Prospero and Pax2 combinatorially control neural cell fate decisions by modulating Ras- and Notch-dependent signaling
© Charlton-Perkins et al; licensee BioMed Central Ltd. 2011
Received: 27 October 2010
Accepted: 3 May 2011
Published: 3 May 2011
The concept of an equivalence group, a cluster of cells with equal potential to adopt the same specific fate, has served as a useful paradigm to understand neural cell type specification. In the Drosophila eye, a set of five cells, called the 'R7 equivalence group', generates a single photoreceptor neuron and four lens-secreting epithelial cells. This choice between neuronal versus non-neuronal cell fates rests on differential requirements for, and cross-talk between, Notch/Delta- and Ras/mitogen-activated protein kinase (MAPK)-dependent signaling pathways. However, many questions remain unanswered related to how downstream events of these two signaling pathways mediate distinct cell fate decisions.
Here, we demonstrate that two direct downstream targets of Ras and Notch signaling, the transcription factors Prospero and dPax2, are essential regulators of neuronal versus non-neuronal cell fate decisions in the R7 equivalence group. Prospero controls high activated MAPK levels required for neuronal fate, whereas dPax2 represses Delta expression to prevent neuronal fate. Importantly, activity from both factors is required for proper cell fate decisions to occur.
These data demonstrate that Ras and Notch signaling are integrated during cell fate decisions within the R7 equivalence group through the combinatorial and opposing activities of Pros and dPax2. Our study provides one of the first examples of how the differential expression and synergistic roles of two independent transcription factors determine cell fate within an equivalence group. Since the integration of Ras and Notch signaling is associated with many developmental and cancer models, these findings should provide new insights into how cell specificity is achieved by ubiquitously used signaling pathways in diverse biological contexts.
A remarkably small number of signaling cascades are used across phyla to control cell specification. Thus, specificity must arise from combinatorial and cross-regulatory interactions among these pathways. Significant progress has been made in identifying the common immediate effectors for many of these conserved pathways, including those used during Ras- and Notch-dependent signaling. However, how these common pathways drive distinct cell fate choices in particular organs remains unclear.
Many of the common components within the Ras/mitogen-activated protein kinase (MAPK) and Notch/Delta signaling pathways have been identified and studied extensively in the developing Drosophila compound eye [1–4]. This is in large part because each of the 20 cells present in the approximately 750 repeating adult eye units (ommatidia) in this organ is stereotypically and sequentially recruited by the reiterative use of these two pathways. First, the R8 photoreceptor neuron is selected through Notch-dependent lateral inhibition, followed by stepwise recruitment of the R2/R5, R3/R4, R1/R6, and R7 photoreceptors (PRs), four cone cells (CCs), two primary pigment cells (PPCs), and approximately nine shared highly pigmented interommatidial cells by use of both Ras and Notch signaling [5–9]. How Ras and Notch are differentially interpreted in these different cell types, however, is not understood.
Specification of the R7 photoreceptor provides a particularly useful model for exploring Ras and Notch signal integration in the fly eye. The R7 and CCs are the last cells to be recruited to ommatidial clusters before pupation, and classic studies have established that prior to their specification, these five cells comprise an 'R7 equivalence group' [10, 11]. All cells within the R7 equivalence group express Notch, the epidermal growth factor (EGF) receptor (EGFR), and Sevenless, a tyrosine kinase receptor that, like EGFR, signals via the Ras/MAPK pathway. The ligands Delta and EGF are made available from the previously specified PRs R1 and R6, but only one cell can directly contact Boss, the Sev ligand, present on the surface of the R8 PR [12–15]. This cell, receiving higher Ras/MAPK levels, is driven towards an R7 fate, while the remaining cells that do not receive Sev signaling become epithelial CCs [2, 16, 17]. These studies have led to the prevailing model that Ras/MAPK signaling is necessary and sufficient for neuronal versus non-neuronal cell fate decisions in this group of cells. However, more recent work in the fly eye has shown that Ras/MAPK levels also indirectly control Delta expression [18, 19], a neurogenic factor in much of the developing nervous system , and mutants affecting Ras or Notch signaling cause only partial R7 versus CC fate switches [2, 10, 12, 17, 19, 21–24]. Thus, it is important to clarify the role of these signaling pathways in R7 versus CC fate choices, as it serves as a useful model for addressing how different levels of signaling mediate cell-specific fate decisions during development.
Two specific downstream targets of Ras and Notch within the R7 equivalence group are the transcription factors Prospero (Pros) and dPax2 (also known as sparkling (spa) or shaven (Sv)) . Pros is expressed in all five cells and is later up-regulated in the presumptive R7 by Sevenless [26, 27]. dPax2, on the other hand, is restricted to the four CC precursor cells, and is later expanded into the PPCs, the next cells to form in the eye . Enhancers partially reflecting these Pros and dPax2 expression patterns have been identified. Pros expression is directly up-regulated by the EGF/Ras/MAPK-activated ETS transcription factor PntP2, and is probably indirectly regulated by Notch signaling [26, 27, 29]. dPax2, instead, is controlled by the combined direct inputs of PntP2 and the Notch/Su(H) activation complex [30, 31]. While these studies provide evidence that Pros and dPax2 are direct transcriptional targets of Ras and Notch signaling, the functional relevance of this regulation remains an unanswered question.
