Abstract
Concentration and DNA-binding kinetics of transcription factors (TFs) are crucial determinants of their function. Using Fluorescence Correlation Spectroscopy (FCS), we have characterized the concentration and cell-to-cell variability of 14 endogenously tagged TFs in live Drosophila imaginal discs. We found that variation in the concentration of different TFs among neighboring cells decreased with increasing average TF concentrations. Among the investigated TFs, Antennapedia (Antp), displayed the lowest average variability and transitioned from a low concentration/high variation state in second instar to a high concentration/low variation state in third instar imaginal discs. Using FCS and temporally resolved gain-and loss-of-function genetic studies we have uncovered that Antp is necessary and sufficient to drive a developmental regulatory switch from a state of auto-activation to a state of auto-repression. This mechanism is controlled by a progressive change in the relative concentrations of preferentially activating and repressing Antp isoforms that bind with different affinities to DNA.
Introduction
In order to understand the mechanisms that control pattern formation and cell fate specification in developing organisms, the intranuclear concentration, DNA-binding kinetics and cell-to-cell variability of relevant TFs need to be quantitatively characterized. TF concentration variability at the tissue level is thought to arise from diverse processes, including protein production and degradation. For example, gene transcription in a given tissue is generally a noisy process. The noise is both intrinsic to the gene expressed (Blake et al., 2003; Elowitz et al., 2002) and also due to variation arising from components of the post-transcriptional cellular machinery (Swain et al., 2002). These can influence the expression and production of functional protein, resulting in protein concentration that exhibits variability among cells.
In undifferentiated tissue or cells, this TF cell-to-cell variability can be the driving force for differentiation. For example, progressive establishment of a Nanog salt-and-pepper expression pattern leads to the formation of primitive endoderm in the mouse preimplantation embryo, whereas loss of the variation results in embryos lacking primitive endoderm entirely (Kang et al., 2013). In Drosophila, Senseless (Sens) expression is required for the establishment of proper number of sensory organ precursors in the ectodermal proneural cluster of cells and unequal concentration among cells is required for their specification (Li et al., 2006). Finally, variation in concentration (rather than its overall average concentration) of the Yan TF causes the transition of developing photoreceptor cells to a differentiated state during Drosophila eye development (Pelaez et al., 2015).
Conversely, in already differentiated tissue or cells, which have acquired a steady state during development, TF variability may need to be counteracted across cells to ensure homogeneity of gene expression patterns and robustness of commitment to a certain transcriptional regime. Such a function has been identified for the Snail (Sna) TF, which is required for the invagination of the mesoderm during Drosophila gastrulation (Boettiger and Levine, 2013), or the Bicoid (Bcd) and Hunchback (Hb) TFs during early embryogenesis (Gregor et al., 2007a; Gregor et al., 2007b; Little et al., 2013). These studies have quantified the tolerable degrees of concentration variability allowing establishment of gene expression territories of remarkable precision in the developing embryo.
Finally, differential fates within the same developmental territory may be induced by TFs deploying different DNA-binding dynamics in between seemingly equipotent cells, despite the existence of very similar concentrations (i.e. low variability). For example, studies on the Oct4 TF in early mouse embryos have shown that differential kinetic behavior of DNA binding, despite equal Oct4 concentration among blastomeres, ultimately dictates an early developmental bias towards lineage segregation (Kaur et al., 2013; Plachta et al., 2011).
So far, variation studies have focused predominantly on monitoring the noise of mRNA production and have dealt almost exclusively with early embryonic patterning TFs and morphogen gradients (Holloway et al., 2011; Holloway and Spirov, 2015; Little et al., 2013; Lucas et al., 2013; Pare et al., 2009). Little information exists about TF variability at the protein level within a tissue, since studies of this sort would require single-molecule sensitivity. Even more importantly, proteins need to be quantified at endogenous levels (Lo et al., 2015).
High sensitivity methods for quantification of proteins of low intracellular abundance, like TFs (Kim et al., 2009; Vaquerizas et al., 2009) usually rely on proteomic analyses based on mass spectrometry. Despite the validity of these methodologies and their evolution to acquire higher accuracy (Brun et al., 2007) and resolution (Hanke et al., 2008), they are inappropriate for studies of variability among cells in the same tissue. They additionally cannot reach the level of single-molecule detection and, most importantly, they cannot be easily applied in developing tissues or cells.
We have previously used methods with single-molecule sensitivity, Fluorescence Microscopy Imaging and FCS, to study Hox TF interactions with nuclear DNA in living salivary gland cells (Papadopoulos et al., 2015; Vukojevic et al., 2010). FCS has been instrumental for quantification of TF dynamics in living cells or tissue in several recent studies (Clark et al., 2016; Kaur et al., 2013; Lam et al., 2012; Mistri et al., 2015; Papadopoulos et al., 2015; Perez-Camps et al., 2016; Szaloki et al., 2015; Tiwari et al., 2013; Tsutsumi et al., 2016). Yet, in these studies, measured protein mobility in overexpressing systems, rather than molecular numbers at endogenous levels.
Recent progress in Drosophila genome engineering led to the establishment of genome-scale resources of endogenously-tagged proteins, modified at the endogenous loci, or by addition of a tagged third copy allele within large genomic fragments (Buszczak et al., 2007; Ejsmont et al., 2011; Ejsmont et al., 2009; Kelso et al., 2004; Morin et al., 2001; Quinones-Coello et al., 2007; Sarov et al., 2016; Venken et al., 2011). These transgenic collections enable, for the first time, systematic studies of TF protein dynamics in vivo at endogenous levels, using methods with single-molecule sensitivity.
In this study, we take advantage of the availability of fly toolkits, in which TFs have been endogenously tagged by different methodologies (6 fosmid lines, 6 BAC lines, 1 FlyTrap line and 1 MiMIC line), to measure the intranuclear concentration of various TFs in vivo by FCS, and their cell-to-cell variability in several fly imaginal discs. The imaginal discs are flat single-layer epithelia comprised of small diploid cells and many TFs are expressed in defined regions within these tissues during development. These TFs were selected on the basis of the following criteria: a) availability of an endogenously-tagged line, b) well characterized expression patterns in imaginal discs, c) representation of most major imaginal discs in our dataset (wing, leg, eye, antennal, genital discs, and also salivary glands) and d) ability to readily visualize them using a standard confocal microscopy setup. Our study identifies Antp as a TF displaying notably lower variability among neighboring cells at the level of protein concentration compared to other TFs and uncovers a mechanism that controls Antp concentration and variation in developing discs.
Results
Characterization of average protein concentrations and cell-to-cell variability of 14 Drosophila TFs
Average concentrations of TFs in neighboring nuclei of third instar imaginal discs were measured by FCS (Figure 1). FCS measurements were preferentially performed in the domains of highest expression in the discs (Figure 1 A-P, white arrows) and have sampled adjacent cell nuclei within these domains. Temporal autocorrelation curves differed among TFs in their amplitude (Figure 1Q), characteristic decay times and the relative contribution of the slow component (Figure 1R). They were fitted with a two-component model of free three-dimensional diffusion and triplet formation (Supplement 1) and interpreted as described in detail in our previous study (Vukojevic et al., 2010). Differences in average concentrations ranging about two orders of magnitude among different TFs, from ~30 nM to ~1.1 μΜ (see Supplement 2 for the calculation) were observed.
FCS analysis of inter-nuclear variability in TF concentration among adjacent nuclei (Figure 2) showed that variation, expressed as variance over squared mean , ranges from ~7 to ~53% (Figure 2 A). We also examined the protein Fano factor values of this dataset (expressed as variance over the mean, ) and obtained values ranging from ~0 to ~20 (Figure 2 A’), in line with previous observations (Sanchez et al., 2011). To evaluate a possible relation of variation to concentration among different TFs, we plotted the variation as a function of average TF concentration (Figure 2 B). We observed elevated levels of variation (CV2 > 0.2) for most TFs, and the variation decreased with increasing average concentrations for all TFs (Newman et al., 2006; Schwanhausser et al., 2011; Taniguchi et al., 2010). But, we also noted that Antp displayed lower variation values for its measured average concentration (CV2 < 0.2), as compared to the other TFs in our dataset (Figure 2 A and B). This distinction prompted us to further examine variation values for average endogenous Antp concentrations among neighboring cells in a collection of cell clusters in the wing and leg discs (Figure 2 C). We observed that the variation decreased steeply with increasing Antp concentration (Figure 2 C), whereas the protein Fano factor increased (Figure 2 C’). This behavior, which is usually indicative of complex regulatory processes at the transcriptional level (Franz et al., 2011; Smolander et al., 2011), led us to further investigate the regulation of Antp transcription genetically.
