As cells proceed along their developmental pathways they make a series of sequential cell fate decisions. Each of those decisions needs to be made in a robust manner so there is no ambiguity in the state of the cell as it proceeds to the next stage. Here we examine the decision made by the Drosophila R7 precursor cell to become a photoreceptor and ask how the robustness of that decision is achieved. The transcription factor Tramtrack (Ttk) inhibits photoreceptor assignment, and previous studies found that the RTK-induced degradation of Ttk was critically required for R7 specification. Here we find that the transcription factor Deadpan (Dpn) is also required; it is needed to silence ttk transcription, and only when Ttk protein degradation and transcriptional silencing occur together is the photoreceptor fate robustly achieved. Dpn expression needs to be tightly restricted to R7 precursors, and we describe the role played by Ttk in repressing dpn transcription. Thus, Dpn and Ttk act as mutually repressive transcription factors, with Dpn acting to ensure that Ttk is effectively removed from R7, and Ttk acting to prevent Dpn expression in other cells. Furthermore, we find that N activity is required to promote dpn transcription, and only in R7 precursors does the removal of Ttk coincide with high N activity, and only in this cell does Dpn expression result.
Development proceeds in a stepwise manner in which cells pass through a series of interim states in the progression from a pluripotent precursor to a fully-specified and final cell fate. Positional signals play a prominent role in this mechanism, with the receipt of the signals promoting the cells to the next state in the series. Each interim state needs to be established robustly so that upon receipt of the subsequent round of signals, the advance to the next state is unambiguous. In this paper we use the R7 photoreceptor of the developing Drosophila eye as a model cell with which to study the molecular mechanisms that occur in the acquisition of interim cell states. Specifically we examine the choice made by the R7 precursor to become a photoreceptor and the mechanisms that cooperate and consolidate that choice. The R7 precursor belongs to a group of cells known as the R7 equivalence group, which gives rise (in addition to R7) to the two R1/6 photoreceptors and the four (non-photoreceptor) cone cells. The fates of the equivalence group cells are determined by the positions they occupy in the growing ommatidial cluster (Fig 1A), and each initially expresses the transcription factor Tramtrack (Ttk88—hereafter referred to as Ttk) which represses the photoreceptor fate. If a cell eliminates Ttk then it becomes a photoreceptor; if not it becomes a cone cell [1,2]. Removal of Ttk is achieved by the activation of the RTK pathway which induces the transcription of phyllopod (phyl) [3,4], a gene encoding an adaptor protein that promotes the Ubiquitination and degradation of Ttk [5] (Fig 1G). The degradation of Ttk in R1/6 precursors is achieved in a relatively simple manner; activation of the RTK pathway by the Drosophila EGF Receptor (DER) removes Ttk (Fig 1G). But the process in the R7 precursor is more complicated because this cell also experiences high N activity which antagonizes the RTK function (Fig 1H), and a much stronger RTK signal is needed in this cell for Ttk removal. This is supplied by the Sevenless (Sev) RTK [7] (Fig 1I) which is expressed at high levels in R7 precursors, and is activated by its ligand Bride of Sevenless (Boss) presented on the adjacent R8 cell [8]. Previously, it was assumed that the Sev-boosted RTK signal simply overcame the N antagonism and promoted the levels of Phyl required for Ttk degradation. In this paper we find that, in addition to the expression of Phyl, a concurrent suppression of ttk transcription is also required for R7 photoreceptor specification, and this is achieved by the action of the Deadpan (Dpn) protein. Dpn is a transcription factor of the helix-loop-helix class [9], closely related to the E (Spl) proteins, and behaves as a direct N response gene in neuroblasts [10] and in the eye [11]. Dpn is expressed in R7 precursors, but not in any other cells of the R7 equivalence group [12] (Fig 1B). This feature led us to investigate the roles it plays in the specification of this cell. In this work, we not only determine that Dpn is required to repress ttk transcription specifically in R7 precursors, but we also establish that ectopic Dpn disturbs the fates of the other cells of the R7 equivalence group, and we infer that Dpn expression needs to be tightly restricted to the R7 precursor. This restriction is established by a two-tier regulation of its gene transcription. First, Ttk represses dpn transcription (in the nascent R7 precursor Ttk levels are high and dpn transcription is repressed). However, as the action of the RTK pathway degrades Ttk, that repression is lost. Second, high N activity is required to promote dpn transcription, and the only cell of the R7 equivalence group that degrades Ttk and has high N activity is R7. Dpn and Ttk act here as mutually repressive transcription factors (Fig 1C), and this relationship lies at the heart of the mechanisms used to silence ttk transcription and to restrict Dpn expression to the R7 