Here, we demonstrate that Pros and dPax2 are expressed at different levels in distinct subtypes of CC precursors, indicating that not all CCs are 'equivalent'. Further, we show that individual mutations in Pros or Pax2 cause minor changes in CC numbers, while their simultaneous removal prevents all CC recruitment, revealing that these factors combinatorially control CC fate determination. Mechanistically, we demonstrate that Pros maintains high-activated MAPK (pERK) levels in the R7 equivalence group, thus promoting neuronal PR fate within this cell population. Conversely, dPax2 transcriptionally suppresses Delta expression in CC precursors, serving as an anti-neuronal factor. Importantly, the integration of Pros and dPax2 functions via Ras and Notch signaling feedback is necessary to fully control neuronal versus non-neuronal cell fate decisions in the fly eye. Since Pros and dPax2 orthologs are co-expressed in many developmental systems requiring Ras/MAPK and Delta/Notch inputs, it is likely that these processes are evolutionarily conserved.
Pros and dPax2 genetically interact to recruit CCs and control lens formation
Pros is known to regulate late aspects of R7 terminal differentiation [26, 32, 33], while dPax2 has been reported to be important for CC shape and organization and PPC recruitment during pupal development [28, 34]. However, the early expression of these factors in response to signals required for R7 versus CC specification suggests that they also may participate in cell fate decisions within the R7 equivalence group.
Summary of photoreceptor and cone cell quantifications from Pros and dPax2 genetic manipulations
pros 17 /pros 17
spa pol /spa pol
pros 17 /+;spa pol /spa pol
sev>dPax2 RNAi + pros RNAi
sev>Pros + dPax2
sev>Pros; spa pol /spa pol
sev>dPax2 + pros RNAi
pros 17 /pros 17
spa pol /spa pol
sev>Pros + dPax2
sev>Pros; spa pol /spa pol
sev>dPax2 + pros RNAi
Prospero and dPax2 define distinct cone cell populations
We next examined the expression of these factors in pros and dPax2 mutants (Figure 2E-G). In dPax2 mutants (Figure 2E), Pros expression is initiated and maintained comparable to wild-type eyes, but Cut is almost undetectable until the last few rows of ommatidia. Therefore, although Cut is present in CCs by pupation in dPax2 mutants (Figure 1G) , CCs lacking Cut are recruited earlier in the imaginal disc (Figure 2E). This is, to our knowledge, the first report showing that CCs can be recruited in the absence of Cut. In pros mutant clones, dPax2 expression is similar to wild-type tissue, but Cut expression is delayed by approximately five rows, specifically in the equatorial and polar CCs (Figure 2F,G and data not shown).
Together, these data suggest that genetically distinct CCs are formed in the eye imaginal disc, that loss of Pros delays equatorial/polar CC recruitment, and that loss of dPax2 prevents Cut expression in all CCs until just prior to pupation.
Ectopic Pros and dPax2 are sufficient to recruit extra cone cells from distinct cell populations
Compared to wild-type eyes (Figure 3A), the ectopic expression of Pros (sev> Pros) frequently leads to ommatidia with one or two extra Cut-positive cells (Figure 3E,N). In addition, in sev>Pros ommatidia with a normal complement of CCs, an extra R7 is occasionally formed (Figure 3E,G,H). We also find a consistent and significant loss of one BarH1-positive PPC per ommatidia in sev> Pros eyes (Figure 3F,N), and this decrease is proportional to the increase in CC and R7 number we observe (Figure 3E,H,N). Ectopic expression of dPax2, on the other hand, causes 26% of ommatidia to form one additional CC, an increase that is concomitant with a reduction in R7 PRs (Figure 3I,K,L,N). We also note that a smaller population of ommatidia (11%) develops only three CCs (data not shown), consistent with previous dPax2 overexpression studies using a multimerized eye-specific enhancerto drive its expression throughout the eye . Interestingly, although dPax2 is necessary for PPC formation , it is not sufficient for PPC development, as we do not find a significant change in PPC number in sev> dPax2 eyes. Moreover, in the few examples where an ectopic PPC does form in sev> dPax2 eyes, an extra CC is also present (Figure 3J), consistent with the fact that PPC number correlates with CC number [18, 39]. Thus, ectopic dPax2 does not lead to ectopic PPC formation, but can generate ectopic CCs.
Together, these gain-of-function (GOF) experiments suggest that Pros can convert PPCs into CCs or R7s, whereas dPax2 can convert R7s into CCs. These findings also indicate that Pros and dPax2 only change fates in cells already endogenously expressing one of these two factors - Pros converts PPCs normally expressing dPax2, while dPax2 converts R7s normally expressing Pros (Figure 3O). To test whether these factors together convert cells into CCs, we co-expressed both Pros and dPax2. Like the individual GOF experiments, this co-expression consistently converts one extra cell into a CC (Figure 3M,N); in contrast, no change in PR or PPC number is observed (Table 1; Additional file 1C,D), indicating that another cell type is being converted to a CC with Pros and dPax2 co-expression. While it is currently unclear what the source of this cell is, it may correspond to a mystery cell, since these cells have been previously described to express sev in the imaginal disc [5, 35, 40]. Alternatively, it is possible that it derives from other non-specified cells in the eye that are usually eliminated by apoptosis during pupal development .
Pros and dPax2 differentially regulate Ras- and Notch-dependent signaling, respectively
R7 and CC fate decisions require input from the Ras and Notch signaling pathways, and Pros and dPax2 are both direct downstream targets of these pathways [26, 27, 29, 30]. Thus, these transcription factors are likely candidates for mediating downstream events and/or feedback regulatory control of these pathways. To test this hypothesis, we analyzed pros and dPax2 loss-of-function (LOF) mutants for factors that lie downstream of Ras (for example, activated MAPK and Yan) and Notch (for example, E(spl) and Delta).