Control of Antp concentration by auto-repression
We started investigating possible regulatory mechanisms of Antp abundance in imaginal discs by examining whether Antp is able to control its own levels (autoregulation). To do this, we first established that the Antp-eGFP MiMIC line, used in our FCS measurements, represents a functional Antp allele. This fly strain has been generated by conversion of the MiMIC insertion MI02272 to a protein trap insert (Venken et al., 2011). Since critical domains for Antp function, such as the homeodomain and YPWM motif, are not interrupted by the introduction of the eGFP artificial exon (Figure 3 – figure supplement 1), we reasoned that the protein should retain a great portion of its full functionality in vivo. This is indeed the case for synthetic Antp (SynthAntp) peptides lacking the long non-conserved N terminus of the protein, which we have functionally investigated previously (Papadopoulos et al., 2011). In fact, flies bearing the MI02272-eGFP protein trap are homozygous viable with adult escapers and only partial lethality at the pupal stage. Because Antp mRNA is not maternally deposited, this indicates that at least the embryonic, larval and pupal functions of Antp are retained in the Antp-eGFP fusion construct. To investigate whether the Antp-eGFP pattern truly reflects the expected Antp protein distribution, we performed immunostainings of the embryonic ventral nervous system and all thoracic imaginal discs (pro-, meso- and metathoracic leg, wing, haltere and humeral discs) of wild type and heterozygous Antp-eGFP larvae. We observed that the Antp-eGFP fusion protein recapitulates the endogenous Antp expression pattern (prominent colocalization of eGFP and Antp, Figure 3 – figure supplement 2). The expression patterns are similar to the ones observed for Antp mRNA, reporter constructs and protein (Engstrom et al., 1992; Levine et al., 1983; Wirz et al., 1986) in third instar imaginal discs. Together, this data support the notion that Antp function is unaltered in Antp-eGFP MiMIC flies.
In order to address whether Antp is able to auto-regulate its own protein levels, we monitored the concentration of endogenous Antp protein upon overexpression of Antp. To distinguish between overexpressed and endogenous protein, we used SynthAntp transgenes fused to eGFP (SynthAntp-eGFP). These transgenes encode the region between the YPWM motif of Antp and its C terminus (but lack the long and non-conserved N terminus of the protein, against which Antp antibodies have been raised). Importantly, their Antp-specific homeotic function has been validated previously (Papadopoulos et al., 2011). Clonal overexpression of SynthAntp-eGFP in the wing disc (Figure 3A-C’) repressed the endogenous Antp protein, indicating that Antp is indeed able to regulate its own protein levels.
Antp protein can repress Antp gene expression at the transcriptional level
We then asked whether this repression occurs at the transcriptional level. To address this we used a previously characterized reporter of the Antp P1 promoter, Antp P1-lacZ (Zink et al., 1991), containing 17.2 kb of the P1 promoter and the first intron of Antp (see Materials and Methods). This reporter has been shown to recapitulate the expression pattern of the Antp mRNA from the P1 promoter ((Engstrom et al., 1992; Zink et al., 1991) and unpublished results). We induced overexpression clones of SynthAntp-eGFP in the wing disc and observed that Antp lacZ-reporter is repressed in the clones (Figure 3D-F). The auto-repressive capacity of Antp was also validated for all leg discs where Antp is strongly expressed (pro-, meso-, and metathoracic leg discs), using SynthAntp-eGFP and three other Gal4 drivers, Distal-less- (Dll), patched- (ptc) and 69B-Gal4 (Figure 3 – figure supplement 3).
In order to rule out that the observed repression is due to putative dominant negative activity of SynthAntp-eGFP, we repeated the experiment in overexpression clones of the full-length untagged Antp protein that is functionally equivalent to the endogenous Antp. Nevertheless, similarly to SynthAntp-eGFP, we observed repression of the Antp reporter (Figure 3G-I’), suggesting that the transcriptional auto-repressive capacity of Antp is not caused by dominant negative effects of the minimal Antp transgene.
Without excluding additional post-transcriptional mechanisms of auto-repression and with no major Antp auto-regulatory capacity having been identified using a P2 promoter reporter (data not shown) (Engstrom et al., 1992; Zink et al., 1991), our results provide evidence that Antp is able to repress itself from the P1 promoter at the transcriptional level.
Antp protein is able to activate Antp gene transcription in imaginal discs
Antp auto-repression capacity offered a plausible explanation for the observed low variation in Antp protein concentration among third instar cell nuclei. However, overexpression of SynthAntp-eGFP from the Distal-less (Dll) (MD23) enhancer surprisingly resulted in ectopic activation of the Antp P1 reporter in distal compartments of third instar discs, where Antp is normally not detected, such as the wing pouch and the distal portion of the antennal disc (Figure 3 J-M, O-R and eGFP controls in N, S and T-V). This finding is opposite to the aforementioned auto-repressive function of Antp and suggested a role in transcriptional auto-activation.
Importantly, the MD23 Dll-Gal4 driver used is not a homozygous viable line and the Dll TF is itself a repressor of Antp during disc development (Emerald and Cohen, 2004). Therefore, to preclude that the observed P1 activation is due to a Dll sensitized background, we induced Dll RNAi-knockdown clones and confirmed that the absence of Dll is not sufficient to induce Antp ectopic expression or alter its capacity to repress in the endogenous expression domain (Figure 3 – figure supplement 4).
We conclude that, in addition to its capacity to repress, Antp is also able to activate its own transcription.
A temporal switch controls the transition of Antp from a state of auto-activation to a state of auto-repression
We reasoned that, since endogenous Antp were able to control its own expression both positively and negatively, this is likely to occur in a temporally resolved manner. To investigate whether auto-regulation of Antp is temporally controlled in development, we induced overexpression clones of full-length untagged Antp either at 26 h (early – first larval instar) or at 60 h (late – second larval instar) of development and harvested third instar wandering stage larvae for analysis (Figure 4 A). Endogenous Antp protein was monitored by means of eGFP visualization of the MiMIC allele. Induction of clonal overexpression of untagged Antp in discs early in development, just after embryonic hatching (first instar), displayed strong ectopic activation of Antp-eGFP (Figure 4 B-D). Notably, in the case of early-induced clones with auto-activation readout, we found no indication of concurrent repression of Antp within its normal expression domain (dashed red line in Figure 4 E and G). Rather, we observed only Antp activation throughout the disc, even in the Antp normal expression domain. We also confirmed that early auto-activation of Antp takes place at the transcriptional level (Figure 4 – figure supplement 1). In contrast, overexpression clones induced during the late second instar stage repressed Antp-eGFP (Figure 4 H-J).
Together, these results clearly pointed to the existence of a switch in Antp auto-regulatory capacity on its own transcription during development. Starting from a preferentially auto-activating state, it changes into an auto-inhibitory one, at later developmental stages. They also suggested that there is no major temporal overlap between auto-activation and auto-repression.
Antp is required for activation and repression of Antp gene transcription during development
Clonal overexpression experiments provided useful insight as to the ability (i.e. the sufficiency) of Antp protein to display opposing regulatory effects on its own transcription during development. We next sought to confirm that the changes in Antp expression actually require Antp protein, and thereby validate the auto regulatory loop. To this end, we first established that Antp can be efficiently knocked down clonally in the wing disc by monitoring the repression of the MiMIC-derived Antp-eGFP protein (Figure 4 K-L’). Then, we induced early Antp RNAi-knockdown clones, monitored the activity of the Antp P1 reporter and found its expression to be efficiently decreased (Figure 4 M-N’ and eGFP controls in Q-R’). This result is in agreement with the results from early clonal overexpression of the full-length protein, which rather triggers activation of Antp P1 transcription (Figure 4 B-G and Figure 4 – figure supplement 1). We conclude that Antp is required for sustaining its own expression, confirming the existence of an Antp auto-regulatory loop.
