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. Author manuscript; available in PMC: 2016 Jun 29.
Published in final edited form as: Curr Biol. 2015 Jun 18;25(13):1784–1790. doi: 10.1016/j.cub.2015.05.039

Dynamics of Inductive ERK Signaling in the Drosophila Embryo

Bomyi Lim 1,2, Carmeline J Dsilva 1, Thomas J Levario 3, Hang Lu 3, Trudi Schüpbach 4, Ioannis G Kevrekidis 1,5, Stanislav Y Shvartsman 1,2,*
PMCID: PMC4675133  NIHMSID: NIHMS705082  PMID: 26096970

Summary

Transient activation of the highly conserved extracellular signal regulated kinase (ERK) establishes precise patterns of cell fates in developing tissues. Quantitative parameters of these transients are essentially unknown, but a growing number of studies suggest that changes in these parameters can lead to a broad spectrum of developmental abnormalities. We provide a detailed quantitative picture of an ERK-dependent inductive signaling event in the early Drosophila embryo, an experimental system that offers unique opportunities for high-throughput studies of developmental signaling. Our analysis reveals a spatiotemporal pulse of ERK activation that is consistent with a model in which transient production of a short-ranged ligand feeds into a simple signal interpretation system. The pulse of ERK signaling acts as a switch in controlling the expression of the ERK-target gene. The quantitative approach that led to this model, based on the integration of data from fixed embryos and live imaging, can be extended to other developmental systems patterned by transient inductive signals.

RESULTS AND DISCUSSION

The ERK pathway is a highly conserved phosphorylation cascade that leads to the activation of the extracellular signal regulated kinase (ERK), an enzyme with multiple substrates, including transcription factors and regulators of the cytoskeleton [1]. Studies in model metazoans established that ERK is used recurrently during development and that loss or severe reduction of ERK activation can cause developmental defects, e.g. [2, 3]. Excessive ERK signaling can also lead to developmental abnormalities. Indeed, activating mutations within the components of the ERK pathway have been recently linked to a large class of human developmental syndromes [4]. Individuals with these mutations display a range of morphological and functional phenotypes, from relatively mild facial asymmetries, to short stature, heart defects, and cognitive delay. Since both reduced and excessive ERK activation can derail normal development, developmental functions of ERK must be understood quantitatively, beyond establishing its necessity in any given process.

Developmental functions of ERK are commonly mediated by transient pulses of ERK activation. The first view of these pulses was provided by Gabay et. al., who used an antibody that recognizes the active, dually phosphorylated form of ERK (dpERK) to visualize ERK activation in the Drosophila embryo [5, 6]. Their analysis revealed that ERK is activated first at the poles of the embryo and then again and again, in patterns of increasing complexity. Each of these patterns is transient and is triggered by a locally activated receptor tyrosine kinase, leading to a long-lasting cell response that is essential for proper embryogenesis. The same scenario, in which a pulse of ERK activation induces a stable response, was subsequently revealed in numerous developmental contexts across species [7–9]. Furthermore, changes in the duration and strength of ERK activation pulses can result in improper assignment of cell fates [10].

What are the quantitative parameters of ERK activation pulses in developing tissues? What controls them and how do they control cell responses? The answers to these questions are largely unknown. Here, we answer them for one of the ERK activation pulses in the early Drosophila embryo, an experimental system that serves as an excellent platform for quantitative experiments and data-driven modeling of developmental dynamics. We analyzed ERK signaling that leads to the activation of the gene intermediate neuroblasts defective (ind), a transcription factor needed for specifying a subset of neurons in the future nerve cord (Figure 1B) [11–14]. Using a combination of imaging and genetic techniques, we reconstructed the ERK-dependent activation of ind with high resolution and proposed a model for ERK dynamics and its transcriptional interpretation.

Figure 1. Kinetics of ERK activation and expression of its target gene.

Figure 1

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(A, B) ERK activation and expression of ind during the 3rd and 4th hour of development. (A) Optical cross-sections of embryos. Time indicates an estimated developmental age of a given snapshot (see Fig. S1, S2). 0 min corresponds to the onset of nuclear cycle 14. The embryo in (B) is positioned with its anterior side to the left, and the dorsal side on top. The arrow indicates the position where optical cross-sections were imaged. (C) ERK activation within the ind expression domain is transient. (D) ind mRNA is induced shortly after ERK is activated and persists after ERK activation decays. ERK activates ind by antagonizing its repression by the uniformly distributed transcriptional repressor Capicua. The time courses of dpERK and ind are plotted at the center of the ind-expression domain, indicated by the arrow in (C). Analysis is based on a dataset from 140 embryos, co-stained with dpERK antibody and ind probe, and imaged together in the same microfluidic device.

