The photoreceptors of the Drosophila compound eye are a classical model for studying cell fate specification. Photoreceptors (PRs) are organized in bundles of eight cells with two major types – inner PRs involved in color vision and outer PRs involved in motion detection. In wild type flies, most PRs express a single type of Rhodopsin (Rh): inner PRs express either Rh3, Rh4, Rh5 or Rh6 and outer PRs express Rh1. In outer PRs, the K50 homeodomain protein Dve is a key repressor that acts to ensure exclusive Rh expression. Loss of Dve results in de-repression of Rhodopsins in outer PRs, and leads to a wide distribution of expression levels. To quantify these effects, we introduce an automated image analysis method to measure Rhodopsin levels at the single cell level in 3D confocal stacks. Our sensitive methodology reveals cell-specific differences in Rhodopsin distributions among the outer PRs, observed over a developmental time course. We show that Rhodopsin distributions are consistent with a two-state model of gene expression, in which cells can be in either high or basal states of Rhodopsin production. Our model identifies a significant role of post-transcriptional regulation in establishing the two distinct states. The timescale for interconversion between basal and high states is shown to be on the order of days. Our results indicate that even in the absence of Dve, the Rhodopsin regulatory network can maintain highly stable states. We propose that the role of Dve in outer PRs is to buffer against rare fluctuations in this network.
The ability of Drosophila to perceive color and motion depends on the specific patterning of several Rhodopsin proteins throughout its retina [1]–[3]. The fly retina is a complex three-dimensional structure that consists of a lattice of approximately 800 simple eyes known as ommatidia [4]. As shown in Figure 1, each ommatidium is a bundle of eight photoreceptor neurons (PRs), with six motion detecting PRs (R1–R6) on the perimeter (“outer” PRs) and two smaller, color detecting PRs (R7 & R8) in the middle (“inner” PRs) [5]–[12]. Beginning at the third instar larva, photoreceptors arise following the passage of a morphogenetic furrow across the eye imaginal disc, a monolayer of epithelial cells. As the furrow passes, cells are recruited to ommatidia in a stereotyped manner wherein the R8 photoreceptor is recruited first and then followed by pairs of outer photoreceptors: R2 and R5, R3 and R4, then R1 and R6. Finally, R7 joins the group of cells. During pupation, photoreceptors express specific Rhodopsins (for details of the process, see [13], [14]). A well-studied genetic network controls Rhodopsin protein expression in the eight PR cell types, and enforces a “one-neuron, one-receptor” rule across the majority of the retina, such that each PR expresses one and only one of five types of Rhodopsin proteins (Rh1, Rh3, Rh4, Rh5, or Rh6) [15]. In outer PRs, each cell expresses Rh1 exclusively. There are two major types of ommatidia: in a random subset consisting of approximately 35% of ommatidia, inner PRs exhibit coupling such that when the R7 cell expresses Rh3, the R8 cell expresses Rh5; in the other 65% of ommatidia, when R7 expresses Rh4, R8 expresses Rh6 (for exception, see [16]). Several regulators of Rhodopsin patterning have been discovered and their regulatory interactions are well-characterized [16]–[21]. The K50 homeodomain protein Defective proventriculus (Dve) was recently shown to enforce the “one-neuron, one-receptor” rule in the outer PRs and in the subset of Rh4-expressing R7 cells [22]. In outer PRs, Dve acts together with the activator Orthodenticle (Otd) in an incoherent feedforward loop motif to repress Rh3, Rh5, and Rh6. In the inner PRs, a second coherent feedforward loop that includes the inner PR factor Spalt (Sal), represses Dve thus allowing Rhodopsin expression. In dve mutants, Rh3, Rh5 and Rh6 are de-repressed in outer PRs at levels that vary among cells. Importantly, at the time of Rh expression, Dve is expressed in outer PR cell types where it represses Rh3, Rh5 and Rh6 (Figure 1). Dve' s effect on Rhodopsin expression, however, is modulated by cell-type specific inputs onto the promoters of each rhodopsin gene (Figure 1 and [22]). Most of these inputs have previously been shown to affect rhodopsin expression in inner photoreceptors only. However, Otd and Hazy/Pph13 (a Q50 homeodomain protein) are expressed in all PRs similarly to Dve [18], [21], [23]. Both Otd and Hazy/Pph13 have been shown to be necessary but not sufficient factors for expression of specific rhodopsins in vivo (Figure 1) and sufficient to activate their expression in vitro [24]. Here, we quantitatively studied a molecular null mutation in dve. Since the variable nature of this phenotype requires a quantitative analysis (Figure 2), we developed image analysis algorithms to identify each ommatidium in the retina and discriminate individual PRs in 3D confocal stacks of retinae. We applied