown 2004). In striking contrast to the retina homeodomain previously (Furukawa et al., 2000). Among all of the transcription factors, only one of the bHLH factors is homeodomain transcription factors examined, XSix3, expressed in a laminar pattern in the central retina, and that XOptx2 (also known as Six6), and XPax6 gave no bias to was XNeuroD in the ONL (Perron et al., 1998). any particular retinal cell types when overexpressed, again These results are basically consistent with previous in line with what has been previously described (Hirsch and studies (Hirsch and Harris, 1997a,b; Perron et al., 1998; Harris, 1997a,b; Zuber et al., 1999). Vignali et al., 2000; Viczian et al., 2003; Poggi et al., 2004). The results of single bHLH misexpression are shown in The fact that some of these transcription factors, especially Fig. 2B. XNeuroD, XNgnr-1, and Xath3 increased photo- the homeodomain ones, are expressed in distinct laminar receptors as observed previously (Yan and Wang, 1998, patterns in the central retina is clearly consistent with the 2004; Morrow et al., 1999; Perron et al., 1999). XNeuroD notion of combinatorial coding shown below. The fact that also increased amacrine cells by itself in accordance with all of these factors are expressed in the CMZ suggests that Morrow et al. (1999). Xath3, Xath5, Xash1, and Xash3 all most cells in this region co-express several, and perhaps all promoted ganglion cell fate (for similar results, see Kanekar of these factors. In fact, previous studies using a double in et al., 1997; Brown et al., 1998; Perron et al., 1999; Wang et situ protocol showed that in the regions of the CMZ where al., 2001; Ohnuma et al., 2002; Yang et al., 2003; Poggi et various homeodomain and bHLH transcription factors are al., 2004). Bipolar cells were decreased with all bHLH expressed, most cells do express most of them at the same factors as was shown earlier (Perron et al., 1999; Morrow et time (Perron et al., 1998). The relative ratios of these factors al., 1999; Ohnuma et al., 2002). Finally, no effects on may clearly be different in different progenitor cells such horizontal and Muller cell numbers were observed with any that particular combinations are more strongly expressed in of the bHLH transcription factors. some of these cells than in others, but the information from These results are all basically consistent with those the RNA localization of each transcription factor provides reported in previous work except that decrease in Muller high possibilities that bHLH and homeodomain factors cells by bHLH factors seen in earlier studies (Takebayashi et could co-exist in the same retinal progenitors and work to- al., 1997; Morrow et al., 1999; Cai et al., 2000; Tomita et gether towards particular cell fate later in the development. al., 2000; Hatakeyama et al., 2001; Sun et al., 2001) was not seen here. The effects of overexpression of single bHLH and homeodomain transcription factors in retinal cell Combinatorial effects of bHLH and homeodomain determination transcription factors

We first repeated and extended previous studies on the These results gave us the confidence and background effects of overexpressing individual transcription factors on data to enable us to launch a large second set of experi- cellular determination in the Xenopus retina. It was ments, in which all 48 possible pair-wise combinations of important to confirm that the factors we are working with the six bHLH and eight HD cDNAs were co-lipofected with here have the activities expected from previous studies from GFP cDNA into the presumptive eye region in stages 16–18 various labs. It was also important to compare the effects of Xenopus embryos. A multiple-way ANOVA was used to each of these factors with each other in the same sets of compare the ratios of each of the six major retinal cell types embryos, using the same protocols and the same counting resulting from the combinatorial action of the two TFs with and statistical methods if we are to appreciate how the the results from retinas lipofected with the cDNAs of each effects of the combined action of these factors differ from of the constituent TFs alone, and with the results from GFP those