precursor. In the nascent R7, Ttk is dominant in the relationship with active transcription and high protein levels, while dpn transcription is repressed and no protein is present (Fig 1D). But once the RTK pathway becomes active this relationship inverts. First Ttk protein is degraded, removing the repression on dpn transcription, and the high N activity then drives dpn gene expression with the resulting Dpn protein acting to inhibit ttk transcription. Thus at the end of the mechanism, Dpn is dominant; with active transcription and high protein levels, whilst ttk transcription is silenced and no protein is present (Fig 1E). Dpn action is required in R7 precursors because the high N activity opposes Ttk degradation. Even with the use of Sev to provide a potent RTK signal in the cell, the degradation mechanism is not able to robustly specify R7 precursors; Dpn repression of ttk transcription is also required. The degradation mechanism removes extant protein, and the transcriptional regulation prevents protein resupply, and the two combine to specify the photoreceptor fate in the R7 precursor.
To determine the roles Dpn plays in R7 specification, we undertook an investigation of its loss-of-function phenotypes. When Dpn is ectopically expressed in R1/6 or cone cell precursors, these cells are re-specified as R7 types (Fig 4A–4C), which highlights the need to restrict Dpn expression in the R7 equivalence group to the R7 precursor. We therefore investigated the mechanisms that restricts Dpn expression, and began with a fine-detailed analysis of its expression pattern. Dpn is first expressed in unidentified cells of very early ommatidial clusters, and then becomes expressed in the R3/4 precluster cells (top bracket Fig 5A' ). As the second-wave-cells are added to the cluster, the expression decays in R3/4, and Dpn is then expressed in R7 precursors (lower bracket Fig 5A' ). This R7 expression is striking in that it is early and ephemeral, and is lost from the cell before any neuronal or R7-specific markers are expressed. This suggested that Dpn expression is likely controlled by the signals that act on the early R7 precursor, and the RTK and N pathways were the most likely candidates. In this section we examine how the RTK pathway and the removal of Ttk control Dpn expression, and in later sections we address the role played by N. When we examined Dpn expression in GMR-ttkRNAi eye discs we saw the normal expression pattern (including R7s) but additionally observed ectopic staining in the cone cell precursors in the more mature ommatidial clusters (cc, Fig 6A). This argues that removal of ttk leads to ectopic expression of Dpn, and we next asked whether Ttk regulates dpn transcription. We began by examining the expression pattern of the dpn. lacZ transgene (Fig 6B), and observed that expression began in the R3/4 and R7 precursors normally, but did not extinguish and persisted into the more mature ommatidial clusters. This reporter appears as a faithful indicator of when dpn transcription is initiated, but whether the later staining is indicative of normal dpn transcription (which we doubt) or whether it is artifactual (perdurance of the lacZ gene product for example) remains unclear. We introduced GMR-ttkRNAi into this background and observed the additional ß-galactosidase staining in cone cell precursors (Fig 6C). The anterior (AC) and posterior (PC) cone cells are the first two cone cells recruited, and the presence of GMR-ttkRNAi re-specifies them both as R7s (as evidenced by Runt staining—red). Interestingly, the ectopic ß-galactosidase staining occurred in PC and not AC (Fig 6C' ). In the next section of the paper we define a critical role for N function in dpn transcription, and note here that N activity terminates first in AC and we suspect that it is the low N activity in this cell that prevents dpn. lacZ expression, and we revisit and expand upon this in the Discussion. The gain of dpn. lacZ expression in cone cells argues that Ttk regulates Dpn expression at the level of transcription. Indeed, if Ttk functions to repress dpn transcription, then the forced expression of Ttk would be expected to prevent the expression of dpn. lacZ in R7 precursors. To this end, we engineered and transformed a UAS. ttk88 transgene and expressed it with GMR. Gal4 in the dpn. lacZ background (Fig 6D). Here we observed the loss of ß-galactosidase staining from the R7 regions of the eye discs (lower bracket) but saw a normal expression at the R3/4 level (upper bracket). Collectively, from the effects of loss and gain of Ttk activity, we infer that Ttk functions to repress dpn transcription. The results above suggest that Ttk degradation is required for the expression of Dpn. But if removal of Ttk were all that was needed for Dpn expression, then following Ttk removal we would expect chronic expression of Dpn in the R7 precursor. This does not happen; the Dpn expression in R7 precursors is short lived. Furthermore, Ttk is degraded in R1/6 precursors and these cells do not express Dpn. We therefore inferred the action of another agency, and since the N pathway is active in the R7 precursor, and ectopic N activation in the eye had already been shown to induce ectopic Dpn expression [11], we began a detailed investigation of the role of N in Dpn regulation.