Since none of the existing techniques for real-time monitoring of ERK activity are working at this time in the embryo, we developed an alternative approach for monitoring the in vivo dynamics of ERK signaling. The main idea is based on the quantitative matching of the morphologies of fixed embryos to a database of live movies of morphogenetic events that coincide with the induction of ind. Because the studied patterns of dpERK and ind are essentially one-dimensional, we focused on one location along the anterior-posterior (AP) axis and collected our data from there, by visualizing the patterns along the dorsoventral (DV) axis (Figure 1A).

ERK induces ind during the 3rd hour of embryogenesis, when the embryo is still a syncytium, with nuclei arranged in a monolayer under the common plasma membrane. During this hour the embryo undergoes cellularization, which transforms the monolayer of nuclei into an epithelial sheet. The embryo then enters gastrulation, a complex process in which the epithelium deforms, paving the way for the formation of the future three-dimensional structures. We monitored both of these processes by live imaging, at the same AP location along the embryo which was used to monitor the patterns of ERK signaling in fixed embryos. Importantly, the Drosophila embryo develops in a highly invariant fashion where both timing and extent of morphological changes are identical from embryo to embryo grown under the same conditions.

The developmental ages of fixed embryos, measured in minutes since the last mitosis in the syncytium, can be estimated by matching their morphological features, such as the extent of cellularization and gastrulation, to the same features in live embryos. Matching is straightforward during cellularization, when the lengths of lateral membranes monotonically increase with time with reproducible kinetics, providing a calibration curve between the age of the embryo and membrane length (Figure S1) [12, 15, 16]. A different strategy is required during gastrulation, when morphological changes cannot be parameterized by a single number, such as membrane length in cellularizing embryos. For this time interval, we constructed a low-dimensional representation of dynamic shape changes, approximating them by the time-dependent superposition of spatial modes, identified as the principal components of a dataset obtained by live imaging of gastrulating embryos (see Supplemental Data for details). The amplitudes of these modes then served as independent variables in a linear regression model that estimated the age of a fixed embryo (Figure S2).

We applied this time estimation protocol to data from 140 fixed embryos, and extracted the signal from a cell where ind is expressed. Our reconstruction revealed a pulse that induces a stable transcriptional response: the superlinear increase of ERK activation is followed by exponential decay with a characteristic time of ~15 minutes (Figure 1C). The ind is expressed within minutes after ERK activation and persists after ERK activation decays to the background level (Figure 1D).

The studied phase of ERK signaling depends on the Epidermal Growth Factor Receptor (EGFR) and is preceded by localized expression of two ligands, Spitz (Spi) and Vein (Vn) [17, 18]. Vn is a secreted neuregulin-like ligand. Spi is a TGFα-like protein, expressed as a transmembrane precursor, which must be processed into active secreted form. This in turn depends on Star, which chaperones Spi out of the endoplasmic reticulum, and Rhomboid (Rho), a protease that cleaves Spi into an active form [19]. All five genes are zygotically expressed: Egfr, spi, and Star are expressed throughout the embryo. vn and rho are expressed in two lateral stripes (Figure 2A, see also Figure S3) [20–23]. We found that ERK activation is abolished in the absence of rho, which is needed to produce active Spi, but is unaffected by removal of vn (Figure 2B). Thus, the EGFR signaling that leads to expression of ind relies only on one ligand, Spi.

Figure 2. Spatiotemporal control of ERK activation.

Figure 2

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(A) Schematic of spatial patterns of the expression of the core components of the EGFR activation system. Egfr, Star, and spi are uniformly expressed, rho and vn are expressed in localized patterns established by the gradient of nuclear Dorsal (see also Fig. S3). (B) Comparison of the ERK activation kinetics between the wild type and mutant embryos reveals that only one ligand contributes to signaling (see also Fig. S3). 36 wild type (WT), 30 vn−/−, and 25 rho−/− embryos were used in the analysis. (C) Joint dynamics of rho and dpERK: ERK activation is delayed with respect to rho expression. Arrows indicate the positions where the signal was analyzed. (D) rho and dpERK at two positions along the DV axis, corresponding to the ventral (light green, light red) and dorsal (dark green, red) borders of the rho pattern. The duration of rho expression decreases from ventral to dorsal boundaries of the domain. Accordingly, ERK activation kinetics changes from a sustained pattern to a transient one. 137 embryos co-stained with rho probe and dpERK antibody were analyzed. Gray shading indicates 5-minute time windows where rho and dpERK are at their peak levels. (E) Average spatial expression of rho and dpERK during the time windows indicated in (D). Spatial expression was plotted along the DV axis (x=0 and 1 indicate the dorsal-most point, x=0.5 indicates the ventralmost point).