of each of the components. In this initial set of alone. These comparisons were run with every combination experiments, we compared the effects of each TF to that of in order to control for variability between embryo batches GFP alone. and to assure that each transcription factor had the same Fig. 2A shows the results with all the homeodomain effects as seen above. Thus, each of the 48 experiments was transcription factors. None caused significant changes in composed of four sets of lipofected embryos: Homeodo- horizontal cells. The percentage of photoreceptors in the main + bHLH + GFP, Homeodomain + GFP, bHLH + GFP, retina was increased with XOtx5b overexpression but and GFP alone. decreased with XOtx2. The opposite effects of these two The results from the 288 categories analyzed (the effects factors were seen in bipolar cells. This confirms the work of of all 48 combinations on each of the 6 retinal cell Viczian et al. (2003). XChx10 promotes bipolar cell fate but types) can be subdivided as shown in Fig. 3 and described not as strongly as previously seen (Hatakeyama et al., 2001; below: Ohtoshi et al., 2004). The predicted reduced production of both photoreceptors and bipolar cells accompanied by an 1. The majority of combinations (214 out of 288) showed increase in ganglion cells and a slight augmentation of no effect on any particular cell types. 106 J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115

Fig. 2. Effects of individual homeodomain (HD) and basic helix–loop–helix (bHLH) transcription factors on retinal cell fate. (A) Among all HD transcription factors, XPax6, XSix3, and XOptx2 have no effects on cell fate by themselves. Photoreceptor (PR) fate is promoted by XOtx5b and inhibited by XOtx2 and XBH. Bipolar cell (BP) fate is increased by XOtx2 and reduced by all except those with no particular effects on determining retinal cell fate. Muller cell (MC) fate is enhanced by XRx1 while XBH promotes both ganglion (GC) and amacrine cell (AC) fates. No obvious changes are observed in horizontal cells (HC). (B) In the case of bHLH transcription factors, all reduce BC generation. PR fate is increased by XNeuroD, XNgnr-1, and Xath3, while GC fate is promoted by all except XNeuroD and XNgnr-1. No effects of these factors are found on HC and MC fates, and the production of AC fate is enhanced only with XNeuroD. All the comparisons are made against GFP. Asterisks indicate that a particular transcription factor can significantly increase or reduce the production of certain cell types. One asterisk indicates 0.01 < P < 0.05; two asterisks indicate 0.001 < P < 0.01; and three indicate P value less than 0.001.

2. Thirty-seven (37) combinations gave the same results on 6. Four (4) combinations produced significantly more of a particular cell types as did the bHLH component alone. given cell type than did the bHLH component alone and 3. Nineteen (19) combinations gave the same results on significantly less than the homeodomain component particular cell types as did the homeodomain component alone. alone. 4. Ten (10) combinations produced significantly more of In terms of combinatorial coding, the vast majority of the a particular cell type than either of the components alone. cases, 274 out of 288 (approximately 96%), those constitut- 5. Four (4) combinations produced significantly less of a ing of categories 1, 2, 3, and 6, seemed relatively particular cell type than either of components alone. uninteresting: the combinations either completely ineffec- J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115 107

cell types in comparison to each other. To our mind, more interesting patterns emerge from this analysis than from the above analysis. Fig. 5 showed the effects of different transcription factors in detail in retinal cell fate specification compared to GFP controls. Symbols ‘‘+’’ and ‘‘À’’ represented increase and decrease, respectively. A single symbol, e.g., ‘‘+’’and a ‘‘À’’, indicated that the effects were significant with a P value between 0.01 and 0.05; ‘‘++’’ and ‘‘ÀÀ’’ for 0.001 < P < 0.01; ‘‘+++’’ and ‘‘ÀÀÀ’’ for P < 0.001. The empty spaces (both white and gray boxes) indicated that no significant effects were observed.