The Drosophila eye is an exquisitely structured photoreceptive organ that requires a precisely orchestrated developmental program to specify the appropriate cells in the correct position, and in this study we examine the mechanisms that ensures that an R7 photoreceptor is generated in every ommatidium. Within the R7 developmental program, the Boss ligand and Sev RTK are engaged to provide a requisite high-level activation of the RTK pathway which promotes the expression of Phyl and elicits the Ubiquitination and degradation of Ttk. Since Ttk is the transcription factor that represses the photoreceptor fate, the RTK pathway plays an indispensible role in specifying the R7 precursor as a photoreceptor. Indispensible as it is, expression of Phyl alone does not appear to be sufficient for the specification of all R7s, since abrogation of dpn gene function leads to ommatidia in which R7s are absent. We find here that Dpn functions to repress transcription from the ttk locus, and cooperates with the degradation mechanism in Ttk removal. We note that Dpn expression ceases early in the life of the R7 precursor and does not therefore function as a long-term mechanism to prevent the resupply of Ttk. Rather, we see it as acting coincidentally with the degradation mechanism, with both required to achieve effective Ttk elimination, and the resulting unambiguous specification of the R7 photoreceptor fate. Both the N and RTK pathways that converge in this mechanism are also short lived, and it remains unclear how the clearing of Ttk from the R7 precursor locks the cell into the photoreceptor pathway. Dpn is both expressed and required in the R7 precursor, and an elegant mechanism utilizing the combined action of the RTK and N pathways ensures that it is selectively expressed in this cell. First we will address the part played by Ttk and argue that it performs two different roles. The primary role of Ttk is to repress the photoreceptor fate (through the regulation of unknown target genes). The secondary role is to prevent the expression of Dpn, and thereby prevent a reduction in its own transcription. This is the function that it plays in cone cell precursors, where in silencing Dpn it prevents the reduction of its own levels that would make it vulnerable to the low-level RTK activity in those cells. Cone cells and nascent R7s are identical in this regard, but as the high level RTK pathway operates in the R7 precursor, a dramatic change ensues resulting in the expression of Dpn and the shutting down of ttk transcription. However, in the cone cell precursors nothing changes, and Ttk persists in repressing Dpn. The other key aspect of Dpn regulation is the role played by N. Consider the R1/6 precursors; they degrade Ttk (and thereby become photoreceptors) but do not express Dpn. Hence, the simple removal of Ttk is insufficient for the expression of Dpn; high-level N activity is also required. N activity is low in R1/6 precursors but high in the R7 (and cone cell) precursors, where it provides the promotion of Dpn expression. Only in the R7 precursor are the requisite conditions for Dpn expression achieved; the repressor (Ttk) is removed, and the promoter (high N activity) is present. Mutually repressive transcription factor interactions normally resolve to one of two bi-stable states in which one dominates the other. In the R7 precursor we can see how a switch between two such states can be regulated by signals entering the cell. In the nascent R7s, Ttk levels are high and the dpn locus is silent. But this situation is altered once the RTK pathway begins Ttk degradation. As the level of Ttk drops below a certain threshold, dpn transcription initiates, and the resulting Dpn protein then feeds back to silence transcription from the ttk locus. Thus the RTK pathway acts to switch the Ttk/Dpn relationship from Ttk-dominant to Dpn-dominant, and in doing this, the clearance of Ttk protein required for photoreceptor specification is achieved. The Enhancer of Split genes are targets of the N pathway, and a number of them encode transcription factors of the helix-loop-helix class. These transcription factors mediate many N activities, but their expression patterns are poorly understood in the developing eye, and which ones mediate N functions in R7 specification has been difficult to determine. However, an enhancer element from the Mdelta gene is expressed selectively in the R7 precursor (not in the R1/6 or cone cell precursors) and ectopic expression of