Conversely, we found that, when Antp was clonally down-regulated by RNAi at later developmental time points (Figure 4 A), the transcription of the reporter was rather up-regulated in the knockdown clones (Figure 4 O and P). Again, this result is in agreement with the corresponding over-expression experiments, which resulted in repression of Antp protein and transcripts (Figure 3 A-I’, 4 B-D and Figure 3 – figure supplement 3). Hence, next to Antp being sufficient, we have shown that it is also required for both repression and activation of Antp P1 transcription in thoracic imaginal discs. Thus, normal patterns of Antp expression require both auto-activation and auto-repression.
Direct correlation between Antp concentration and homeotic function
With Antp having been identified to possess opposing roles on the regulation of its transcription and the observation of prominent auto-activation in some overexpression assays (Figure 3 J-V and Figure 4 – figure supplement 1), but no obvious auto-activation in others (Figure 3 – figure supplement 3), we reasoned that these differences might be due to a) changes in concentration (i.e. auto-repression and auto-activation might occur at different concentrations of Antp) and b) differences in DNA-binding affinities associated with activation and repression.
To examine the effect of concentration on Antp function, we expressed the same SynthAntp-eGFP construct in the antennal disc, using four different Gal4 driver lines, Dll (MD23)-, ptc-, Dll- (MD713)- and 69B-Gal4. We monitored the concentration of SynthAntp-eGFP by imaging (Figure 5 A-D) and FCS (Figure 5 I and I’), while also assessing its capacity to induce homeotic transformations in the antenna (Figure 5 E-H). We found that the Dll (MD23) line is indeed the strongest driver among the four (Figure 5 A). It caused complete transformations of the distal antennal segments into a tarsus (Figure 5 E), whereas the remaining Gal4 drivers all induced transformations to different extents, progressively weaker for decreasing concentrations (Figure 5 B-D and F-H). The concentration range between the strongest and weakest Gal4 driver was more than eightfold (Figure 5 A-D, I and I’). FCS analysis also showed that, within the experimental error of our measurements, there were no differences among Antp FCS measurements upon induction by ptc-Gal4, Dll-Gal4 and 69B-Gal4 (Figure 5 J), as evident from the overlap of the temporal autocorrelation curves shown in Figure 5 I after their normalization to the same amplitude, Gn(τ) = 1 at τ = 10 μs (Figure 5 J). This indicated that Antp DNA-binding behavior is not altered by overexpression.
Taken together, these results show that different Gal4 drivers yield highly different concentrations of overexpressed Antp (Figure 5 I and I’). Moreover, the homeotic activity of Antp readily depends on, and directly correlates with, the concentration triggered by ectopic activation (Figure 5 E-H).
Antp can repress and activate its transcription at endogenous concentrations
If Antp auto-activation is readily observed only in conditions of very high overexpression (3.8 μΜ), more than 7 times higher than the average endogenous concentration of Antp-eGFP in the wing disc measured by FCS (0.5 μΜ, Figure 1 K and Q, light grey autocorrelation curves), it is possible that Antp auto-repression and auto-activation do not occur at all at physiological Antp concentrations. To analyze whether this is the case, we carefully examined the capacity of Antp to repress and ectopically activate its own expression using the 69B-Gal4 driver. This line triggered concentrations of overexpressed Antp of 473 nM in average (Figure 5 D), similar to endogenous Antp levels (501 nM, Figure 1 K and Q). We confirmed that in the wing disc, endogenous Antp protein is repressed at the base of the wing blade, when it is co-expressed with the 69B-Gal4-driven SynthAntp (Figure 5 K-L, negative controls in M-N and Figure 3 – figure supplement 3). However, we did not observe any ectopic Antp P1 reporter activation caused by 69B-Gal4-mediated overexpression using antibody staining against β-galactosidase (Figure 3 – figure supplement 3 and Figure 3 – figure supplement 4, E-F’). By performing more sensitive X-gal stainings of Antp P1-lacZ activity however, we detected weak but definite induction of Antp P1 expression in the antennal disc (Figure 5 O and negative control in P). We also confirmed the ability of the remaining two Gal4 10/27/2018 drivers (the MD713 Dll-Gal4 and ptc-Gal4) to activate Antp transcription ectopically (Figure 5 – figure supplement 1). We conclude that Antp is able to repress and activate its own transcription at physiologically relevant concentrations, comparable to its normal concentration in the wing disc. Moreover, our data suggest that, for equal Antp concentrations, auto-repression is a more efficient process than auto-activation.
Antp switches from a low-concentration/high-variation to a high-concentration/low-variation state
The Antp gene is able to switch between states of auto-activation and auto-repression and it is likely that the auto-repressive state limits the variation of Antp protein concentration among neighboring cells late in development, in third instar imaginal discs. If so, variation would be higher during earlier stages, when auto-repression does not operate. To test this hypothesis, we monitored the endogenous expression levels and cell-to-cell variation of Antp nuclear concentration in second instar wing and leg discs by FCS, using the Antp-eGFP MiMIC allele (Figure 6 A-D). We observed significantly lower average concentrations of Antp protein in both second instar wing and leg discs, as compared to their third instar counterparts (Figure 6 C and D, compare the amplitudes of the average autocorrelation curves – the amplitude is inversely proportional to the number of molecules). This indicates that endogenous Antp concentration changes from second to third instar in thoracic discs (Figure 6 C). Importantly, variation in concentration among neighboring nuclei in second instar discs was roughly double compared to third instar discs (Figure 6 C), indicating that the increase in concentration is accompanied by a suppression of concentration variability.
FCS measurements were performed either on endogenous Antp in second and third instar wing and leg discs (Figure 6 D and E); or on endogenous and overexpressed Antp in the wing and antennal discs, respectively (Figure 6 F and G). Temporal autocorrelation curves recorded in third instar discs, when normalized to the same amplitude, Gn(τ) = 1 at τ =10 μs (Figure 6 E), displayed a notable shift towards longer characteristic times, as compared to measurements performed in second instar discs. Fitting of the autocorrelation curves (see Supplement 1 for details) identified two average characteristic decay times, τ1 = (800 ± 400) μs and , whereas in the third instar average τ2 was found to be significantly longer, (Student’s two-tailed t-test, p =3·10−7). This observation indicates that endogenous Antp is initially fast moving in the nucleus and participates in considerably fewer interactions with the DNA. However, in later discs, it appears to display much longer decay times, reflected by the shift of τ2 to longer times, indicating longer-lasting interactions with the DNA. In contrast, when FCS measurements were compared between endogenous third instar and overexpressed Antp (Figure 6 F and normalized autocorrelation curves in G) we found indistinguishable decay times, as evidenced from the overlap of the normalized temporal autocorrelation curves, despite more than sevenfold concentration difference (0.5 μΜ, as compared to 3.8 μΜ). Fitting of the autocorrelation curves (see Supplement 1 for details) identified average , similar to the average value obtained from fitting third instar FCS data (Student’s two-tailed t-test, p = 3.3. 10−1). Moreover, the characteristic decay times were found to be independent of the concentration of Antp-eGFP over the whole concentration range examined (Figure 6 – figure supplement 1, A and B), showing that the underlying fluorescence intensity fluctuations are generated by diffusion.
Our results show that Antp starts being expressed at relatively low and highly variable levels in early developing discs, and with the capacity to auto-activate its own transcription. Later in development, it reaches a state of higher average concentrations and considerably lower variation between neighboring cells.
Calculation of the endogenous apparent dissociation constant of Antp from FCS measurements
With concentration of Antp not being sufficient to explain the observed differences in Antp dynamics in the second and the third instar leg and wing discs (compare Figure 6 E with G), we sought to investigate the underlying differences at the level of Antp-DNA binding.