Photoreceptors Fig. 3. Pie chart showing the proportion of combinatorial effects of each Combinations consisting of XNeuroD, XNgnr-1, Xath3, specific functional category when compared to its corresponding individual and XOtx5b are particularly powerful enhancers of photo- controls. Multiple-way ANOVA followed by post hoc statistical tests was receptor fates. While XOtx5b has strong activity in used to identify which cases might be assigned to each category, using a increasing photoreceptors by itself, its effect is further criterion of P < 0.05 to indicate significance. strengthened in combination with the other three factors, which themselves also promotes this cell fate. Interestingly, tive on a particular cell type, or the effect was no different the positive effects of XNeuroD and XNgnr-1 on photo- than one of the two components, or the effect was receptor fate are diminished when they are co-expressed intermediate, an averaging of the two phenotypes. The with either XBH, a strong ganglion cell-promoting factor, remaining 14 (4%) results were more interesting and or XChx10, which may be more involved in bipolar cell represented possible combinatorial coding in retinal cell fate. The interference on photoreceptor fate driven by fate specification. These were the combinations that NeuroD and XNgnr-1 might be either due to suppressive produced increases or decreases beyond what was expected effects of these homeodomain factors or to a more direct from the individual components. These 4% could be further effect these factors may have in promoting other fates. broken down into those that showed enhanced effects in the Combinations involving Xash1, XRx1, XSix3, XOptx2, same direction as both of the two components, those that and XPax6 are all capable of inducing photoreceptors in showed enhanced effects in the same direction as one of the various degrees. As none of the five factors mentioned have components, and those that affected cell types that were known effects on this cell type, these results suggest that unaffected by either of the components. there may be interactions between these bHLH and The first category contained Xath3, Xath5, or Xash3 + homeodomain proteins. XBH, which all increased ganglion cells by itself and A major reduction in photoreceptors is observed in the promoted further production of this cell type when co- XBH + Xath5 combination. Since both factors by themselves expressed. For example, Xath5 + XBH produced more strongly promote ganglion cells at the expense of photo- ganglion cells at the expense of photoreceptors and bipolar receptors, the further reduction in this cell type could simply cells than either factor alone (Fig. 4A). These results be a consequence of their combined individual functions. supported those of Poggi et al. (2004). The second category included Xath3 or Xath5 + Rx1. Fig. 4B showed that Xath3 Bipolar cells by itself decreased bipolar cells while XRx1 had no effect Bipolar cells present a very different picture. Almost all on this cell type. However, a further reduction in bipolar combinations, we found, cause an overall decrease of this cells was found when the two factors were co-lipofected. cell type. The exceptions consist of combinations involving Finally, all the other combinations among the interesting XChx10. It may be that bipolar cells are highly specified results were in the third group, the combinations that had and very sensitive to particular levels of various tran- functions different from either of the components. Repre- scription factors. Thus, this fate is compromised by the sentative of this class was Xash3 + XOptx2 (Fig. 4C). While overexpression of almost any of these transcription factors. Xash3 promoted ganglion cell fate and XOptx2 had no bias Moreover, even though both XOtx2 and XChx10 have the on retinal cell fate decision, the combination of these two potential to promote this cell fate by themselves, they factors caused an increase in horizontal cells. function differently in the context of bHLH factors. All the bHLH factors tested decrease bipolar cells, and XChx10, Breakdown of combinatorial function by cell type but not XOtx2, is able to rescue this cell population in combination with most of the proneural genes. The results It is informative to contextualize the combinatorial results suggest that XChx10 and XOtx2 play different roles in of this paper on a cell type by cell type basis, by comparing bipolar cell development and that a unique cellular context how effective all the combinations are at promoting specific may be required for each. 108 J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115

Fig. 4. Fluorescent images taken from stage 41 Xenopus retinas, each representing an example of one of the three major interesting results. (A) This combination shows additive effects compared to either of its components alone. (B) This combination gives stronger effect of one component but not the other. (C) This combination increases a retinal cell type that neither of the factors alone affects. Particular cell types were determined from their shapes and their laminar positions in the retina, ONL (outer nuclear layer), INL (inner nuclear layer), and GCL (ganglion cell layer). The bar graph next to each category panel shows how significant the difference is with the presence of asterisks. Asterisks represent significance levels as in Fig. 2.