this gene in R1/6 precursors re-specifies them as R7 types (albeit at a modest frequency) [15,16]. Since Dpn is closely related to the E (Spl) family of proteins, we suggest that it likely has the same ability to re-specify R1/6 precursors as R7s. This begs the question of whether Dpn normally performs this function in R7, or whether it mimics the activity of one or more of the E (Spl) proteins. We cannot currently answer this question, but we note that Dpn is expressed in R7 and not R1/6 and so has the spatial restriction needed to perform that function. We also note that dpn loss of function does not switch the fate of R7s to R1/6s (although this may simply result from redundancy with other factors). Although we remain agnostic on the question of whether Dpn functions to specify the R7 photoreceptor type, the fact that ectopic Dpn expression in R1/6 precursors changes their fate, highlights the fact that are now two reasons why Dpn expression is exclusive to R7 precursors in second wave cells. First, as described above, if it were expressed in cone cell precursors, their lowered Ttk levels might lead them to be specified as photoreceptors. Second, we now see that although Dpn expression in R1/6 precursors would not disturb their specification as photoreceptors (their Ttk is already degraded), it would disturb which type of photoreceptor they would be specified as. When Ttk activity is reduced in cone cell precursors (GMRG4. ttkRNAi), although both the anterior and posterior cone cells (AC and PC) were (as expected) re-specified as R7s (as evidenced by Runt expression) there was a striking asymmetry in the de-repression of dpn. lacZ activity; it occurred in PC and not AC. We infer that this relates to the different N activities in these two cells. Sev expression is a simple meter of N activity in the R7 equivalence group [6], and we noticed many years ago that Sev staining decayed in AC before PC [17]. From this we infer that N levels fall first in this cell, and since dpn. lacZ critically requires high N activity for its expression, the failure to de-repress dpn. lacZ in AC simply results from the absence of the requisite high N activity. In addition to the roles played by Ttk and N in regulating Dpn, we also infer an additional (Phyl-independent) role played by the RTK pathway. Consider the expression of Phyl in a sev mutant background. In one experiment we show that this is not sufficient to restore all R7s (Fig 2C), requiring the co-expression of Dpn (Fig 2E) for full rescue. Yet, in another experiment we show that expression of Phyl in a sev mutant background promotes that requisite Dpn expression (Fig 5D). The simplest explanation here is that the RTK pathway promotes dpn transcription through two independent mechanisms, and only when both are active is a sufficient level of Dpn generated. One mechanism is the process we have described in which RTK activity promotes phyl expression that results in the removal of Ttk. The other mechanism is unknown, but one possibility is that the dpn gene is a direct target of the RTK pathway. In this scenario we envisage the RTK pathway to directly promote dpn transcription, and to indirectly de-repress its expression through promotion of phyl transcription. There is an enigma at the heart of N activity in the R7 precursor; it both promotes and opposes the removal of Ttk from the cell. This enigma likely reflects the evolutionary history of the ommatidium, and suggests that the role of N in opposing Ttk removal represents an ancient function designed to prevent the promiscuous specification of photoreceptors [18], and that in repurposing the cell in the R7 position to become a photoreceptor, the high N activity was used to activate new mechanisms to counteract the primary function. We previously described the transcription of sev as one of these new mechanisms, and now recognize dpn transcription as another. Indeed, we now list four distinct N functions in the R7 precursor; the ancient role that opposes Ttk removal, the two functions that promote Ttk elimination (sev and dpn transcription), and the role that specifies the R7 versus the R1/6 fate once Ttk is degraded. All four functions occur roughly simultaneously, highlighting the impressive, and counterintuitive ways that N is employed in R7 specification, and the R7 photoreceptor is emerging not just as a classic model for studying cell specification, but also becoming as an excellent system to probe a cell’s evolutionary history.