Live imaging of Antp-eGFP in the wing disc (Figure 6 H and I) revealed that Antp is not evenly localized in cell nuclei, it is excluded from the nucleoli and forms bright sites of accumulation in different nuclear compartments (Figure 6 K and L). These are presumably sites of pronounced DNA-binding and transcriptional regulation. Taking advantage of the variation in Antp-eGFP concentration among wing disc nuclei, we constructed DNA-binding curves and calculated the apparent endogenous dissociation constant for Antp in third instar discs (Figure 6 M). We observed that the fraction of DNA-bound Antp, which is reflected by the relative amplitude of the slow component measured by FCS, scales linearly with the total amount of TF (Figure 6 M and Figure 6 – figure supplement 1, A), yielding the following regression equation: y = 0.38x – 5.78. Using the previously described simple two-step model of TF-DNA binding, in which non-specific interactions are faster and precede the specific interactions (Vukojevic et al., 2010), we have calculated the endogenous apparent dissociation constant for specific third instar Antp binding to nuclear DNA to be , (see Supplement 3 for the calculation of the specific dissociation constant and Supplement 4 for the approximation of the number and concentration of specific binding sites for Antp). Importantly, we arrived at a very similar value of the apparent dissociation constant, when it was calculated from the sum of FCS measurements on third instar endogenous and overexpressed Antp (Figure 6 H-J, 209 measurements), namely (Figure 6 N and Figure 6 – figure supplement 2, B, regression equation: y = 0.46x – 7.29, see Supplement 5 for the calculation). Fluorescence Recovery After Photobleaching (FRAP) analysis (Figure 6 – figure supplement 1, C) showed that the dissociation rate of specific Antp-DNA complexes is low . Together, FCS and FRAP measurements indicate that Antp binds with high affinity to its specific binding sites in third instar disc cells (Figure 6 – figure supplement 1, C), as evidenced from the apparent dissociation constant that is in the order of 10 nM. In contrast, the dissociation constant calculated from FCS measurements performed in second instar discs was (Figure 6 N and Figure 6 – figure supplement 2, C, regression equation: y = 0.44x – 2.6, see Supplement 6 for the calculation), indicating considerably lower affinity of Antp binding to the DNA in early discs.
We conclude that Antp binding behavior to the DNA changes from second to third instar discs, transitioning from a state of low affinity to a state of stronger binding on the DNA.
Dynamic control of Antp auto-regulation by different Antp isoforms
The changing binding behavior of Antp on DNA from second to third instar discs and the developmental transition from an auto-activating to an auto-repressing state suggested a causal relationship between the two parameters. However, it provided no explanation as to how this could be regulated molecularly. We, therefore, sought to identify additional regulatory mechanisms that could link the observed differences in DNA-binding affinities to Antp auto-activation and repression.
We have previously established that the transcriptional activation and repression capacities of the Antp TF are favored or disfavored by naturally occurring isoforms differing in as little as four amino acids in the linker between the YPWM motif and the homeodomain (Papadopoulos et al., 2011). Long linker isoforms (8 amino acids) favor transcriptional activation of Antp target genes, whereas short linker ones (4 amino acids) favor repression (Figure 7 A). We, therefore, examined whether the linker length is also responsible for differences in auto-regulation. Ectopic expression of SynthAntp-eGFP peptides featuring a long linker displayed significantly weaker repression capacity on endogenous Antp, as compared to their short linker counterparts (Figure 7 B-E’ and quantified in J). This finding indicates that, also in the case of auto-repression, short linker isoforms function as more potent repressors. Interestingly, the YPWM motif was found to be to a great extent dispensable for auto-repression. Constructs bearing mutations of the YPWM motif to alanines (Figure 7 A) could still repress endogenous Antp (Figure 7 F-I’ and quantified in J). We conclude that different Antp isoforms display differential auto-repressive capacity and that this function resides in a small C-terminal portion of the protein, which includes the homeodomain, whereas the long N terminus and the YPWM motif are dispensable.
If Antp auto-repression is differently regulated by short and long linker isoforms, this may be reflected in the relative abundance of their corresponding mRNA variants. We, therefore, investigated the expression of long and short variants in thoracic imaginal discs by quantifying their relative abundance. We found that both transcript variants are expressed from embryonic to third instar development (Figure 7 K). However, a major redistribution of their relative amounts occurs during this time, starting with excess of long linker transcripts and ending with excess of short linker ones in third instar wing and leg imaginal discs, in line with previous observations (Stroeher et al., 1988).
With short linker isoforms showing enhanced repressor capacity and long linker ones showing enhanced activating capacity, our findings suggest that the switch between auto-activation and auto-repression is controlled at the level of transcript variant abundance (without excluding additional mechanisms).
Relative changes in Antp transcript variant concentration, differential efficiency of their encoding isoforms to repress or activate the Antp gene, the developmental switch of the Antp gene from auto-activation to repression and the different mobilities of Antp between second and third instar imaginal discs all point towards the hypothesis that the two isoforms have different properties in their modes of interaction with the DNA. To investigate this hypothesis, we expressed the two isoforms in third instar wing and antennal discs from the 69B enhancer, which we established to result in Antp concentrations close to (if not below) endogenous levels (Figure 5 D). We performed FCS measurements, sampling cells with similar expression levels of short or long linker Antp-eGFP isoforms (Figure 7 I’). We observed that the short linker isoform displayed shorter characteristic decay times, as evidenced by the shift of average temporal autocorrelation curves towards longer characteristic decay times and the higher relative contribution of the second component as evident from its increased relative amplitude (Figure 7 L’). Similar to our comparative analysis between second and third instar discs (Figure 6 D and E), this finding indicates that the short linker isoform binds to the DNA with considerably higher affinity than its long linker counterpart. Plotting the fraction of the slow FCS component (reflecting the DNA-bound fraction of Antp), as a function of the total Antp concentration (as in Figure 6 M-O) allowed us to calculate the apparent equilibrium dissociation constants for the two isoforms (see Supplement 7 for the calculation) to be (Figure 7 M and Figure 7 – figure supplement 1, D, regression equation: y = 0.34x – 5.31) and (Figure 7 N and Figure 7 – figure supplement 1, E, regression equation: y = 0.24x – 3.28). The tenfold difference in the dissociation constants indicates a tenfold higher binding affinity of the short linker isoform on the DNA, in line with our observations during the developmental transition from second to third instar discs (Figure 6 M-O).
Taken together, our experiments on endogenous and synthetic Antp and on short and long linker variants indicate that differences during disc development can be largely attributed to differences in the affinity of the investigated isoforms.
We have also addressed the efficiency of short and long linker variants in Antp auto-activation during early stages (in the embryo) (Figure 7 – figure supplement 2) and found that the long linker isoform displayed stronger activation capacity of the Antp P1 reporter than its short linker equivalent, but no ectopic activation was observed upon mutation of the YPWM motif (Figure 7 – figure supplement 2).
Taken together, the switch of Antp from an auto-activating to an autorepressive state and the alteration of its DNA-binding behavior during disc development can be largely explained by a developmental regulatory shift in the relative concentrations of preferentially activating and repressing Antp isoforms, which themselves display distinct properties in their modes of interaction with the DNA.
Discussion
In this work, we have characterized the endogenous molecular numbers (concentration) and cell-to-cell variation in the concentration of 14 Drosophila TFs in imaginal discs. With single-point FCS being routinely used for measurements of the concentration of fluorescent molecules and their dynamics in live cells, and with novel FCS methodology for larger scale measurements across a whole tissue or animal constantly emerging (Krieger et al., 2015; Papadopoulos et al., 2015), we expect that the study of variation in protein levels will become readily accessible, complementing the established methods to quantify mRNA variation.
We focused our in-depth mechanistic study on Antp and our findings are summarized in Figure 8. Antp is initially expressed in thoracic imaginal discs, producing (at least) two functionally distinct isoforms, one being a preferentially auto-activating and the other a preferentially auto-repressing species. The two isoforms differ in the mode of interactions they undergo with the DNA, as well as their affinities of DNA-binding, with the activating isoform characterized by low binding affinity and the repressing isoform showing higher binding affinity on the DNA. The differences in affinities between the two isoforms could be owed to a) trans modifications (one isoform being a more efficient DNA-binder than the other), or b) to cis modifications of the corresponding DNA-binding sites at the chromatin level (binding sites being more receptive to binding by one Antp isoform and not the other). Moreover, the two isoforms appear to be initially present in the nuclei in unequal concentrations (higher concentration of the activating isoform). For simplicity, the binding of Antp isoforms to the Antp promoter is depicted as direct, although an indirect mechanism, involving induction or repression of intermediate genes, is also possible. Moreover, binding sites of activating or repressing isoforms may or may not be the same. The aforementioned state is characterized by overall low average concentrations of Antp and high variability of total concentration among neighboring cells. High variation in endogenous Antp protein concentration suggests a high level of transcriptional noise introduced in the regulation of Antp target genes. In fact, it has been well established that increased affinity of TF binding to its target sites readily increases transcriptional noise (Dadiani et al., 2013; Shimoga et al., 2013; Suter et al., 2011).