The limiting component for Spi secretion, rho is first expressed in a broad lateral domain that later shrinks from the dorsal side to a thin ventrolateral stripe, reflecting the decreasing levels of maternal activators responsible for rho expression (Figure 2C). As a consequence, cells at the center of the future neural ectoderm region express rho for shorter periods of time than cells at the ventral border, resulting in clear differences in the duration of ERK activation (Figure 2D). At all positions, the peak levels of rho and dpERK transients are shifted in time. For instance, at the center of the ind-expression domain, rho peaks at ~10 minutes after the start of nuclear cycle 14 and the peak of dpERK is detected ~45 minutes later (Figure 2D). However, the spatial profiles of rho and dpERK at these two times strongly overlap (Figure 2E), indicating that the peak of rho precisely predicts the peak of ERK, which suggests that Spi is a short-ranged ligand.

These observations lead to the following model for ERK activation: Consider a field of cells, each of which produces receptors at a spatially uniform rate VR. Concurrently, cells are secreting a short-ranged ligand, with a rate that depends on the position within the tissue VL(x). Because the ligand is short-ranged, the model neglects its diffusion from the site of production. New complexes are formed at a rate proportional to the product of ligand and receptor concentrations. If the rates of ligand and receptor production do not depend on time, the concentrations of both free ligands and free receptors grow linearly with time. Then, with zero initial conditions (due to zygotic expression of both receptor and ligand), the rate of complex formation can be approximated as: ∂C(x,t)/∂t~VRVL(x)t2. This implies that the concentration of complexes increases as the cube of time: C(x,t)~VRVL(x)t3 (Figure 3A). Assuming that the level of signaling is proportional to the number of complexes, the model predicts that signaling levels also increase as the cube of time. We found that the rising phase of the ERK activation pulse is indeed accurately described by a power law function of time, whose exponent lies between 3 and 4 (Table S1).

Figure 3. Model for kinetics of ERK activation.

Figure 3

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(A) Schematic of the simple ligand-receptor binding model; the spatial pattern of ligand production along the DV axis is shown on the right. (B) Removal of sna leads to ectopic ERK activation on the ventral side of the embryo. (C) ERK activation in the wild type and sna−/− embryos measured at the ventral-most cell (left) and at the ind-expressing cell (right). The location of the measured cells is indicated by the arrows in (B). 57 WT and 47 sna−/− embryos were analyzed. (D) ERK activation is uniformly reduced in the Egfr+/− embryos. (E) ERK activation was measured at the middle of the ERK expression domain (left) and in the ind-expressing cells (right). ERK activation in the Egfr+/− embryos follows the same kinetics as in the wild type, but with uniformly reduced amplitude (see also Table S2). The location of the measured cells is indicated by the arrows in (D). 66 WT and 67 Egfr+/− embryos were analyzed.

In addition to describing the wild type activation kinetics, this model makes a number of predictions. First, if the ligand is indeed short-ranged, then ectopically produced ligand should affect signaling only very locally and should not perturb the power-law of activation kinetics. To test this prediction, we examined signaling in embryos where ligand is ectopically produced in the ventral cells, where rho is normally repressed by the transcriptional repressor Snail (Figure 3B). In snail mutants rho is expressed in its normal lateral domain as well as throughout the ventral domain [20]. We quantified the ERK activation kinetics at two positions – the ind-expressing cell and the ventralmost cell of the embryo. Consistent with the model, ventrally produced ligand did not affect ERK signaling in lateral cells. Importantly, the kinetics of the endogenous and ectopic ERK activation were identical (Figure 3C, Table S2).