Ganglion cells or XBH. For Xath5 and XBH, since each of these is a A strong increase of GCs is observed in almost all strong promoting agent for GCs, the combinatorial results combinations where one of the partners is Xath5, XPax6, may simply reflect the individual function of these J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115 109

Fig. 5. Matrices of combinatorial significance by cell types. This figure consists of 6 matrices, each associated with a specific cell type, photoreceptor (PR), bipolar cells (BC), ganglion cell (GC), horizontal cells (HC), amacrine cells (AC), and Muller cells (MC). Red cells in each matrix indicate a significant increase in this cell type compared against the relative GFP controls, whereas blue cells indicate a decrease. The +, ++, +++ and À, ÀÀ, ÀÀÀ symbols, along with the cells increasing color saturation of the tables’ cells indicate increasing levels of significance [+/À for 0.01 < P < 0.05 (pale); ++/ÀÀ for 0.001 < P < 0.01 (medium); +++/ÀÀÀ for P < 0.001 (saturated)]. White (individual transcription factors compared to GFP) and gray boxes (combinations compared to GFP) represent that no significant difference were observed after statistical tests. transcription factors, which are also dominant to the effects Xath3 is another ganglion-promoting transcription factor. of any partners with which they were paired. XPax6, on In contrast to Xath5, none of homeodomain TFs, except the other hand, does not influence any retinal cell types by XBH, affects GC fate when combined with Xath3. The itself (Hirsch and Harris, 1997a,b). However, its expression difference between Xath5 and Xath3 may be related to the in both retinal progenitors and mature ganglion cells (see dual roles of Xath3 in promoting both photoreceptor and Fig. 1) suggests that XPax6’s influence on GC identity ganglion cells versus the more restricted role of Xath5 in requires bHLHs. promoting GCs. 110 J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115

Interestingly, all bHLH genes, even Xath5, when result of this triple lipofection was compared to each of its combined with XChx10, do not affect GC percentages. component doubles: Xath5 + XBH, Xath5 + Xash3, and This might be due to a key function of XChx10 in bipolar Xash3 + XBH. cells (see above) or a role of XChx10 in promoting cell A total of 8 triple combinations were examined. For proliferation and delaying cell cycle exit (Burmeister et al., photoreceptors, we tried three triple combinations using the 1996). following genes: XNeuroD, Xath3, XNgnr-1, and XOtx5b. For ganglion cells, we also tried four triples: Xath5 + XBH Horizontal cells plus either Xath3, Xash1, Xash3, or XPax6. For horizontal Very few combinations seem to influence the generation cells, we tried Xash1 + XPax6 + XOptx2. Unfortunately, of horizontal cells. Only the combinations of Xash1 or none of these experiments showed any further enhancement Xash3 plus either XPax6 or XOptx2 affects horizontal cell of the respective retinal cell type. Instead, we observed that numbers. Surprisingly, none of these transcription factors the double transfection of XNeuroD + XOtx5b gave most take part in this horizontal cell determination by themselves. photoreceptors, Xath5 + XBH gave the most ganglion cells, and that Xash1 + XPax6 or XOptx2 gave the most Amacrine cells horizontal cells. These results indicate that misexpressing Even fewer combinations affect the determination of additional bHLH and HD transcription factors does not amacrine cells. Only Xath3 with XRx1 causes an increase in necessarily strengthen the intrinsic programs for retinal cell this cell type. Again, similarly to horizontals, neither of determination, and suggest that these transcription factors these factors on its own affected amacrine cell fate. In mice, by themselves are not sufficient to determine retinal cell amacrine cells increase when NeuroD is combined with type. Extrinsic cues and cell cycle regulators might work in Six3 or Pax6 (Inoue et al., 2002). However, there was no conjunction with these factors to specify vertebrate retinal obvious alteration in amacrine cells in combinations with fate (Ohnuma et al., 2002). XNeuroD, although XNeuroD is known to participate in amacrine cell fate by itself. Are Xash3, XPax6, and XOptx2 necessary for the determination of horizontal cell fate? Muller cells No dramatic changes are detected in Muller cells, the Overexpression studies like the ones above show that late-born intrinsic glial cells of the retina, in most combinatorial coding of TFs can be sufficient to push combinations. However, a decrease in this cell type is misexpressing