With Antp transcription being induced in the majority of the cells (without ruling out repression in some of them), the total amount of TF increases and (by means of an unknown regulatory mechanism) the relative distribution of preferentially activating and repressing Antp species is progressively shifted towards the production of more repressing isoforms. Therefore, above a certain concentration threshold, the Antp promoter gradually switches from an activated configuration to a repressed one, as development proceeds. Because this switch is concentration-dependent, it results in higher homogeneity of Antp concentration among cells. Therefore, variation becomes progressively suppressed.
The gradual repression of Antp at the transcriptional level provides a plausible explanation for the measured high variation in concentration at early, and the low variation at later, stages. At the same time, our measurements suggest that no steady state in Antp concentration has been reached until, at least, early third instar development. With the repressor isoforms displaying stronger and longer-lasting binding to the DNA, this state persists, allowing the control of variation, while maintaining Antp at desired levels.
Taken together, our quantitative measurements of protein concentrations, cell-to-cell variation, DNA-binding affinities and differential regulatory capacities of isoforms suggest a simple qualitative model of auto-regulation in the Antp locus. This model should explain how Antp transits from an initial state of low concentration/high variation to a state of high concentration/low variation. Future experiments, in combination with quantitative stochastic modeling, will test whether the proposed model is indeed sufficient to explain the establishment of a stable, high concentration/low variation state of a TF such as Antp.
Is Antp maintenance required for proper development of thoracic discs? In our overexpression experiments in leg and wing discs, we showed that increased Antp concentration during disc development leads to loss of Antp transcripts and protein, which is normally expressed in defined domains throughout disc development. Despite this, flies strongly overexpressing SynthAntp by Dll-Gal4 (MD23) are viable and fertile and can be kept as a stock. The lack of adult Antp loss-of-function phenotypes (due to repression of endogenous Antp) in flies strongly overexpressing SynthAntp requires explanation. One possibility is that Antp function is dispensable during late disc development. A second possibility is that, despite repression of the endogenous Antp gene, SynthAntp rescues its function. We addressed this question and found that SynthAntp can indeed compensate to a great extent for repression of endogenous Antp (Figure 8 – figure supplement 1). Our results indicate that Antp maintenance is required for normal leg and notum development and suggest that endogenous auto-repression within the Antp normal expression domains is in fact an inefficient process, which takes place upon concentration increase only (for example, as a cell-autonomous response to increased variation).
In our dataset of 14 TFs, we have measured values of variation in TF concentration ranging from 40% to 60% in imaginal discs and determined their corresponding protein Fano factor values. Having measured variation of molecular numbers, the obtained data represent true Fano factor values, devoid of scaling factors (Raj and van Oudenaarden, 2009; Sanchez et al., 2011). Our study shows that Fano factors values for all TFs measured are in the range of 0-20, in line with Fano factor values of other TFs determined previously to lie between 0 and 30 (Sanchez et al., 2011). Moreover, the majority of TFs examined show high Fano factor values, Ff > 1, providing strong evidence that transcriptional bursting is likely to be a significant source of the observed cell-to-cell variability.
Contrary to Sens, Fkh and Antp TFs, which displayed Ff > 10, we also identified TFs with low Fano factor values, such as Atonal (Ato), Eyeless (Ey) and Spalt Major (Salm).
Ato acts as a differentiation factor for R8 photoreceptor development in the eye disc and its levels are subject to Notch-Delta signaling and auto-regulation (Baker et al., 1996; Jarman et al., 1995). This mechanism might be sufficient to explain the low variation observed.
For Ey, no mechanism of auto-regulation has been determined yet, but cells displaying low or suppressed noise are thought to reside in an undifferentiated state (Kumar et al., 2014), as assessed for pluripotent stem cells. This view is compatible with the low variation observed for Ey in undifferentiated eye disc cells (anterior to the morphogenetic furrow), and the low Fano factor value Ff ≈ 1 suggests that the transcription of this gene is likely to be a continuous process that occurs with a constant probability and according to the statistics of a Poisson process.
Interestingly, Salm was the only TF examined for which Ff < 1. Given that the distribution of Salm is constrained by the Decapentaplegic (Dpp) signaling pathway that controls growth and patterning during imaginal disc development (Organista et al., 2015), it is perhaps not surprising that Salm displays a pattern of occurrence that is more regular than the randomness associated with a Poisson process.
Finally, we have measured Ff = 10 for Sex combs reduced (Scr). This value is equal to the value measured for Scr mRNA transcripts in the embryo (Boettiger and Levine, 2013; Pare et al., 2009). Quantification of Scr mRNA (and also the ones of Ubx and Deformed (Dfd)) in stage 11 embryos exhibited a very high degree of variation (expressed as standard deviation over the mean) among very closely related cells in the Scr expression domain. Although mRNA levels of these TFs may not necessarily reflect protein concentration, comparison of variation in mRNA levels and the corresponding variation in protein concentration of a collection of TFs identified a linear relationship between the two, in a system of simple repression architecture (Sanchez et al., 2011). Taken together, these findings demonstrate that some Hox TFs display increased variation, whether at the level of mRNA or protein concentration, but no generic rule applies (Antp displays comparatively low variation for its concentration).
Our FCS measurements of Antp in diverse tissues, variable concentrations, different developmental time points using distinct isoforms have allowed us to calculate the endogenous apparent dissociation constant for specific Antp-DNA binding to be and the average number of Antp molecules per nucleus to be roughly 7000. The measured Kd value is similar to the apparent dissociation constant calculated for Scr in salivary gland cells (Vukojevic et al., 2010), . The similarity of the values could be explained by the fact that these two TFs are, in terms of their homeodomains and hence DNA-binding properties, the most closely related Hox TFs in flies. Conversely, the roughly threefold stronger binding of Scr could be explained by the fact that Scr homodimerizes upon DNA binding, whereas Antp does not (Papadopoulos et al., 2012). Previous studies examined binding of a recombinant Antp homeodomain peptide to an 18 base pair DNA probe of the BS2 binding site (GAGAAAAAGCCATTAGAG) of a ftz enhancer. These studies derived dissociation constants in the range of 1 – 2 nM (Kd = 1.6 nM) (Affolter et al., 1990; Muller et al., 1988). The tenfold difference between the in vitro binding constant and the apparent in vivo dissociation constant in the two datasets can be explained by: a) the lack of a full-length protein in the in vitro binding assay, b) interactions with other proteins in the densely packed nuclear environment (including, but not limited to, cofactors), which are not present in the in vitro binding constant, or c) the existence of a collection of binding sites with different affinities for Antp.
We have demonstrated that the relative efficiencies of auto-activation and repression rely considerably on the type of Antp isoform engaged in the regulation of the P1 promoter (without excluding additional mechanisms). The size of the linker between the YPWM motif and the homeodomain is important, with short linker isoforms behaving as more efficient repressors than their long linker counterparts. We have previously studied the regulatory capacity (transcriptional activation and repression) of long and short linker Antp isoforms and found that the long linker ones behave as more efficient activators, whereas the short linker ones as effective repressors (Papadopoulos et al., 2011). The observed efficiencies for auto-activation and repression are in line with our previous findings. Moreover, we have demonstrated that auto-repression relies on a small portion of the protein, which contains the homeodomain and its C terminus. The YPWM motif, which binds cofactors, such as Extradenticle (Exd), is dispensable for auto-repression. Whether Exd retains some other role in the auto-repression mechanism described remains to be investigated. In contrast, the YPWM motif is required for at least part of the auto-activating function of Antp, since mutations of it did not allow auto-activation to take place, as compared to the wild type TF. Nevertheless, the question remains as to how the linker size (the three-dimensional spacing between the homeodomain and the YPWM motif) could establish preferences on transcriptional activation or repression, even in the absence of a functional YPWM motif (at least for the repressive function). Our study indicates additional functions of the linker region (or parts of it), independent of the YPWM motif. These could be due to interactions with other cofactors or to differential binding affinities of the homeodomain on a subset of binding sites in the presence of a short or a long amino acid stretch.