Since pERK phosphorylates Yan and blocks the nuclear accumulation of this ETS-related transcriptional repressor , as a further test that Pros effects pERK levels, we next examined Yan expression in pros LOF and GOF tissues. Reflective of the fact that Yan directly represses Pros expression , Yan levels decrease at the time that Pros levels increase in wild-type tissue (Figure 4E). However, in pros mutant clones, nuclear Yan levels increase (Figure 4F), and in sev>Pros tissue, Yan levels decrease (Figure 4G). This provides additional support for a role for Pros in up-regulating pERK activity. We also functionally tested a role for Pros in up-regulating pERK activity by taking advantage of the fact that R7 specification fails in sev flies due to insufficient Ras/pERK signaling [2, 10, 16, 24]. We reasoned that if Pros is sufficient to up-regulate pERK, then misexpressing Pros in sev mutants should rescue R7 differentiation. Indeed, sev; sev> Pros flies develop an R7 in >90% of ommatidia, whereas no R7 forms in sev mutants alone (Table 1; Additional file 1E,F).
In many systems, Delta is repressed in Notch signal-receiving cells to prevent receptor-ligand co-expression and facilitate unidirectional cell-cell signaling . Since Delta expression is up-regulated in dPax2 mutants, we also analyzed Notch activation in these mutants by examining the expression of another Notch target gene, E(spl) . This shows that E(spl) expression is significantly and cell-autonomously reduced in RNAi-mediated dPax2 knockdown tissue compared with control tissue (Figure 5E,F). Due to the extensive feedback between Notch and Delta, determining whether dPax2 is necessary for directly repressing Delta and/or promoting Notch activity requires further tests. Nevertheless, our experiments provide evidence that dPax2 controls at least some aspects of Notch signaling, likely by repressing Delta, and that Pros helps mediate downstream processes for Sev and/or EGF signaling by maintaining increased pERK levels.
Pros and dPax2 combinatorially control neuronal versus non-neuronal cell fate decisions
Here, we present evidence that Pros and dPax2 together specify neuronal versus non-neuronal fates by combined feedback into the Ras/MAPK and Delta/Notch signaling pathways, respectively. Specifically, we demonstrate that Pros and dPax2 are differentially expressed in distinct CC subpopulations, and synergistically contribute to CC recruitment and lens formation. Moreover, we show that within the R7 equivalence group, Pros is required for obtaining the high pERK levels necessary for sensory (R7) cell fate determination, whereas dPax2 is critical for repressing Delta expression in response to Notch to block sensory cell specification. Thus, in one context (that is, CC recruitment), Pros and dPax2 function cooperatively, whereas in another (that is, R7 versus CC fate choice), they function antagonistically. These studies ultimately reveal that Pros and dPax2 are mediators of the same signaling pathways that initiate their expression, and that cells previously considered equivalent are instead differentially sensitized to EGF and Notch signaling based on the expression levels of two distinct transcription factors. Interestingly, cell lineages in many other sensory organs also make use of reiterative Notch and Ras signaling, express dynamic levels of dPax2 and Pros, and require dPax2 and Pros for distinct cell fate decisions [45–48]. Thus, Pros and dPax2 are likely to perform similar functions in other regions of the Drosophila nervous system. Moreover, as we discuss later, these functions may be evolutionarily conserved in other animals and developmental contexts.
Neuronal versus non-neuronal cell fate decisions by the combinatorial actions of Pros and dPax2
One of the best-studied systems related to breaking 'equivalence' in a group of cells is lateral inhibition during neurogenesis. In this process, a field of neuroepithelial cells initially expresses uniform and low levels of a member of the proneural family of basic helix-loop-helix (bHLH) transcription factors. bHLHs activate Delta, whereas Notch activation suppresses bHLH and Delta expression. Thus, through a feedback system between Notch and Delta, bHLHs and Delta become progressively restricted to one cell within the proneural field. This cell differentiates into a neuron, whereas its neighboring cells receive high levels of anti-neural Notch signaling and remain epithelial .
Becoming non-equivalent within the R7 equivalence group by Pros and dPax2 expression
R7 versus cone cells
This study suggests that at least two subclasses of CCs are specified in the eye based on differing relative expression levels of Pros and dPax2/Cut. Importantly, the eye-regulatory enhancers for both Pros and dPax2 give a 'salt-and-pepper' expression pattern to reporter genes [29, 31] (MCP and TC, unpublished), suggesting that these differences are controlled transcriptionally. Given our findings that Pros and dPax2 feedback into the same pathways known to control their expression, these transcription factors may also have some auto-regulatory functions in this system. Notably, however, even though Pros and dPax2 expression requires input from Ras/pERK and Notch-dependent signaling, and both pathways are required for R7 and CC fate, Pros is expressed in all five cells of the R7 equivalence group whereas dPax2 is restricted to CC precursors. This observation raises the possibility that Boss-Sevenless signaling not only activates Pros expression, but may also actively suppress dPax2 expression in the presumptive R7. Such a scenario would also help explain recent findings by Swanson and colleagues  showing that disrupting the dPax2 CC enhancer often causes inappropriate expression in PRs - perhaps these artificial enhancers lack or disrupt Sev-dependent repression elements. Since expressing an activated Sev receptor leads to a higher average number of ectopic R7 cells than expressing constitutively active Ras in the same cells (2.8 per ommatidia with sev-SevS11) versus approximately 0.65 for sev-RasV12 (Table 1) [10, 17, 24, 55], and because misexpressing Pros only weakly converts CCs into R7 cells unless dPax2 is also removed (Figure 6), it is unlikely that repression of dPax2 in the presumptive R7 is mediated through Ras/pERK. Thus, we propose that while Pros mediates the Ras output of the Sev receptor, another factor activated in the presumptive R7, possibly Phyllopod , suppresses dPax2 in R7 cells. These two events together would then help define differences between the R7 and CC precursors within the R7 equivalence group.