Protocols for adult eye sectioning and antibody staining have been described previously [6,19]. In all data analyses the R8 photoreceptors were individually identified to ensure that they were not mistaken as supernumerary R7s. Primary antibodies: rabbit anti-β galactosidase (Cappel); rabbit anti-GFP, mouse anti-GFP IgG2a (Molecular Probes); guinea pig anti-Runt (gift of J. Reinitz, Stony Brook University, NY, USA); rabbit anti-Runt (gift of Andrea Brand, Gurdon Institute, Cambridge, UK); guinea pig anti-Dpn (gift of James Skeath, Washington University School of Medicine, Washington, USA); mouse anti-Svp (gift of Y. Hiromi, National Institute of Genetics, Japan); rat anti-Elav, mouse anti-Cut (Developmental Studies Hybridoma Bank). Alexa Fluor 488,555,647 conjugated secondary antibodies were used (Molecular Probes). For amplification of the β-galactosidase staining, Donkey anti-rabbit HRP conjugated secondary antibodies were used. Amplification was conducted using Tyramide Signal Amplification (TSA) systems (PerkinElmer) following manufacturer’s protocol. sev[d2], sev. N[act], sev. sev[act], Nts, sev. Su (H) enR, GMR. sev, sev. phyl, ttk. lacZ, sev. Gal4, GMR. Gal4, (see [6,20]. dpn1, UAS. dpn (gifts of Sarah Bray), UAS ttkRNAi [TRiP stock HMS03008], UAS dpnRNAi [TRiP stock HMC03154] (Bloomington Stock Centre). UAS phylRNAi [GD12579 v35469] VDRC, >tub. Gal80> (gift of Gary Struhl). Sev. Su (H) EnR clones were induced as previously described[6]. Clones expressing sevG4-dpn were induced in a hs. flip; >tub. Gal80>/dpn. lacZ; Sev. Gal4/UAS. dpn background by 37°C heat shocks given to larvae at 24-48hrs AEL. dpn1 clones were generated in w, hs. flip; FRT42D, dpn1/FRT42D, w+, M (2) 53C flies by 37°C heat shocks delivered 24-72hrs AEL. Nts flies were reared at 18°C and shifted to 30°C in the third instar for 24 h followed by dissection and immunohistochemistry. The coding sequence for ttk88 was amplified from the cDNA provided by Craig Montell (John Hopkins University School of Medicine) using appropriate PCR primers and cloned into the attB UAS vector using KpnI-EcoRI restriction enzymes, and transformed using standard protocols.
Animals are made from a vast diversity of different cell types, and understanding how they are specified is a major goal of developmental biology. In this study we use the Drosophila R7 photoreceptor as a model system for understanding how cell fate specification occurs. We examine the step when the R7 precursor cell adopts the photoreceptor fate, and ask how the signaling pathways active in the cell are integrated to provide an unambiguous directive to become a photoreceptor. The transcription factor Tramtrack (Ttk) represses the ability of the cell to become a photoreceptor, and how it is removed is the focus of this study. Previous work identified a protein degradation mechanism, and here we describe the role of the transcription factor Deadpan (Dpn) in repressing ttk transcription. We find that both the protein degradation mechanism and transcriptional silencing are required for efficient Ttk removal. Dpn expression needs to be restricted to the R7 precursor and we describe how the mutual antagonism between Ttk and Dpn and the action of the Notch signaling pathway are integrated to ensure that Dpn is selectively expressed in the cell.
medicine and health sciences
gene regulation
regulatory proteins
precursor cells
social sciences
dna-binding proteins
cloning
neuroscience
dna transcription
gene function
transcription factors
molecular biology techniques
eyes
research and analysis methods
specimen preparation and treatment
staining
animal cells
proteins
gene expression
sensory receptors
head
molecular biology
biochemistry
signal transduction
cellular neuroscience
psychology
cell staining
cell biology
anatomy
neurons
genetics
photoreceptors
biology and life sciences
cellular types
ocular system
afferent neurons
sensory perception
R7 Photoreceptor Specification in the Developing Drosophila Eye: The Role of the Transcription Factor Deadpan