Throughout this study, we have used the P1 promoter reporter to dissect the regulatory behavior of Antp on its own expression. The reason for this has been that P1 expression patterns have been thoroughly characterized previously in imaginal discs, (Engstrom et al., 1992; Zink et al., 1991) and unpublished results. Whether additional auto-regulation of Antp occurs also from the proximal P2 promoter in discs, remains to be examined, but our initial experiments provided negative evidence of such regulation, taking place in imaginal discs (data not shown). Auto-activation of the Antp P2 promoter has previously been suggested to take place in the embryonic nervous system and this mechanism has been invoked to explain why no Antp P2 reporter transcription was observed in neuronal cells in Antp mutant embryos (Appel and Sakonju, 1993), whereas global auto-regulation of Hox gene complexes has been shown to be in effect also in mammalian limb development (Sheth et al., 2014). These pieces of evidence suggest evolutionarily conserved mechanisms for establishing (auto-activation) or limiting (auto-repression) Hox levels and variation in different developmental contexts. Nevertheless, at least in the case of the 17.2 kb P1 reporter, for which auto-regulation was shown in imaginal discs (this study), rigorous cis-element analyses should be able to identify relevant enhancers and silencers responsible for Antp auto-regulation. These might well be the regulatory elements identified to function in S2 cultured cells (Saffman and Krasnow, 1994; Winslow et al., 1989), although this hypothesis still needs to be examined.
Negative auto-regulation has been identified as a frequently deployed mechanism for the reduction of noise (cell-to-cell variability) and the increase of regulatory robustness in various systems (Becskei and Serrano, 2000; Dublanche et al., 2006; Gronlund et al., 2013; Nevozhay et al., 2009; Shimoga et al., 2013; Thattai and van Oudenaarden, 2001). Auto-repression has been described for Ultrabithorax (Ubx) in haltere specification (Crickmore et al., 2009; Garaulet et al., 2008) or in Ubx promoter regulation in Drosophila S2 cells (Krasnow et al., 1989), whereas an auto-activating mechanism is responsible for the maintenance of Deformed expression in the embryo (Kuziora and McGinnis, 1988). We have established that an auto-inhibitory mechanism, that is analogous to that of Ubx, exists for Antp. Similar to our observations with Antp, only a small portion of Ubx, containing the homeodomain and C-terminus, is sufficient to mediate Ubx repression (Crickmore et al., 2009).
Materials and Methods
Fly stocks used
The Antp-eGFP MiMIC line has been a kind gift from Hugo J. Bellen. The atonal (VDRC ID 318959), brinker (VDRC ID 318246), spalt major (VDRC ID 318068), yorkie (VDRC ID 318237), senseless (VDRC ID 318017) and Sex combs reduced (VDRC ID 318441) fosmid lines are available from the Vienna Drosophila Resource Center (VDRC) and have been generated recently in our laboratory (Sarov et al., 2016). The fork head (stock 43951), grainy head (stock 42272), Abdominal B (stock 38625), eyeless, (stock 42271), spineless (transcript variant A, stock 42289), and grain (stock 58483) tagged BACs were generated by Rebecca Spokony and Kevin P. White and are available at the Bloomington Stock Center. For the scalloped gene, a GFP-trap line was used (Buszczak et al., 2007), a kind gift from Allan C. Spradling laboratory (line CA07575), with which genome-wide chromatin immunoprecipitation experiments have been performed (Slattery et al., 2013). For the spineless gene, Bloomington stock 42676, which tags isoforms C and D of the Spineless protein has been also tried in fluorescence imaging and FCS experiments, but did not yield detectable fluorescence in the antennal disc, rendering it inappropriate to be used in our analysis. Therefore, we resided to stock 42289, which tags the A isoform of the protein. For the eyeless gene, the FlyFos015860(pRedFlp-Hgr)(ey13630::2XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT line (VDRC ID 318018) has been tried also in fluorescence imaging and FCS experiments, but did not yield detectable fluorescence in the eye disc for it to be used in our analysis. The act5C-FRT-yellow-FRT-Gal4 line used for clonal overexpression or knock down has been described (Ito et al., 1997). The UAS-Antp lines (synthetic and full-length, wild type or modified), as well as UAS-Scr constructs have been previously described (Papadopoulos et al., 2011; Papadopoulos et al., 2010). The Dll-Gal4 (MD23 and MD713) lines have been a kind gift of Ginés Morata (Calleja et al., 1996). 69B-Gal4 and ptc-Gal4 have been obtained from the Bloomington Stock Center. The Antp P1-lacZ has been previously described (Engstrom et al., 1992; Zink et al., 1991) and spans the region between 9.4 kb upstream of the P1 promoter transcription initiation site and 7.8 kb downstream into the first intron, including the first exon sequences and thus comprising 17.2 kb of Antp regulatory sequences (pAPT 1.8). The line used has been an insertion of the pAPT 1.8 vector bearing the P1 promoter regulatory sequences upstream of an actin-lacZ cytoplasmic reporter and has been inserted in cytogenetic location 99F on the right chromosomal arm of chromosome 3. The Dll- and Antp-RNAi lines have been from VDRC, lines GD4607 and KK101774, respectively. UAS-eGFP stock was a kind gift of Konrad Basler. We are indebted to Sebastian Dunst for generating the ubi-FRT-mCherry(stop)-FRT-Gal4(VK37)/CyO line, which drives clonal overexpression upon flippase excision, while simultaneously marking cells by the loss of mCherry. For red-color labeling of clones the act5C-FRT-CD2-FRT-Gal4, UAS-mRFP1(NLS)/TM3 stock 30588 from the Bloomington Stock Center has been used. The MS243-Gal4; UAS-GFP/CyO line was a kind gift from the laboratory of Ernesto Sánchez-Herrero.