Cone cell subtypes
Our findings that different CC subtypes exist during their early recruitment provide a useful paradigm for interpreting previous experiments focused on R7 versus CC specification. For instance, despite the widely held paradigm that EGF and Notch are 'equivalent' in all cells within the R7 equivalence group, the majority of mutants tested to support this model generally only affect a subset of cells [10, 17, 19, 21, 23, 26, 30, 55–57]. One explanation for this is that these different mutants differentially participate in the formation of distinct CCs. Thus, re-examining these mutant phenotypes may help define other targets associated with CC subtype specification and/or differentiation and help us understand the biological relevance of having two CC subtypes.
Potential parallel functions for Pros/Prox1 and dPax2/Pax2 in other systems
To date, whether Pax2 or Pros are involved in regulating Notch or MAPK signaling in other systems has not been directly tested. However, Pros has been implicated in regulating Ras signaling via the EGFR ligand Vein in Drosophila glioblast formation , and several components within the Ras and Notch signaling pathways have been identified as potential pros targets by microarray analysis [59, 60]. Similarly, the vertebrate ortholog of Pros, Prox1, is known to act downstream of tyrosine kinase-dependent Ras signaling in differentiating lens fiber cells [61, 62]. In addition, Notch signaling has recently been implicated in vertebrate lens development, and the required Notch ligand is supplied by both cell populations that express Prox1: epithelial lens progenitors and differentiating primary fiber cells [63–65]. Thus, it is possible that the ability of Pros to affect Ras and Notch signaling may be evolutionarily conserved. Further supporting this hypothesis, Prox1 has recently been shown to reduce Notch activity to induce neurogenesis . Likewise, similar to Drosophila Pax2, vertebrate Pax2 is frequently a target of Notch signaling in sensory systems, including the inner ear, and has been proposed to mediate at least some of the Notch-dependent functions important for hair cell development . Pax2 and Notch signaling are also critical regulators of kidney development, and both are potential therapeutic targets in many renal cancers [68–72]. Therefore, we are optimistic that the present findings will provide the theoretical framework and a tractable genetic system for advancing our understanding of developmental processes in a wide range of biological systems.
Materials and methods
Generation of RNAi lines
Two UAS-dPax2 RNAi lines were generated using the following primers against the Sv cDNA cpx1 : forward 1, CTGAGAATTC ATGCTTATAATGGATATACAGACATCG (Eco RI), reverse 1, CTGATCTAGA GTTGTATTCCTAATATTCCATTTATGC (Xba I); forward 2, CTGAGAATTC AATTGTAAGGAATAAAGCCGCCGAG (Eco RI), reverse 2, CTGATCTAGA GTTGTATTCCTAATATTCCATTTATGC (Xba I).
Inverted repeats were ligated with Eco R1, and then cloned into pMF3 (Vienna Drosophila RNAi Center) with Xba I. A UAS-Pros RNAi line was generated by amplifying from ProsS cDNA  using the primers AAGGATCC CGGCTGCCATGTTCCAGGCGC (Bam HI) and CCTGCGCA ATGGCGCTTCTTCTTTGGTGTC (Mlu I). (Italics represent the restriction enzyme sites that appear in parenthesese following the sequence. Inverted repeats were ligated with Mlu I, subcloned with Bam HI into pBSIIKS(-) in Sure Cells (Invitrogen, Carlsbad, CA, USA), and cloned into pUAST  using Xba I and Xho I. Transgenic lines were generated in yw 67 flies using standard procedures (Rainbow Transgenics, Camarillo, CA, USA). Multiple lines for each construct were tested and those phenocopying spa pol or pros 17 mutants were retained for further analysis.
FRT82Bubi-GFPnls, RpS3/TM6B, FRT82Bubi-GFP/TM6B, sev, spa pol , Dl-LacZ, sev- Rasv12, sev-Gal4, mirr-GAL4, and UAS-CD8::GFP flies were from the Bloomington Stock Center. Other lines were: UAS-prosS and FRT82B-pros 17 , ey flp3.5 (provided by Claude Desplan), UAS-dPax2, and UAS-ProsKKRNAi (Vienna Drosophila RNAi Center). prospero mitotic clones were analyzed in ey flp3.5 ; Sp/CyO; FRT82ubi-GFP/FRT82B-pros17 flies, whereas Minute clones to generate eyes almost entirely mutant for pros were generated from ey flp3.5 ; Sp/CyO; FRT82ubi-GFPnls, RpS3/FRT82B-pros17 flies. Other genotypes were: yw 67 ; Sp/CyO; TM2/TM6B ('wild-type'), yw 67 ; Sp/CyO; TM2/TM6B; spa pol /spa pol (d Pax2 LOF), yw 67 ; Sp/CyO; FRT82B-pros17/TM6B; spa pol /spa pol (pros/+, dPax2 LOF), UAS-d Pax2RNAi; sev-GAL4/CyO; UAS-Pros RNAi/TM6B (sev>dPax2 RNAi + Pros RNAi ), yw 67 ; sev-Gal4; UAS-Pros/TM6B (sev>pros), yw 67 ; sev-Gal4; UAS-dPax2/TM6B (sev>dPax2), yw 67 ; sev-Gal4; UAS-dPax2/UAS-Pros (sev> Pros/dPax2), sev; Sp/CyO; TM2/TM6B (sev), sev; sev-Gal4/CyO; UAS-Pros/TM6B (sev; sev> Pros), yw 67 ; sev-Gal4/CyO; UAS-Pros/TM6B; spa pol /spa pol (Pros GOF, dPax2 LOF), and yw 67 ; Sp/sev-Rasv12, CyO; TM2/TM6B (sev-Rasv12), yw 67 ; sev-GAL4/UAS-prosKKRNAi; UAS-dPax2/TM2 (sev>dPax2 + Pros RNAi ), yw 67 ; UAS-CD8::GFP; mirr-GAL4 (mirr>GFP), UAS-d Pax2RNAi; UAS-CD8::GFP/Sp (or CyO); mirr-GAL4/TM2 (mirr>GFP,d Pax2RNAi). Flies were raised on standard cornmeal/yeast/molasses/agar media at 25°C.