cells towards particular fates. To test whether observed when Xash3 + XPax6 or XOptx2 or XBH are such combinations are essential for these fates, it is overexpressed. Interestingly, most combinations involving necessary to inhibit the function of these TFs. To test this, XChx10 also decrease Muller cells. This effect may reflect a we made VP16-Activator and Engrailed-Repressor (EnR) positive influence of XChx10 on the decision of late fusion constructs of Xash3, Pax6, and XOptx2. Previous progenitors to become bipolar rather than Muller cells. studies suggest that Xash3 and XPax6 acts as activators. Although our earlier results with single lipofections did not Therefore, the VP16 fusion constructs with these genes show that bHLH transcription factors reduced Muller cells should mimic the normal activity and the EnR fusion significantly on their own, we note in this table that most constructs should have a dominant negative or anti-morphic bHLH genes in combination with XRx1 inhibited the effect. XOptx2 functions as a repressor during retina function of this homeodomain protein in increasing this development (Zuber et al., 1999). In this case, the VP16 cell type. fusion should be anti-morphic while the EnR fusion should mimic the native function. The combinations of Xash3+ Combinatorial effects of three transcription factors XPax6 and Xash3+XOptx2 promote horizontal cell fate when overexpressed (Fig. 4C). Very much in accord with Our results showed that some of combinations of bHLH the identified transcriptional activities of these genes and and HD TFs interacted to enhance particular fates but that our previous combinatorial effects, we found an increase of no combination was sufficient to instruct more that 65% of horizontal cells when VP16-Xash3 was co-lipofected with misexpressing progenitors to undertake any particular cell either VP16-XPax6 or EnR-XOptx2 (Fig. 6); these combi- fates. This suggested to us that more factors might be nations mimic the activity of the native TFs when overex- involved. We therefore asked if the choice of particular fates pressed in combination. The other 6 fusion construct could be further enhanced when three rather than two combinations, however, failed to generate a significant transcription factors were simultaneously misexpressed. Our increase of HCs indicating that the increased production of strategy was to use the above results to devise mixtures of this cell type requires both factors to be transcriptionally TFs that all have similar effects on retinal cell fate decisions. functional. There was, in fact, a significant reduction in For example, Xath5 + XBH strongly promotes ganglion cell horizontal cells with some of these other combinations in determination and Xash3 also favors the production of which one or other TF was acting in a dominant negative ganglion cells, so we tested Xath5 + XBH + Xash3. The way, suggesting that the individual transcriptional activities J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115 111

Fig. 6. Anti-morphic combinations of Xash3 with either XPax6 or XOPtx2 testing whether these combinations are both sufficient and necessary for the production of horizontal cells. (A) Structures of pCS2-VP16 and pCS2-Engrailed cDNA-containing plasmids, which were used in the study. (B) A total of8 combinations were performed to test the effect of the transcriptional activities of Xash3 + XPas6 or XOptx2 on horizontal cell fate. VP16-Xash3 + VP16- XPax6 and VP16-Xash3 + EnR-XOptx2 were able to increase the proportion of this cell type compared to GFP controls. In contrast, the other 6 combinations either gave no significant proportional change in horizontal cells compared to GFP controls or actually decreased the percentage of horizontal cells, suggesting that the normal transcriptional activities of individual factors are necessary for the combinatorial effects. Significance levels of asterisks are as in previous figures, though here, an unpaired t test was used to compare each combination against the GFP control data. of some of these factors were important for the normal kind, we chose to overexpress all possible combinations of production of horizontal cells. a set of bHLH and homeodomain transcription factors, which have previously been shown to be expressed in the developing retina. Our results reveal new combinations of Discussion bHLH and homeodomain transcription factors that influence the fate of particular cell types through unidentified Extensive studies on the roles of single transcription mechanisms in the vertebrate retina, but they also factors in retinal progenitors have led to the realization