Fly genotypes of fluorescence images
Figure 1 A: FlyFos018487(pRedFlp-Hgr)(ato37785::2XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT
Figure 1 B: FlyFos024884(pRedFlp-Hgr)(brk25146::2XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT
Figure 1 C: FlyFos030836(pRedFlp-Hgr)(salm30926::2XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT
Figure 1 D: FlyFos029681(pRedFlp-Hgr)(yki19975::2XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT
Figure 1 E: w1118; PBac(fkh-GFP.FPTB)VK00037/SM5
Figure 1 F: sd-eGFP (FlyTrap, homozygous)
Figure 1 G: w1118; PBac(grh-GFP.FPTB)VK00033
Figure 1 H: FlyFos018974(pRedFlp-Hgr)(Scr19370::2XTY1-SGFP-V5-preTEV-
BLRP-3XFLAG)dFRT
Figure 1 I: FlyFos015942(pRedFlp-Hgr)(sens31022::2XTY1-SGFP-V5-preTEV-
BLRP-3XFLAG)dFRT
Figure 1 J and K: Antp-eGFP (MiMIC) homozygous (line MI02272, converted to an artificial exon)
Figure 1 L: w1118; PBac(Abd-B-EGFP.S)VK00037/SM5 Figure 1 M: w1118; PBac(ey-GFP.FPTB)VK00033
Figure 1 N: w1118; PBac(ss-GFP.A.FPTB)VK00037
Fi.g 1 O and P: w1118; PBac(grn-GFP.FPTB)VK00037
Figure 3 A-C’: hs-flp; act5C-FRT-yellow-FRT-Gal4/+; UAS-SynthAntp-eGFP/+
Figure 3 D-F: hs-flp; act5C-FRT-yellow-FRT-Gal4/+; UAS-SynthAntp-eGFP/Antp P1- lacZ
Figure 3 G-I’: hs-flp/+; act5C-FRT-yellow-FRT-Gal4, UAS-eGFP/+; UAS-Antp (full-length, untagged)/Antp P1-lacZ
Figure 3 J-M and O-R: Dll-Gal4 (MD23)/+; UAS-SynthAntp-eGFP/Antp P1-lacZ
Fig 3 N, S and T-V: Dll-Gal4 (MD23)/+; UAS-eGFP/Antp P1-lacZ
Figure 4 B-J: hs-flp/+; ubi/-FRT-mChery-FRT-Gal4/+; Antp-eGFP (MiMIC)/UAS-Antp (full-length, untagged)
Figure 4 K-L’: hs-flp/+; UAS-Antp-RNAi/+; Antp-eGFP (MiMIC)/act5C-FRT-CD2-FRT-Gal4, UASmRFP1(NLS)
Figure 4 M-P: hs-flp/+; UAS-Antp-RNAi/+; Antp P1-lacZ/act5C-FRT-CD2-FRT-Gal4, UASmRFP1(NLS)
Figure 4 Q-R’: hs-flp/+; act5C-FRT-yellow-FRT-Gal4/+; Antp P1-lacZ/UAS-eGFP
Figure 5 A and E: Dll-Gal4 (MD23)/+; UAS-SynthAntp-eGFP/+
Fig 5 B and F: ptc-Gal4/+; UAS-SynthAntp-eGFP/+
Figure 5 C and G: Dll-Gal4 (MD713)/+; UAS-SynthAntp-eGFP/+
Figure 5 D, H and K, L, O: 69B-Gal4/UAS-SynthAntp-eGFP
Figure 5 M, N and P: 69B-Gal4/UAS-Egfp
Figure 6 A, B, H, I, K and L: Antp-eGFP (MiMIC) homozygous
Figure 6 J: Dll-Gal4 (MD23)/+; UAS-SynthAntp-eGFP/+
Figure 7 B-C’: ptc-Gal4/+; UAS-SynthYPWM,LLAntp-eGFP/+
Figure 7 D-E’: ptc-Gal4/+; UAS-SynthYPWM,SLAntp-eGFP/+
Figure 7 F-G’: ptc-Gal4/+; UAS-SynthAAAA,LLAntp-eGFP/+
Figure 7 H-I’: ptc-Gal4/+; UAS-SynthAAAA,SLAntp-eGFP/+
Figure S2: Antp-eGFP (MiMIC)/TM3, dfd-YFP
Figure S3 upper panel: Dll-Gal4 (MD23)/+; UAS-SynthAntp-eGFP/+ or ptc-Gal4/+; UAS-SynthAntp-eGFP/+ and Dll-Gal4 (MD23)/+; UAS-eGFP/+ or ptc-Gal4/+; UAS-eGFP/+ in control stainings
Figure S3 lower panel: Dll-Gal4 (MD23)/+; UAS-SynthAntp-eGFP/Antp P1-lacZ and Dll-Gal4 (MD23)/+; UAS-eGFP/Antp P1-lacZ in control stainings
Figure S4 A-C’’: hs-flp/+; act5C-FRT-yellow-FRT-Gal4/+; UAS-SynthAntp-eGFP/UAS-Dll-RNAi
Figure S4 D and E: hs-flp/+; act5C-FRT-yellow-FRT-Gal4/+; UAS-eGFP/UAS-Dll-RNAi
Figure S5: hs-flp; act5C-FRT-yellow-FRT-Gal4/+; UAS-SynthAntp-eGFP/Antp P1-lacZ
Figure S6 A-D: Dll-Gal4 (MD713)/+; UAS-SynthAntp-eGFP/Antp P1 -lacZ Figure S6 E-F’: 69B-Gal4/UAS-SynthAntp-eGFP
Figure S6 G-H’: ptc-Gal4/+; UAS-SynthAntp-eGFP/+
Figure S6 I-J’: ptc-Gal4/+; UAS-eGFP/+
Fig S9 A and B: ptc-Gal4/+; Antp P1-lacZ/UAS-YPWM,LLAntp (full-length, untagged)
Figure S9 C and D: ptc-Gal4/+; Antp P1-lacZ/UAS-YPWM,SLAntp (full-length, untagged)
Figure S9 E and F: ptc-Gal4/+; Antp P1-lacZ/UAS-AAAA,LLAntp (full-length, untagged)
Figure S9 G: Antp P1-lacZ/TM3, dfd-YFP
Figure S10 A upper row: Dll-Gal4 (MD23)/+; UAS-SynthAntp-eGFP/+
Figure S10 A lower row, B-C’, H and I: Dll-Gal4 (MD23)/+; UAS-mCitrine-SynthScr/+
Figure S10 D-E: ptc-Gal4/+; UAS-mCitrine-SynthScr/Antp P1-lacZ
Figure S10 F and G: Dll-Gal4 (MD23)/+; UAS-mCitrine-SynthScr/Antp P1-lacZ
Figure S10 J-K’ and M: MS243-Gal4/+; UAS-mCitrine-SynthScr/Dr
Figure S10 L and N-O’: MS243-Gal4/+; UAS-SynthAntp-eGFP/Dr
Figure S10 P and Q: MS243-Gal4; UAS-eGFP
Preparation of second and third instar imaginal discs for FCS measurements
For FCS measurements, imaginal discs (eye-antennal, wing, leg, humeral and genital) and salivary glands were dissected from third instar wandering larvae, or wing and leg discs from second instar larvae, in Grace’s insect tissue culture medium (ThermoFisher Scientific, 11595030) and transferred to 8-well chambered coverglass (Nunc® Lab-Tek™, 155411) containing PBS just prior to imaging or FCS measurements. Floating imaginal discs or salivary glands were sunk to the bottom of the well using forceps.
Immunostainings in embryos and larval imaginal discs
Embryonic stainings were performed using the precise protocol from (Grieder et al., 2000). Larval imaginal discs were stained according to (Papadopoulos et al., 2010). Stainings for the endogenous Antp protein have been performed using a mouse anti-Antp antibody (Developmental Studies Hybridoma Bank, University of Iowa, anti-Antp 4C3) in a dilution of 1:250 for embryos and 1:500 for imaginal discs. eGFP, or eGFP-tagged proteins have been stained using mouse or rabbit anti-GFP antibodies from ThermoFisher Scientific in a dilution of 1:500 in imaginal discs and 1:250 in embryos. mRFP1 was stained using a Chromotek rat anti-RFP antibody. A rabbit anti-β-galactosidase antibody (Cappel) has been used for Antp P1 promoter stainings in a dilution of 1:1000 in the embryo, whereas for stainings in imaginal discs we used the mouse anti-β-galactosidase 40-1a antibody from Developmental Studies Hybridoma Bank, University of Iowa in a dilution of 1:50. Confocal images of antibody stainings represent predominatly Z-projections and Zeiss LSM510, Zeiss LSM700 or Zeiss LSM880 Airyscan confocal laser scanning microscopy systems with an inverted stand Axio Observer microscope were used for imaging. Image processing has been performed in Fiji (Schindelin et al., 2012). For optimal spectral separation, secondary antibodies coupled to Alexa405, Alexa488, Alexa594 and Cy5 (ThermoFischer Scientific) were used.
Colocalization of wild type and eGFP-tagged MiMIC Antp alleles in imaginal discs
To examine whether the pattern of the MiMIC Antp-eGFP fusion protein recapitulates the Antp wild type expression pattern in both embryo and larval imaginal discs, we performed immunostainings of heterozygous Antp-eGFP and wild type flies to visualize the embryonic (stage 13) and larval expression of Antp and eGFP. In this experiment, we 1) visualized the overlap between eGFP and Antp (the eGFP pattern reflects the protein encoded by the MiMIC allele, whereas the Antp pattern reflects the sum of protein produced by the MiMIC allele and the allele of the balancer chromosome) and 2) compared the eGFP expression pattern to the Antp expression pattern in wild type discs and embryos.
Induction of early and late overexpression and RNAi-knockdown clones in imaginal discs
Genetic crosses with approximately 100 virgin female and 100 male flies were set up in bottles and the flies were allowed to mate for 2 days. Then, they were transferred to new bottles and embryos were collected for 6 hours at 25°C. Flies were then transferred to fresh bottles and kept until the next collection at 18°C. To asses Antp auto-activation, the collected eggs were allowed to grow at 25°C for 26 h from the midpoint of collection, when they were subjected to heat-shock by submersion to a water-bath of 38°C for 30 min and then placed back at 25°C until they reached the stage of third instar wandering larvae, when they were collected for dissection, fixation and staining with antibodies. To assess Antp auto-repression, the same procedure was followed, except that the heat-shock was performed at 60 h of development after the midpoint of embryo collection. Whenever necessary, larval genotypes were selected under a dissection stereomicroscope with green and red fluorescence filters on the basis of deformed (dfd)-YFP bearing balancer chromosomes (Le et al., 2006) and visual inspection of fluorescence in imaginal discs.