these methods to quantify cell-specific effects in dve mutants in thousands of cells by measuring relative protein levels for the three Rhodopsins in the eight different PR types over a time course of four weeks. We measured a wide cell-to-cell variability in Rhodopsin expression. Our ability to precisely quantify Rhodopsin levels enables detection of subtle differences among the outer PR cell types, which manifest in the de-repression of Rhodopsins. We use stochastic models to understand the underlying causes of the observed Rhodopsin distributions. This allows us to attribute differences among cells to the rates of molecular processes such as protein and mRNA synthesis and degradation rates. On the basis of our modeling, we propose a functional role for Dve in outer PRs as a buffer against rare fluctuations in the Rhodopsin regulatory network.
Thirty dve mutant retinae collected at four different developmental time points were analyzed by confocal microscopy and automated image analysis algorithms (see Methods). We developed algorithms that automatically analyze three-dimensional confocal stacks of entire retinae and computationally extract ommatidia from the stacks (Figure 3). Within each ommatidium, the algorithms identified photoreceptor cells and assigned the correct photoreceptor type. For most retinae, our algorithms identified >500 ommatidia, and the number that were sufficiently well-resolved to allow automatic identification of individual photoreceptors was typically in the range 70–200 ommatidia per retina. We quantified Rhodopsin protein levels using a local relative intensity measure (Il) across an interval of z slices identified by the algorithm to maximize both the number of ommatidia with well-resolved PRs and the number of slices used for quantification (Figure S1 and Text S1). This intensity measure, Il, is calculated in each z slice such that each PR' s intensity value (Ipr) is normalized by the local ommatidia background (Iomma), and averaged over the z slices: Il = <Ipr/Iomma>. In order to quantitatively compare Rhodopsin protein levels across individual retinae, we overcame several technical challenges resulting from imaging within the complex retinal tissue (see Methods and Figure 4). Using Il to quantify protein levels, we demonstrated that Rhodopsin distributions are reproducible across replicates (see Methods and Figure S2). We applied our quantification approach using Il to measure cell-specific Rhodopsin expression in dve mutants across several time points. By comparing the measured distributions for different Rhodopsins in different PRs, we observed PR specific effects in de-repression. The distribution of Rhodopsin protein levels we measured should be informative of the processes of mRNA and protein production and degradation for different rhodopsin genes. Previous studies have derived the form of the equilibrium distributions for several different models [26]–[28]. In the simplest model, a constitutively active promoter produces transcripts that are translated into protein [27]. Assuming that protein lifetimes are significantly longer than mRNA lifetimes, the equilibrium distribution of protein levels is a gamma distribution, , with shape parameter α and scale parameter β. The two parameters are related to the rates of mRNA production () and degradation (), and of protein production () and degradation () as follows: , . We expect this model to be appropriate when transcriptional activity is uniform across cells. For example, since wild-type flies strongly repress Rh3, Rh5, and Rh6 in outer PRs, their very low expression levels in those cells should be well-modeled by a uniform basal transcription rate. To test this, we measured Rhodopsin distributions in wild-type flies. We found excellent fits to gamma distributions for all three Rhodopsins, shown in Figure 7 (insets) and Table 1. The difference between Rh3 and Rh5 was pronounced in their values of α (they had identical values of β), indicating differences in their basal promoter activities or protein turnover rates, rather than differences in translation or RNA stability. In contrast to these results from wild-type retinae, the distributions in dve retinae were not fit as well by this simple model (see Figure S3). While in some cases the fit was good at the tails of the distributions, the fit at the modes exhibited significant deviations. The simple model, therefore, predicts fewer cells expressing low levels of Rhodopsin than observed. Comparing dve and wild-type distributions in Figure 7, appears that the mode corresponds to the subpopulation of cells that exhibit basal expression. These deviations from the simple model indicate that protein expression is strongly non-uniform across cells. One possibility is that Rhodopsin promoters can interconvert reversibly between on and off states, and mRNA is produced only when the promoter is on. In several different regimes of this promoter on/off interconversion model, protein