demonstrate that this particular coding system is neither that, in many instances, they influence but do not by extensive nor efficient in determining retinal cell fates. The themselves determine cell fate. It therefore seems likely molecular coding of retinal cell fate is probably much that the multitude of different retinal types arise by a more more complex. complex molecular process than a ‘‘single transcription Before we discuss our results on pairs of transcription factor–single cell fate’’ mechanism. There is accumulating factors, it is worth considering the effects of our results from evidence that combinations of bHLH and homeodomain single lipofections. Most of these results were consistent transcription factors have synergistic roles in the gener- with previous observations in Xenopus. For example, Xath5 ation of particular types of retinal cells (Hatakeyama et al., promotes GCs (Kanekar et al., 1997; Poggi et al., 2004); 2001; Inoue et al., 2002). In an attempt to understand the Xath3 promotes both PRs and GCs (Perron et al., 1999); extent and effectiveness of a combinatorial code of this XNgnr-1 promotes PRs (Perron et al., 1999); XNeuroD 112 J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115 promotes PRs and amacrines (Kanekar et al., 1997); XBH al., 1998; Perron et al., 1999; Cai et al., 2000; Sun et al., promotes GCs (Poggi et al., 2004); XOtx2 promotes bipolar 2001; Inoue et al., 2002). One possibility is that, in the cells (Viczian et al., 2003); and XOtx5b promotes PRs absence of a marker, we may have occasionally counted (Viczian et al., 2003). These results not only gave us Muller glia as bipolar cells as they share many morpho- confidence for exploring the combined effects of two factors logical attributes. This might also help explain why we together, but also provided some new findings. failed to see a significant increase of bipolar cells when Our single lipofections provide new information on the overexpressing XChx10. effects of known transcription factors, such as Xash1 and Our results on combinatorial coding must first be put Xash3, which had not yet been tested in this assay. into context of other studies that show clear interactions Overexpression of either Xash1 or Xash3 led to an increase between bHLH and homeodomain transcription factors on in GCs. This is supported by the work of Kanekar et al. cellular determination in the retina. Xath5 and XNeuroD (1997), although a technical error in that study precluded the work with Brn3 in promoting ganglion cell differentiation direct demonstration that Xash3 activated XNeuroD which and survival (Hutcheson and Vetter, 2001), while the co- is upstream of Xath5 (see erratum, Kanekar et al., 1998). expression of Xath5 and XBH enhances ganglion cell Interestingly, Matter-Sadzinski et al. (2001) provided production more than either factor alone (Poggi et al., evidence that Cash1 antagonizes the activation of the Cath5 2004). Rx1, Pax6, and Six3 also work with various bHLH promoter. In situ hybridization reveals that Cash1 expres- in supporting context-dependent cell determination (Brown sion starts at stage 24 in the chick retina (Jasoni et al., 1994), et al., 1998; Ashery-Padan and Gruss, 2001; Tessmar et while Xash1 expression in the Xenopus retina is much al., 2002; Andreazzoli et al., 2003). The case for earlier (Perron et al., 1998). In mice, it has been suggested considering combinatorial coding as an important feature that Mash1 is involved in opsin expression in photo- of retinal cell determination was made most dramatically receptors (Ahmad, 1995; Ahmad et al., 1998), but it has by Inoue et al. (2002) who showed an increased also been shown to promote bipolar cell determination in a production of amacrine cells when Math3 or NeuroD are context-dependent manner (Hatakeyama et al., 2001). The co-expressed with Pax6 or Six3, while the generation of variation in observations across different animal models horizontal cells requires the combination of Math3 or suggests that the function of these Ash type transcription NeuroD with Pax6. This is particularly interesting because factors, at least with respect to which cell type in the retina the misexpression of Pax6 or Six3 alone does not influence they promote, might be poorly conserved evolutionarily. retinal fates, and either Math3 or NeuroD alone only This poor conservation may have less to do with the promotes rod fate (supporting the previous finding that structure of the bHLH transcription factors than it does with NeuroD interacts with Crx to promote photoreceptor fates; the temporal expression, i.e., cross-species differences may Furukawa