Primary antibodies were generated by Cocalico Biologicals (Reamstown, PA, USA) against denatured His-tagged proteins produced from pET-28a (+) as previously described . Rat anti-BarH1 was created against a full-length protein made from the BarH1 cDNA present in pGEX-4T-BarH1, guinea pig anti-Pros was produced against amino acids 399 to 972 from a Sac I fragment from ProsS , and anti-dPax2 antibodies were produced against amino acids 308 to 512 from a Bam H1/Eco RI fragment from pGEX-dPax2. Specificity was determined by comparison with previously described staining patterns, and in spa pol or pros 17 mutants. Guinea pig anti-Pros immunolocalization was further confirmed with two previously described antibodies: mouse MR1A against amino acids 1,196 to 1,320 , and rabbit 89E against amino acids 409 to 438 .
Histology and microscopy
Adult lenses were visualized by SEM using anesthetized flies mounted on carbon tabs and directly analyzing them with a Hitachi S-3400N. For thin sections, eyes were fixed for 15 minutes in 4% paraformaldehyde/phosphate-buffered saline, washed twice with 0.1% Triton X/phosphate-buffered saline (PBT), serially dehydrated in ethanol, incubated in 1:1 ethanol/LR-White resin (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour and 100% resin for 1 hour, then polymerized with one drop per milliliter of accelerator. Sections (2 μm) were dried for 15 minutes on a slide warmer and stained with 1% toluidine blue for 10 minutes and mounted in Entellan (EMS), or rehydrated in PBT overnight followed by antibody staining and mounting as previously described . Imaginal discs and retinas dissected from pupa 45 hours after puparium formation (45% pupation) were immunostained as previously described for whole mount adult retinas . Antibodies were from the Developmental Studies Hybridoma Bank unless indicated otherwise, and diluted as follows: Cut (mouse, 1:100), Pros (rabbit , 1:1,000), Pros (mouse, 1:10), Pros (guinea pig, this paper, 1:1,500), dPax2 (rabbit , 1:50; rabbit, this paper, 1:1500; guinea pig, this paper, 1;1,500), Elav (mouse or rat, 1:200), Eya (mouse, 1:50), BarH1 (rat, this paper, 1:200; rabbit , 1:50), Dlg (mouse, 1:50), E-cadherin (rat, 1:20; rabbit, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:50), GFP (chicken, Abcam, Cambridge, MA, USA, 1:500), β-gal (chicken, Sigma-Aldrich, St. Louis, MO, USA, 1:1,000), Otd (guinea pig , 1:750), Rh3 (chicken , 1:40), Rh4 (rabbit, gift from C Zuker/N Colley, 1:150), Delta (mouse, 1:50), E(spl) (mouse , 1:1), pERK (rabbit, Sigma-Aldrich, 1:20), m(Espl) (1:2 ) and N-Cad (rat, 1:20). Secondary antibodies were conjugated to Alexa Fluor 488, 555, 647 and 750 nm (goat, Invitrogen) or Cy2, 3 or 5 (donkey, Jackson Immunoresearch, West Grove, PA, USA), and diluted 1:500. Polymerized actin was detected using AlexFluor 488-conjugated phalloidin (Invitrogen), which was added to the secondary antibody dilutions at 1:20 according to the manufacturer's suggestion. Samples were imaged with a Zeiss Apotome, deconvolved with Axiovision 4.6 or Zeiss LSM 700 confocal and processed in Adobe Photoshop 7.0.
Cell type quantification
Flies were raised at 25°C, pupal retinas were dissected at 45 hours after puparium formation (45% pupation), and samples were stained with Otd, Cut, and BarH1 to identify PRs, CCs, and PPCs, respectively. Individual ommatidia were defined by co-staining with either E-cadherin or Dlg. Counting from a minimum of 100 ommatidia from at least for separate mutant eyes was performed, and compared to wild-type values using one-way ANOVA (Microsoft Excel; StatPlus). For pros center versus border clonal analysis, clone borders were defined as being within at least one ommatidium of any GFP-expressing cell, while clone centers were at least two ommatidial spaces away from any GFP-expressing cell.
epidermal growth factor
epidermal growth factor receptor
green fluorescent protein
mitogen-activated protein kinase
primary pigment cell
scanning electron microscopy.
We are grateful to S Barolo, S Bray, S Britt, R Carthew, N Colley, C Desplan, Y-N Jan, T Kojima, M Noll, and C Zuker for flies and reagents, as well as the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology for providing antibodies contributed by G Rubin, N Bonini, T Umura, and A Peden. We also thank Ross Cagan and Don Ready for sharing their extensive knowledge with us regarding cone cell development. This work was supported by T32-HD046387 (DLK), RO1-GM079428 (BG), Research to Prevent Blindness, the Ziegler Foundation for the Blind, and R01-EY017907 (TAC).