levels are predicted to exhibit a gamma distribution. For example, if the gene' s inactivation rate is much larger than both the activation rate and the mRNA degradation rate, then mRNA levels will have a gamma distribution; hence, if protein levels closely track mRNA, the gamma distribution will be observed [26]. Likewise, if the promoter state interconverts significantly faster than proteins are degraded, a gamma distribution is predicted [28]. Our data exhibits significant deviations from both gamma distributions (Figure S3) as well as the general solution obtained in [28] (data not shown). Our data are much more consistent with a two-state network model in which proteins can be produced at either a low, basal rate (off state) or a high rate (on state), and interconversion between the two states is much slower than all other processes. We emphasize that these two states are not necessarily different states of the promoter alone – they may result from differences in other processes and thus correspond to the overall state of the network that regulates Rhodopsin production. Due to the slow interconversion between states, proteins levels in each state exhibit a gamma distribution; the overall distribution is a mixture of two gamma distributions, with parameter pon denoting the fraction of cells in the on state. Fits to the two-state model are shown in Figure 7, where the blue line indicates the distribution for cells in the on state (parameter values are given in Table 1). While such mixture models can suffer from over-fitting, here we have avoided this problem by explicitly measuring the wild-type distributions, which correspond to the off state (Figure 7, inset). The wild-type off state is similar in its position and shape to the dve off state across time points, indicating that the fits are reliable. The model allows us to infer the fraction of cells in the on state at each time point (Table 1). For Rh5, we see initially a very small fraction of cells are on (pon = 0. 02); over the course of 2 weeks, cells get induced until approximately 2/3 of cells are on, a value that is maintained through the 4 week time point. For Rh3, pon remains at ∼0. 3 until 4 weeks, when it decreases to 0. 2. For Rh6, pon fluctuates in the range 0. 2–0. 3 over the time course. The difference between the on and off states of cells, and further implications of our modeling, are addressed in detail in Discussion. The existence of common regulatory mechanisms acting on different genes can often be inferred by measuring correlations of their expression levels within cells. Although Otd regulates three different rhodopsin genes, the presence of Dve in wild-type retinae maximally represses these genes in outer PRs, and correlations cannot be observed. In dve mutants, Rhodopsin expression is revealed in outer PRs. If Otd were acting alone, we would expect that fluctuations in Otd levels would lead to positive correlations between different pairs of Rhodopsins. However, the presence of other regulators in different outer PR cells could modulate the strength or even the sign of correlations. To test this, we plotted the distribution of relative levels for pairs of Rhodopsins that were co-stained, Rh3–Rh6 and Rh5–Rh6 (Figure 8). In the pooled data, across all replicates and photoreceptors, we found statistically significant positive correlations for Rh3–Rh6 at the last time point (Spearman' s ρ≈0. 3, p-value<10−4) and negative correlations for Rh5–Rh6 (Spearman' s ρ≈−0. 35, p-value<10−4) (Figure S4, upper panels). To verify these, we examined correlations within each replicate and within each photoreceptor (Figure S4, lower panels). For Rh3–Rh6, although several replicates exhibit statistically significant correlations, there is no consistent pattern, although there is a strong tendency for positive correlations at the fourth week time point. Some positive correlations may be spurious, e. g. while the measure Il normalizes for variations in local mean intensity across the retina, higher-order intensity variations might account for weak positive correlation between Rh3–Rh6. For Rh5–Rh6, on the other hand, we find reproducible anti-correlation across all three replicates at the 4 week time point, which are consistent across PR types. One explanation for anti-correlation could be exclusion of different Rhodopsins due to limited space and dense packing within the rhabdomere. In that case, we would expect a cloud of points slightly elongated along a line of negative slope. Figure 8 is not consistent with this scenario. Instead, along the increasing Rh5 axis in the 4 week panel, we see a spread of points, with decreasing density in the Rh6 direction. Cells that highly express one Rhodopsin, tend to express the other type at a lower level, and there are few cells that co-express both Rhodopsins at high levels. We conclude the Rh5–Rh6 anti-correlation is the result of factors other than Otd acting in the outer photoreceptors (see Discussion).