et al., 1997). Another study from the same be of a heterochronic rather than molecular nature. This is laboratory showed that Chx10 and Math3 or Mash1 suggested by the fact that in Drosophila, bHLH factors together are involved in bipolar cell generation though appear to have redundant functions (Jimenez and Campos- neither factor alone influences this cell type (Hatakeyama Ortega, 1987; Hinz et al., 1994), and in vertebrates have et al., 2001). One naturally wonders if these are examples stage-dependent effects on the promotion of different cells of a more extensive but comprehensible combinatorial types in the retina (Moore et al., 2002). code or if they are scattered individual instances of It is also important to mention the few cases where we functional interactions between such factors in cell fate found different results with single factor overexpression regulation. It was with this question in mind that we tested than previously published. XRx1 is one example. We found a complete matrix of eight retinal homeodomain and six a small but significant increase in Muller glia in contrast to bHLH genes on each of the six major retinal cell types. Casarosa et al. (2003), who found that this factor promotes Among the 288 sets of results, most (214) are progenitor cell proliferation without affecting cell fate uninteresting in the sense that no qualitative or quantitative choices. Our results are more consistent with an increase effects are observed at all. The 56 results that showed in Muller cells observed by overexpression of XRx1 effects different from one factor but not the other are also homologues in the mouse and chick (Fisher and Reh, not particularly interesting. Such results may simply mean 2001, 2003; Furukawa et al., 2002). The differences that either these factors do not function together or other between our results and those of Casarosa et al. (2003) players, not contained in the lipofection mixture, are may be purely a statistical artifact. In this study, we were required for an effective interaction. 18 of the total results particularly interested in minor differences among all cell are clearly more interesting in terms of the combinatorial types, whereas the previous studies were more on the hypothesis—i.e., the combination does something that lookout for dramatic effects on single cell type. We may, neither factor alone can do. Some of these, however, are therefore, have flagged minor differences unnoticed in these predictable, such as when the phenotype is intermediate previous studies. Perhaps it is surprising then, that we failed between the two factors (such as Xath3 + XChx10 for to find a reduction of Muller cells by most bHLHs as several bipolar cells). The most interesting cases were the others have previously seen (Tomita et al., 1996; Brown et combinations that produce a phenotype that is in the same J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115 113 direction but stronger than either factor alone (such as same parent plasmids, the results should be comparable Xath5 + XBH for GCs) (Poggi et al., 2004), or the cases across the matrix and can be assumed to reflect what of truly unexpected effects, for instance, the production of happens when retinal progenitors express these two tran- extra horizontal cells by the combination of Xash1 + scription factors at approximately equal levels. However, XPax6. Some, but not all of these results, are consistent we were unable to monitor the actual concentrations of the with previous studies. We failed, for example, to observe transcription factors in the lipofected cells and we did not the result obtained in mice that combinations of XNeuroD test the possibility that specific but unequal rather than or Xath3 with XSix3 or XPax6 lead to an increase in equal proportions of transcription factors are required to amacrine and horizontal cells (Inoue et al., 2002) and that direct cell fate efficiently. Another problem with an the combination of Xash1 or Xath3 and XChx10 promotes overexpression study as opposed to a knock-out or a bipolar cell fate (Hatakeyama et al., 2001), although we knock-down study is that we cannot judge the necessity of did find that XChx10 rescues the reduction of bipolar fates particular combinations of factors in retinal cell determi- caused by several bHLH factors. These differences in the nation. However, it would be a huge and expensive results could be due to non-conservation of the combina- enterprise to do a similar matrix of knock-outs using torial code between species, the types of constructs used, mice, and in mice, and even in Xenopus or zebrafish where the overexpression