- Kumar JP: Signalling pathways in Drosophila and vertebrate retinal development. Nat Rev Genet. 2001, 2: 846-857. 10.1038/35098564.View ArticlePubMed
- Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM: Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell. 1991, 67: 701-716. 10.1016/0092-8674(91)90065-7.View ArticlePubMed
- Frankfort BJ, Mardon G: R8 development in the Drosophila eye: a paradigm for neural selection and differentiation. Development. 2002, 129: 1295-1306.PubMed
- Voas MG, Rebay I: Signal integration during development: insights from the Drosophila eye. Dev Dyn. 2004, 229: 162-175. 10.1002/dvdy.10449.View ArticlePubMed
- Wolff T, Ready DF: Pattern Formation in the Drosophila Retina. 1993, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
- Cagan R: The signals that drive kidney development: a view from the fly eye. Curr Opin Nephrol Hypertens. 2003, 12: 11-17. 10.1097/00041552-200301000-00003.View ArticlePubMed
- Doroquez DB, Rebay I: Signal integration during development: mechanisms of EGFR and Notch pathway function and cross-talk. Crit Rev Biochem Mol Biol. 2006, 41: 339-385. 10.1080/10409230600914344.View ArticlePubMed
- Charlton-Perkins M, Cook TA: Building a fly eye: terminal differentiation events of the retina, corneal lens, and pigmented epithelia. Curr Topics Dev Biol. 2010, 93: 129-173.View Article
- Bao S: Two themes on the assembly of the Drosophila eye. Curr Topics Dev Biol. 2010, 93: 85-127.View Article
- Dickson B, Sprenger F, Hafen E: Prepattern in the developing Drosophila eye revealed by an activated torso--sevenless chimeric receptor. Genes Dev. 1992, 6: 2327-2339. 10.1101/gad.6.12a.2327.View ArticlePubMed
- Tomlinson A, Bowtell DD, Hafen E, Rubin GM: Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila. Cell. 1987, 51: 143-150. 10.1016/0092-8674(87)90019-5.View ArticlePubMed
- Reinke R, Zipursky SL: Cell-cell interaction in the Drosophila retina: the bride of sevenless gene is required in photoreceptor cell R8 for R7 cell development. Cell. 1988, 55: 321-330. 10.1016/0092-8674(88)90055-4.View ArticlePubMed
- Tomlinson A, Struhl G: Delta/Notch and Boss/Sevenless signals act combinatorially to specify the Drosophila R7 photoreceptor. Mol Cell. 2001, 7: 487-495. 10.1016/S1097-2765(01)00196-4.View ArticlePubMed
- Freeman M: Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell. 1996, 87: 651-660. 10.1016/S0092-8674(00)81385-9.View ArticlePubMed
- Cooper MT, Bray SJ: R7 photoreceptor specification requires Notch activity. Curr Biol. 2000, 10: 1507-1510. 10.1016/S0960-9822(00)00826-5.View ArticlePubMed
- Tomlinson A, Ready DF: Sevenless: a cell-specific homeotic mutation of the Drosophila eye. Science. 1986, 231: 400-402. 10.1126/science.231.4736.400.View ArticlePubMed
- Basler K, Christen B, Hafen E: Ligand-independent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye. Cell. 1991, 64: 1069-1081. 10.1016/0092-8674(91)90262-W.View ArticlePubMed
- Nagaraj R, Banerjee U: Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye. Development. 2007, 134: 825-831. 10.1242/dev.02788.View ArticlePubMed
- Tsuda L, Nagaraj R, Zipursky SL, Banerjee U: An EGFR/Ebi/Sno pathway promotes delta expression by inactivating Su(H)/SMRTER repression during inductive notch signaling. Cell. 2002, 110: 625-637. 10.1016/S0092-8674(02)00875-9.View ArticlePubMed
- Artavanis-Tsakonas S, Rand MD, Lake RJ: Notch signaling: cell fate control and signal integration in development. Science. 1999, 284: 770-776. 10.1126/science.284.5415.770.View ArticlePubMed
- Bhattacharya A, Baker NE: The HLH protein Extramacrochaetae is required for R7 cell and cone cell fates in the Drosophila eye. Dev Biol. 2009, 327: 288-300. 10.1016/j.ydbio.2008.11.037.PubMed CentralView ArticlePubMed
- Van Vactor DL, Cagan RL, Kramer H, Zipursky SL: Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell. Cell. 1991, 67: 1145-1155. 10.1016/0092-8674(91)90291-6.View ArticlePubMed
- Zheng L, Carthew RW: Lola regulates cell fate by antagonizing Notch induction in the Drosophila eye. Mech Dev. 2008, 125: 18-29. 10.1016/j.mod.2007.10.007.PubMed CentralView ArticlePubMed
- Fortini ME, Simon MA, Rubin GM: Signalling by the sevenless protein tyrosine kinase is mimicked by Ras1 activation. Nature. 1992, 355: 559-561. 10.1038/355559a0.View ArticlePubMed
- Garcon L, Lacout C, Svinartchouk F, Le Couedic JP, Villeval JL, Vainchenker W, Dumenil D: Gfi-1B plays a critical role in terminal differentiation of normal and transformed erythroid progenitor cells. Blood. 2005, 105: 1448-1455. 10.1182/blood-2003-11-4068.View ArticlePubMed
- Kauffmann RC, Li S, Gallagher PA, Zhang J, Carthew RW: Ras1 signaling and transcriptional competence in the R7 cell of Drosophila. Genes Dev. 1996, 10: 2167-2178. 10.1101/gad.10.17.2167.View ArticlePubMed
- Xu C, Kauffmann RC, Zhang J, Kladny S, Carthew RW: Overlapping activators and repressors delimit transcriptional response to receptor tyrosine kinase signals in the Drosophila eye. Cell. 2000, 103: 87-97. 10.1016/S0092-8674(00)00107-0.View ArticlePubMed