In this paper, we established the Drosophila compound eye as a quantitative system for studying cell-specific gene expression. We developed methods to image the complex three-dimensional tissue and automatically identify ommatidia and their constituent PRs. We quantified the cell-specific effects of removal of dve, a key transcriptional repressor that regulates Rhodopsin patterning. In wild type outer PRs, which exclusively express Rh1, Dve represses the expression of Rh3, Rh5, and Rh6. Our data shows that removal of Dve leads to a continuous and wide distribution of expression levels (Figure 7). This cell-to-cell variability exhibits PR-specific differences (Figure 6) suggesting that each cell type may express different levels of Rhodopsin regulators (Figure 1). Importantly, we could only detect these differences by comparing distributions since mean Rhodopsin levels exhibit little change among outer PRs. The fact that Rh3 repression is greatest in R3 and R4 cells is consistent with the fact that the R3/R4 pair is known to undergo additional differentiation after PRs are recruited [29]. Thus, our analysis has revealed that cell fate differences in R3 and R4 yield distinct Rh3 expression in dve mutants. Furthermore, other co-recruited pairs (R1/R6 and R2/R5) also appear to regulate Rh3 levels similarly, suggesting that each pair may have similar levels of Rh3 regulators. Surprisingly, however, such similarities between co-recruited pairs are not detected for Rh5 and Rh6. This observation suggests that outer PRs exhibit differences in the amount and types of Rhodopsin regulators they contain. For example, the R3 cell exhibits a strong tendency to repress Rh5, which is distinct from all other PRs (Figure 6). These differences in Rhodopsin regulation in outer PRs have not previously been shown. While some examples of differential gene expression among outer PR types are known [30]–[32], further study of Rhodopsin regulation in outer PRs is needed to identify the molecular basis for the differences we observe. We showed that Rhodopsin distributions in outer PRs are consistent with a two-state model in which Rhodopsin production occurs at either high or basal rates, which we call respectively the on and off states (Figure 7). To understand the biological basis for these two states, it is instructive to compare the model parameters α and β between them, across time points, for each Rhodopsin (Table 1). In all cases, we find and. The most pronounced differences are between β values, which are an order of magnitude or more larger in the on state than the off state at most time points. Since is the ratio of protein translation to mRNA degradation rates, the model suggests that differences between the two states of cells in the dve mutant may stem from post-transcriptional regulation. The on state could be associated with stabilization of the transcript (reduction of) and/or increased translation (increase of). Differences in Rhodopsin trafficking to the rhabdomere may comprise additional post-transcriptional differences between the states. Because our experiments measure protein level distributions, they do not provide information about the transcription rate independently of, i. e. only the ratio can be determined. Thus, transcriptional differences between on and off states cannot be inferred. However, under the reasonable assumption that, the observation that implies that Rhodopsin turnover rates (discussed below) increase significantly in the on state. Our model assumes that on ↔ off switches occur infrequently compared to the equilibration timescale of the gene expression and protein translation dynamics. This timescale was shown to be of order [28], and can be estimated from measured values of Rhodopsin turnover rates. The best measurements are of Rh1 in the blowfly, Calliphora, a dipteran with the same compound eye organization and patterning of Drosophila. The half-life of Rh1 depends on exposure to light: photo-activated Rh1 has a half-life of 2 hours, while in the dark its half life is 5 days [33]. Subsequent studies suggest that half-lives are longer in Drosophila, e. g. photo-activated Rh1 half-life is ∼13 hours [25]. Our flies were raised in 12 h light-dark cycles, hence 1 day is an approximate upper bound for the equilibration timescale. The rate constant for on ↔ off switching is therefore predicted to be significantly slower than 1 event per day per cell, a result that is fully consistent with our observation that changes in pon occur over the timescale of weeks (Table 1). The timescale for on ↔ off switches, which is comparable to the organismal lifespan, is strikingly slow in view of other systems where stochastic activation occurs on the order of minutes [34], [35]. To some extent, this difference could result from the fact that photoreceptors are post-mitotic cells with overall slower metabolic processes than the actively dividing cells used in previous studies. More important in our view, however, is the fact that the default state of rh3, rh5, and rh6 genes in