techniques involved, and, of course, the morpholino technology is possible; it would be necessary stage of misexpression. It is important to note that the to find ways to make conditional knock-outs as earlier loss cases here of combinatorial effects, although statistically of many of these genes interferes with optic vesicle significant, are uniformly not very dramatic. Our most development, which in turn prevents cell type analysis. efficient combination increased the GCs from 25% to 65%. Here, we used activation (VP16) and repression (EnR) Other combinatorial effects were clearly weaker. The fusion proteins to provide anti-morphic or dominant previous reports of such combinatorial effects (Hata- negative data on the necessity of the transcriptional keyama et al., 2001; Inoue et al., 2002; Poggi et al., function of these proteins in horizontal cell determination. 2004) have also shown that particular combinations do not The results suggested that the normal transcriptional lead all the misexpressing cells to the same fate. Rather, all activity of two identified combinations is sufficient for the studies to date, including this one, seem to agree that horizontal cell fate, and that the normal activity of such combinations alter fate probability distribution rather individual components of the combination was necessary than explicitly coding for one fate versus another. for this fate. Perhaps the way that bHLH and homeodomain tran- While this study demonstrates some possibly intrinsic scription factors work together is better illustrated in our combinatorial coding, it provides no clear evidence analysis of how significantly the factors on their own or in concerning the mechanisms whereby the two transcription combination affect the normal distribution of retinal cell factors work together. One possibility is that there is direct types. Our results suggest that where each of the factors protein–protein interaction resulting in conformational has a positive affect on a particular cell type, it is likely changes and alternation in DNA binding specificity that the combination of the two factors will produce a (Brown et al., 1998; Ashery-Padan and Gruss, 2001; more significant result in the same direction. This type of Hutcheson and Vetter, 2001; Tessmar et al., 2002; analysis also shows that both photoreceptors and GCs are Andreazzoli et al., 2003). Alternatively, our results using largely positively regulated while bipolar and Muller cells VP16 and EnR fusion constructs in some combinations are largely negatively regulated by these factors either suggest that the TFs function as individual transcription alone or in combination. The fact that RGCs and photo- factors. If so, the profile of gene expression resulting from receptors are early cell types and Muller cells and bipolar the activity of both factors would be necessary to bias cells are late cell types might be significant in this respect, RPCs towards specific cell fates. Much more work is especially as the bHLH factors have stage-dependent required to resolve this problem, though our systematic effects (Moore et al., 2003). If the entire experiment were matrix study provides as a good starting point. repeated with later transfections, the results might have Our basic conclusion is an extension of that reached been different. Very few combinations affected the about single transcription factors, i.e., that combinations of percentages of horizontal and amacrine cells, cell types one bHLH and one HD transcription factor might not be born in the middle of retinogenesis. Perhaps these cell enough to determine distinct cell types in the vertebrate types require more specific combinations of factors to retina; just as there is no ‘‘one transcription factor–one cell influence their fates. type’’ rule, neither is there a simple bHLH/HD combina- An overexpression study of this type has the advantage torial code that specifies particular cell types. The few of allowing us to screen the matrix efficiently, and to judge triple lipofections we tried did not show any further if certain combinations are sufficient to promote specific enhancements. Our results suggest that we have been cell fates intrinsically in an otherwise normal in vivo thinking too simplistically and that if a transcriptional code environment. Moreover, by using the same amount of each exists, then several additional factors are likely to be DNA in the total mixture and the same promoters on the involved and it will be much more complicated. 114 J.C.-C. Wang, W.A. Harris / Developmental Biology 285 (2005) 101–115

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