- Fu W, Noll M: The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye. Genes Dev. 1997, 11: 2066-2078. 10.1101/gad.11.16.2066.PubMed CentralView ArticlePubMed
- Hayashi T, Xu C, Carthew RW: Cell-type-specific transcription of prospero is controlled by combinatorial signaling in the Drosophila eye. Development. 2008, 135: 2787-2796. 10.1242/dev.006189.PubMed CentralView ArticlePubMed
- Flores GV, Duan H, Yan H, Nagaraj R, Fu W, Zou Y, Noll M, Banerjee U: Combinatorial signaling in the specification of unique cell fates. Cell. 2000, 103: 75-85. 10.1016/S0092-8674(00)00106-9.View ArticlePubMed
- Swanson CI, Evans NC, Barolo S: Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer. Dev Cell. 2010, 18: 359-370. 10.1016/j.devcel.2009.12.026.PubMed CentralView ArticlePubMed
- Cook T, Pichaud F, Sonneville R, Papatsenko D, Desplan C: Distinction between color photoreceptor cell fates is controlled by Prospero in Drosophila. Dev Cell. 2003, 4: 853-864. 10.1016/S1534-5807(03)00156-4.View ArticlePubMed
- Morey M, Yee SK, Herman T, Nern A, Blanco E, Zipursky SL: Coordinate control of synaptic-layer specificity and rhodopsins in photoreceptor neurons. Nature. 2008, 456: 795-799. 10.1038/nature07419.PubMed CentralView ArticlePubMed
- Shi Y, Noll M: Determination of cell fates in the R7 equivalence group of the Drosophila eye by the concerted regulation of D-Pax2 and TTK88. Dev Biol. 2009, 331: 68-77. 10.1016/j.ydbio.2009.04.026.View ArticlePubMed
- Bowtell DD, Kimmel BE, Simon MA, Rubin GM: Regulation of the complex pattern of sevenless expression in the developing Drosophila eye. Proc Natl Acad Sci USA. 1989, 86: 6245-6249. 10.1073/pnas.86.16.6245.PubMed CentralView ArticlePubMed
- Blochlinger K, Bodmer R, Jan LY, Jan YN: Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes Dev. 1990, 4: 1322-1331. 10.1101/gad.4.8.1322.View ArticlePubMed
- Vandendries ER, Johnson D, Reinke R: orthodenticle is required for photoreceptor cell development in the Drosophila eye. Dev Biol. 1996, 173: 243-255. 10.1006/dbio.1996.0020.View ArticlePubMed
- Higashijima S, Kojima T, Michiue T, Ishimaru S, Emori Y, Saigo K: Dual Bar homeo box genes of Drosophila required in two photoreceptor cells, R1 and R6, and primary pigment cells for normal eye development. Genes Dev. 1992, 6: 50-60. 10.1101/gad.6.1.50.View ArticlePubMed
- Miller DT, Cagan RL: Local induction of patterning and programmed cell death in the developing Drosophila retina. Development. 1998, 125: 2327-2335.PubMed
- Cagan RL, Ready DF: Notch is required for successive cell decisions in the developing Drosophila retina. Genes Dev. 1989, 3: 1099-1112. 10.1101/gad.3.8.1099.View ArticlePubMed
- Rusconi JC, Hays R, Cagan RL: Programmed cell death and patterning in Drosophila. Cell Death Differ. 2000, 7: 1063-1070. 10.1038/sj.cdd.4400767.View ArticlePubMed
- Rebay I, Rubin GM: Yan functions as a general inhibitor of differentiation and is negatively regulated by activation of the Ras1/MAPK pathway. Cell. 1995, 81: 857-866. 10.1016/0092-8674(95)90006-3.View ArticlePubMed
- Parks AL, Turner FR, Muskavitch MA: Relationships between complex Delta expression and the specification of retinal cell fates during Drosophila eye development. Mech Dev. 1995, 50: 201-216. 10.1016/0925-4773(94)00336-L.View ArticlePubMed
- Jennings B, Preiss A, Delidakis C, Bray S: The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo. Development. 1994, 120: 3537-3548.PubMed
- Kavaler J, Fu W, Duan H, Noll M, Posakony JW: An essential role for the Drosophila Pax2 homolog in the differentiation of adult sensory organs. Development. 1999, 126: 2261-2272.PubMed
- Manning L, Doe CQ: Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage. Development. 1999, 126: 2063-2071.PubMed
- Reddy GV, Rodrigues V: Sibling cell fate in the Drosophila adult external sense organ lineage is specified by prospero function, which is regulated by Numb and Notch. Development. 1999, 126: 2083-2092.PubMed
- Lai EC, Orgogozo V: A hidden program in Drosophila peripheral neurogenesis revealed: fundamental principles underlying sensory organ diversity. Dev Biol. 2004, 269: 1-17. 10.1016/j.ydbio.2004.01.032.View ArticlePubMed
- Doe CQ, Chu-LaGraff Q, Wright DM, Scott MP: The prospero gene specifies cell fates in the Drosophila central nervous system. Cell. 1991, 65: 451-464. 10.1016/0092-8674(91)90463-9.View ArticlePubMed
- Vaessin H, Grell E, Wolff E, Bier E, Jan LY, Jan YN: prospero is expressed in neuronal precursors and encodes a nuclear protein that is involved in the control of axonal outgrowth in Drosophila. Cell. 1991, 67: 941-953. 10.1016/0092-8674(91)90367-8.View ArticlePubMed
- Nagaraj R, Banerjee U: Regulation of Notch and Wingless signalling by phyllopod, a transcriptional target of the EGFR pathway. EMBO J. 2009, 28: 337-346. 10.1038/emboj.2008.286.PubMed CentralView ArticlePubMed
- Hassan B, Li L, Bremer KA, Chang W, Pinsonneault J, Vaessin H: Prospero is a panneural transcription factor that modulates homeodomain protein activity. Proc Natl Acad Sci USA. 1997, 94: 10991-10996. 10.1073/pnas.94.20.10991.PubMed CentralView ArticlePubMed