outer PRs is off, and the strong Rhodopsin-specific activators are not expressed in these cells (Figure 1). Thus, the very slow timescale we infer for on ↔ off interconversion suggests that even in the absence of Dve, the Rhodopsin regulatory network in outer PRs can maintain two extremely stable states of Rhodopsin expression. We therefore propose that the functional role of Dve in these cells is to buffer against rare fluctuations in the Rhodopsin regulatory network. While the mechanism by which Dve buffers against fluctuations is not known, we provide a simple toy network model in which buffering operates directly through Dve' s known behavior as transcriptional repressor of rhodopsin genes. The mathematical model presented in Text S1 is constructed by analogy with well-known bistable networks such as the lac operon [36], [37]. The capacity of photoreceptors to produce large amounts of Rhodopsin protein requires up-regulation of the protein production machinery, which could be induced by Rhodopsin protein itself within a positive-feedback loop. In this case, once Rhodopsin levels increase beyond some threshold, protein production would kick into “high gear”, with concomitant increase in degradation pathways to allow for efficient turnover. Depending on the Rhodopsin mRNA level, the system can be either bistable or monostable, as we show in Text S1. If Rhodopsin mRNA concentration is very low (or very high), the system has a single stable fixed point, corresponding to low (or high) production. In an intermediate range of mRNA levels, the system exhibits bistability (see Figure S5). In the bistable regime, a cell that is in the low production state remains stably in that state. An increase of Rhodopsin levels by rare fluctuation is required to drive the system into the high production state. Once there, it remains stably in high gear. Within this network, the role of Dve is to buffer against fluctuations [38] by ensuring that the system remains in the monostable regime. Removal of Dve increases mRNA levels, moving the system into the bistable range. Thus, in this model Dve exhibits the quintessential hallmark of a buffer: it controls the stability of the system, not its state (see Text S1). Our toy network provides a plausible mechanism of buffering by Dve, but other scenarios are clearly possible, e. g. via additional regulatory interactions. The key point indicated by our results is that Rhodopsin production is not entirely determined by transcript levels. Removal of Dve renders the system poised for activation, making it susceptible to fluctuations. While such fluctuations can in principle occur without activators, it is known that the specific Rhodopsin activators Otd and Hazy/Pph13 are expressed in outer PRs [18], [21], [23]. The levels of these regulators and any others would thus be major determinants of the rate of fluctuations that drive the system from one stable state to the other. Using our approach, we measured correlations between levels of different Rhodopsin proteins. While we observed weak but significant positive and negative correlations between both Rh pairings (Figure S4), only the negative correlations of Rh5 and Rh6 at the 4 week time point were consistent across replicates and cell types. It is noteworthy that in wild-type flies in the inner R8 cell, Rh5 and Rh6 expression is strongly bimodal due to the presence of a double negative feedback loop between warts and melted [19]. Transcriptional reporters of warts [19] and melted (D. Jukam, personal communication) are expressed in low levels in subsets of outer PRs, suggesting that the major effectors of this negative feedback loop are present in outer PRs. The presence of these regulators could result in the anti-correlations of Rh5 and Rh6 revealed in dve mutants. Moreover, recent work has shown that in R8 photoreceptors, Rh6 acting through an uncharacterized pathway has the capacity to inhibit Rh5 expression [39]. Removal of Rh6 leads to progressive expression of Rh5 in R8 PRs, which becomes apparent only after 2 weeks. Our finding of Rh5–Rh6 anti-correlation in dve outer PRs, which develops only after 2 weeks, suggests that a similar Rh6-mediated repression could be active in the outer PRs and revealed in the dve mutant. Our results demonstrate the power of applying quantitative approaches to the study of systems-level problems in developmental biology. Our measurement of cell-specific Rhodopsin distributions enables detection of subtle differences among outer PR types, which were previously unknown. Our modeling of the distributions reveals that post-transcriptional processes play a major role in stochastic de-repression of Rhodopsins in outer PRs in the absence of Dve. More generally, we infer that the cellular state corresponding to basal Rhodopsin production is stably maintained by the Rhodopsin regulatory network even without Dve. On the basis of these findings, we conclude that Dve' s role in outer PRs may be to act as a buffer against fluctuations in the genetic network that controls Rhodopsins.