Measurement of Antp transcript variant abundance
The linker between the Antp YPWM motif and the homeodomain encodes the sequence RSQFGKCQE. Short linker isoforms encode the sequence RSQFE, whereas long linker isoforms are generated by alternative splicing of a 12 base pair sequence encoding the four amino acid sequence GKCQ into the mRNA. We initially designed primer pairs for RT-qPCR experiments to distinguish between the short and long linker mRNA variants. For the short linker variant, we used nucleotide sequences corresponding to RSQFERKR (with RKR being the first 3 amino acids of the homeodomain). For detection of the long linker variant we designed primers either corresponding to the RSQFGKCQ sequence, or GKCQERKR. We observed in control PCRs (using plasmid DNA harboring either a long or a short linker cDNA) that primers designed for the short linker variant still amplified the long linker one. Moreover, with linker sequences differing in only four amino acids, encoded by 12 base pars, primer pairs flanking the linker could also not be used, since both variants would be amplified in RT-qPCR experiments with almost equal efficiencies. Therefore, we used primer pairs flanking the linker region to indiscriminately amplify short and long linker variants, using non-saturating PCR (18 cycles) on total cDNA generated from total RNA. We then resolved and assessed the relative amounts of long and short linker amplicons in a second step using Fragment Analyzer (Advanced Analytical). RNA was extracted from stage 13 embryos, second instar larvae at 60 h of development, and leg or wing discs from third instar wandering larvae using the Trizol® reagent (ThermoFischer Scientific), following the manufacturer’s instructions. Total RNA amounts were measured by NanoDrop and equal amounts were used to synthesize cDNA using High-Capacity RNA-to-cDNA™ Kit (ThermoFischer Scientific), following the manufacturer’s instructions. Total cDNA yields were measured by NanoDrop and equal amounts were used in PCR, using inhouse produced Taq polymerase. 10 ng of plasmid DNA, bearing either a long or a short transcript cDNA were used as a control. PCR product abundance was analyzed both by agarose gel electrophoresis and using Fragment Analyzer (Advanced Analytical).
Fluorescence Microscopy Imaging of live cells and FCS
Fluorescence imaging and FCS measurements were performed on two uniquely modified confocal laser scanning microscopy systems, both comprised of the ConfoCor3 system (Carl Zeiss, Jena, Germany) and consisting of either an inverted microscope for transmitted light and epifluorescence (Axiovert 200 M); a VIS-laser module comprising the Ar/ArKr (458, 477, 488 and 514 nm), HeNe 543 nm and HeNe 633 nm lasers and the scanning module LSM510 META; or a Zeiss LSM780 inverted setup, comprising Diode 405 nm, Ar multiline 458, 488 and 514 nm, DPSS 561 nm and HeNe 633 nm lasers. Both instruments were modified to enable detection using silicon Avalanche Photo Detectors (SPCM-AQR-1X; PerkinElmer, USA) for imaging and FCS. Images were recorded at a 512×512 pixel resolution. C-Apochromat 40x;/1.2 W UV-VIS-IR objectives were used throughout. Fluorescence intensity fluctuations were recorded in arrays of 10 consecutive measurements, each measurement lasting 10 s. Averaged curves were analyzed using the software for online data analysis or exported and fitted offline using the OriginPro 8 data analysis software (OriginLab Corporation, Northampton, MA). In either case, the nonlinear least square fitting of the autocorrelation curve was performed using the Levenberg–Marquardt algorithm. Quality of the fitting was evaluated by visual inspection and by residuals analysis. Control FCS measurements to asses the detection volume were routinely performed prior to data acquisition, using dilute solutions of known concentration of Rhodamine 6G and Alexa488 dyes. The variation between independent measurements reflects variations between cells, rather than imprecision of FCS measurements. For more details on Fluorescence Microscopy Imaging and FCS, refer to Supplement 1.
Sample size, biological and technical replicates
For the measurement of TF molecular numbers and variation (Figures 1 and 2), 7-10 larvae of each fly strain were dissected, yielding at least 15 imaginal discs, which were used in FCS analysis. For the Fkh TF, 7 pairs of salivary glands were analyzed and for AbdB, 12 genital discs were dissected from 12 larvae. More than 50 FCS measurements were performed in patches of neighboring cells of these dissected discs, in the regions of expression indicated in Figure 1 by arrows. Imaginal discs from the same fly strain (expressing a given endogenously-tagged TF) were analyzed on at least 3 independent instances (FCS sessions), taking place on different days (biological replicates) and for Antp, which was further analyzed in this study, more than 20 independent FCS sessions were used. As routinely done with FCS measurements in live cells, these measurements were evaluated during acquisition and subsequent analysis and, based on their quality (high counts per molecule and second, low photobleaching), were included in the calculation of concentration and variability. In Figure 2 A, n denotes the number of FCS measurements included in the calculations.
For experiments involving immunostainings in imaginal discs to understand the auto-regulatory behavior of Antp (Figures 3 and 5–7 and their figure supplements, except for the temporally-resolved auto-activating and repressing study of Antp in Figure 4, as discussed above), 14-20 male and female flies were mated in bottles and 10 larvae were selected by means of fluorescent balancers and processed downstream. Up to 20 imaginal discs were visualized by fluorescence microscopy and high resolution Z-stacks were acquired for 3-5 representative discs or disc regions of interest per experiment. All experiments were performed in triplicate, except for the temporal analysis of Antp auto-regulatory behavior in Figure 4, which was performed 5 times and the quantification of repression efficiency of short and long linker Antp isoforms in Figure 7, which was performed 4 times.
For the quantification of transcript variant abundance in Figure 7 K, RNA and thus cDNA was prepared from each stage 3 independent times (biological replicates) and the transcript abundance per RNA/cDNA sample was also analyzed 3 times.
For the experiment on the requirement of Antp maintenance for proper leg development in Figure 8 – figure supplement 1, A, more than 100 adult flies have been analyzed and this experiment has been performed more than 10 times independently.
Acknowledgements
We are deeply saddened by the unexpected passing of Prof. Walter J. Gehring, at the very inception of this work, when the project was still in the planning and preliminary data gathering stage. Prof. Gehring was an extraordinary human being and a scientific giant, whose work will continue to educate and inspire generations to come. The authors are indepted to Sonal Nagarkar Jaiswal, Paolo Mangahas, and Hugo J. Bellen for creating and sharing with us the Antp-eGFP line. DKP has been supported by a long-term fellowship from the Swiss National Science Foundation (PBBSP-138700) and a long-term fellowship from the Federation of European Biochemical Societies (FEBS) at initial stages of this project. VV has been supported by the Knut and Alice Wallenberg foundation and Karolinska Institute Research Funds. DKP would like to express his gratitude to PT for outstanding scientific, and uninterrupted financial, support. DKP is indebted to Markus Burkhardt, head of the imaging platform at the Center for Regenerative Therapies Dresden (CRTD), for help and discussions regarding FCS experiments in Dresden; Sylke Winkler and the DNA sequencing facility of the Max-Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) for providing assistance with Antp transcript variant quantification; as well as the Light Microscopy facility of MPI-CBG. DKP is also grateful to KS for numerous discussions and support throughout the implementation of this work.
Footnotes
Author contributions: DKP and VV, designed and performed the binding study of Antp, analyzed the data; DKP and KS, designed and performed the study of Antp relative transcript abundance, analyzed the data; YE, provided the Antp P1 reporter line; DKP and PT, conceived the comparative analysis of TFs and the examination of Antp variation in second instar imaginal discs; DKP, conceived the project, designed and performed all remaining experiments, analyzed the data, prepared figures, wrote the manuscript; DKP, KS, YE, LT, RR, VV and PT critically reviewed the manuscript and approved it for submission.