- Shilo BZ: Signaling by the Drosophila epidermal growth factor receptor pathway during development. Exp Cell Res. 2003, 284: 140-149. 10.1016/S0014-4827(02)00094-0.View ArticlePubMed
- de Celis JF, Bray S: Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development. 1997, 124: 3241-3251.PubMed
- Matsuo T, Takahashi K, Kondo S, Kaibuchi K, Yamamoto D: Regulation of cone cell formation by Canoe and Ras in the developing Drosophila eye. Development. 1997, 124: 2671-2680.PubMed
- Lai ZC, Rubin GM: Negative control of photoreceptor development in Drosophila by the product of the yan gene, an ETS domain protein. Cell. 1992, 70: 609-620. 10.1016/0092-8674(92)90430-K.View ArticlePubMed
- Hayashi T, Kojima T, Saigo K: Specification of primary pigment cell and outer photoreceptor fates by BarH1 homeobox gene in the developing Drosophila eye. Dev Biol. 1998, 200: 131-145. 10.1006/dbio.1998.8959.View ArticlePubMed
- Griffiths RL, Hidalgo A: Prospero maintains the mitotic potential of glial precursors enabling them to respond to neurons. EMBO J. 2004, 23: 2440-2450. 10.1038/sj.emboj.7600258.PubMed CentralView ArticlePubMed
- Guenin L, Raharijaona M, Houlgatte R, Baba-Aissa F: Expression profiling of prospero in the Drosophila larval chemosensory organ: Between growth and outgrowth. BMC Genomics. 2010, 11: 47-10.1186/1471-2164-11-47.PubMed CentralView ArticlePubMed
- Choksi SP, Southall TD, Bossing T, Edoff K, de Wit E, Fischer BE, van Steensel B, Micklem G, Brand AH: Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Dev Cell. 2006, 11: 775-789. 10.1016/j.devcel.2006.09.015.View ArticlePubMed
- Burgess D, Zhang Y, Siefker E, Vaca R, Kuracha MR, Reneker L, Overbeek PA, Govindarajan V: Activated Ras alters lens and corneal development through induction of distinct downstream targets. BMC Dev Biol. 2010, 10: 13-10.1186/1471-213X-10-13.PubMed CentralView ArticlePubMed
- Zhao H, Yang T, Madakashira BP, Thiels CA, Bechtle CA, Garcia CM, Zhang H, Yu K, Ornitz DM, Beebe DC, Robinson ML: Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation. Dev Biol. 2008, 318: 276-288. 10.1016/j.ydbio.2008.03.028.PubMed CentralView ArticlePubMed
- Duncan MK, Cui W, Oh DJ, Tomarev SI: Prox1 is differentially localized during lens development. Mech Dev. 2002, 112: 195-198. 10.1016/S0925-4773(01)00645-1.View ArticlePubMed
- Rowan S, Conley KW, Le TT, Donner AL, Maas RL, Brown NL: Notch signaling regulates growth and differentiation in the mammalian lens. Dev Biol. 2008, 321: 111-122. 10.1016/j.ydbio.2008.06.002.PubMed CentralView ArticlePubMed
- Saravanamuthu SS, Gao CY, Zelenka PS: Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation. Dev Biol. 2009, 332: 166-176. 10.1016/j.ydbio.2009.05.566.PubMed CentralView ArticlePubMed
- Kaltezioti V, Kouroupi G, Oikonomaki M, Mantouvalou E, Stergiopoulos A, Charonis A, Rohrer H, Matsas R, Politis PK: Prox1 regulates the notch1-mediated inhibition of neurogenesis. PLoS Biol. 2010, 8: e1000565-10.1371/journal.pbio.1000565.PubMed CentralView ArticlePubMed
- Riley BB, Chiang M, Farmer L, Heck R: The deltaA gene of zebrafish mediates lateral inhibition of hair cells in the inner ear and is regulated by pax2.1. Development. 1999, 126: 5669-5678.PubMed
- Gokden N, Gokden M, Phan DC, McKenney JK: The utility of PAX-2 in distinguishing metastatic clear cell renal cell carcinoma from its morphologic mimics: an immunohistochemical study with comparison to renal cell carcinoma marker. Am J Surg Pathol. 2008, 32: 1462-1467. 10.1097/PAS.0b013e318176dba7.View ArticlePubMed
- Gokden N, Kemp SA, Gokden M: The utility of Pax-2 as an immunohistochemical marker for renal cell carcinoma in cytopathology. Diagn Cytopathol. 2008, 36: 473-477. 10.1002/dc.20842.View ArticlePubMed
- Ozcan A, Zhai J, Hamilton C, Shen SS, Ro JY, Krishnan B, Truong LD: PAX-2 in the diagnosis of primary renal tumors: immunohistochemical comparison with renal cell carcinoma marker antigen and kidney-specific cadherin. Am J Clin Pathol. 2009, 131: 393-404. 10.1309/AJCPM7DW0XFHDHNY.View ArticlePubMed
- Tamimi Y, Ekuere U, Laughton N, Grundy P: WNT5A is regulated by PAX2 and may be involved in blastemal predominant Wilms tumorigenesis. Neoplasia. 2008, 10: 1470-1480.PubMed CentralView ArticlePubMed
- Beland M, Bouchard M: [PAX gene function during kidney tumorigenesis: a comparative approach]. Bull Cancer. 2006, 93: 875-882.PubMed
- Brand AH, Perrimon N: Targeted gene-expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993, 118: 401-415.PubMed
- Xie B, Charlton-Perkins M, McDonald E, Gebelein B, Cook T: Senseless functions as a molecular switch for color photoreceptor differentiation in Drosophila. Development. 2007, 134: 4243-4253. 10.1242/dev.012781.View ArticlePubMed
- Cook T: Cell diversity in the retina: more than meets the eye. Bioessays. 2003, 25: 921-925. 10.1002/bies.10356.View ArticlePubMed
- Spana EP, Doe CQ: The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila. Development. 1995, 121: 3187-3195.PubMed
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