All retinae were dissected from dve186 flies, a molecular null deficiency [22]. Flies were raised on standard corn meal-molasses-agar medium and grown at 25°C. Flies were staged and then dissected at 4 time points after eclosion: 0 weeks (+/−1 day), 1 week (+/−1 day), 2 weeks (+/−1 days), 4 weeks (+/−1 days). All retinae were stained with Alexa-488 conjugated phalloidin, which binds actin and is used to visualize the actin-dense rhabdomeres. Additionally, two antibodies are used to simultaneously visualize Rhodopsins: retinae were co-stained with one of two pairs, either mouse anti-Rh3 (1∶10) and rabbit anti-Rh6 (1∶2000), or mouse anti-Rh5 (1∶200) and rabbit anti-Rh6 (1∶2000). The fluorophores conjugated to secondary antibodies were Alexa 568 (Rh3), Alexa 568 (Rh5), and Alexa 633 (Rh6). Retinae were dissected and fixed for 15 minutes with 4% formaldehyde at 25°C. Retinae were rinsed twice, washed for at least 2 hours in PBX and then incubated overnight with the primary antibodies diluted in PBX. Retinae were then rinsed twice, washed in PBX for more than 4 hours and incubated overnight with secondary antibodies. Retinae were mounted in Prolong Gold following two additional rinses and a 2+ hour wash. All retinae were cured for at least 5 days but no more than 7 days. Prolong Gold exhibits the best refractive index matching between mounting media and objective oil. Images were acquired using confocal scanning laser microscopy (Leica SP5) with a 40× oil immersion objective (NA = 1. 25). Retinae were mounted on glass slides under a cover slip in Prolong Gold. Optical sections were collected every 250 nm, with 8-bit depth and a pixel size of 160 nm×160 nm. Each channel was scanned separately and images were line averaged. Retinae are approximately 100 microns deep with a maximal diameter of approximately 500 µm. Individual ommatidia radii are in the range 3–5 µm, and the distance between neighboring ommatidial centers is 12–15 µm. The centers of PRs within an ommatidium are approximately 2 µm apart, while the spacing between each PR is less than 250 nm, and thus just within the resolution limit. A single image stack typically consisted of ∼300 optical slices and saved as 3–4 GB of data (Figure 2A).
Complex networks of genetic interactions govern the development of multicellular organisms. One of the best-characterized networks governs the development of the fruit-fly retina, a highly organized, three-dimensional organ composed of a hexagonal grid of eight types of photoreceptor neurons. Each photoreceptor responds to a particular wavelength of light depending on the Rhodopsin protein it expresses. We present novel computational methods to quantify cell-specific Rhodopsin levels from confocal microscopy images. We apply these methods to study the effect of the loss of a key repressor that ensures each photoreceptor expresses only one Rhodopsin. We show that this perturbation has cell-specific effects. Our measurement of the cell-type specific Rhodopsin distributions reveals differences between photoreceptor cells, which could not otherwise be detected. Using mathematical models of gene expression, we attribute this variability to stochastic events that activate Rhodopsin production.
systems biology
developmental biology
biology
computational biology
genetics and genomics
Stochastic De-repression of Rhodopsins in Single Photoreceptors of the Fly Retina