Another prediction is that the signaling level should be uniformly affected by the change in the receptor production rate. We tested this prediction in embryos with a single copy of the Egfr gene (Figure 3D). By analyzing the ERK activation kinetics in the ind-expressing cell and in the middle of the ERK activation domain, we found that ERK activation in Egfr heterozygous embryos follows the same kinetics as in wild type embryos in both domains (Table S2). The amplitude of dpERK, however, was approximately halved in the heterozygous embryos in both positions (Figure 3E). The spatially uniform change of signaling dynamics in response to this perturbation is in contrast to a nonuniform change observed in response to a variation in the pattern of ligand production.

With a small number of modifications, such as including the effects of the termination of ligand production and degradation of ligand-receptor complexes, our model can account for both the rising and falling phases of ERK activation (results not shown). Thus, we propose that the EGFR-dependent pulse of ERK signaling in the embryo reflects the time scales of ligand production and receptor expression. Transient ERK activation by EGFR was examined in a large number of studies with cultured cells and the observed dynamics were commonly explained by models with complex feedbacks within the intracellular part of the ERK cascade, e.g. [24–27]. Our results suggest that a much simpler model, in which the intracellular cascade acts a mere sensor of receptor activation, can account for ERK signaling in a developing embryo (Figure 4A).

Figure 4. Switch-like regulation of ind expression by ERK.

Figure 4

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(A) Dynamics of inductive signaling: a pulse of rho expression translates into a pulse of ERK activation and step-like induction of ind. Both steps in this cascade (rho/dpERK) and (dpERK/ind) rely on multiple components that control multiple aspects of signal reception and interpretation. (B) Lateral views of ERK activation and ind expression at the onset of gastrulation (~3h 10min). ERK activation is significantly reduced in csw-RNAi embryos (Right). ind is induced in a domain that is more narrow and variable along the AP axis. (C) (Left) ERK activation in the csw-RNAi embryos is reduced to about a quarter of the wild type level. The activation is still transient. (Right) The induction of ind is delayed in time. 40 WT and 112 csw RNAi embryos were analyzed. See also Figure S4.

Our quantitative assays can be further used to probe how ERK signaling is affected by multiple components involved in ERK activation and its transcriptional effects (Figure 4A) [28]. For instance, one can ask what aspects of ERK signaling are important for activation of ind? Is the entire transient important? Alternatively, the signal may work as a switch, where ERK activation needs to cross some critical threshold. These questions are relevant for all systems that rely on transient ERK signaling.

Note that ind is induced well before the levels of ERK activation reach its peak (Figure 1C, D). According to the switch-like scenario, ind could be activated by significantly reduced ERK levels, but with a delay. This prediction is borne out by the analysis of embryos in which the ERK activation is strongly reduced by using RNAi against Corkscrew (csw), a tyrosine phosphatase that acts as a positive regulator of the ERK cascade (Figure 4B) [29, 30]. We found that the maximal level of ERK activation in csw-RNAi embryos is reduced to ~25% of the wild type level. Consistent with the switch model, the ind induction is strongly delayed (Figure 4C, Figure S4). Remarkably, ind is induced at approximately the same levels of dpERK in both backgrounds, which fully accounts for the observed induction delay in the csw-RNAi embryos.

Importantly, the power law of the activation kinetics in the csw-RNAi embryos is the same as in the wild type, providing further support for the model in which the time course of ERK activation reflects the ligand/receptor dynamics, with the intracellular part of the pathway acting as a linear signal transducer (Table S1). If the intracellular pathway was significantly nonlinear, then the strong reduction of the amplitude of the signal would have been accompanied by significant distortions of the kinetics of ERK phosphorylation, beyond the observed effect on the amplitude [31, 32]. Thus, our model accounts for both the wild type kinetics of ERK activation and for its changes in mutants. In the future, our model, which is based on the reconstruction of signaling dynamics from snapshots, should be validated by live imaging, using recently developed sensors of ERK signaling [33, 34]. A similar approach, based on the quantitative comparison of signaling in the wild type and mutant embryos, can be used to dissect the precise biochemical effects of mutations identified by studies of developmental abnormalities associated with deregulated ERK signaling [4].

EXPERIMENTAL PROCEDURES

Fly strains, staining, and microscopy

Oregon-R (OreR) flies were used as the wild-type strain. Histone-RFP, Spaghetti squash (Sqh)-GFP, vnL6 [35], rho7M [17], snaIIG05 [36], EgfrCO [37], cswHMS04072 RNAi [38] alleles were used in the experiments. Immunostaining, in situ hybridization, and image processing protocols followed the procedures described in our earlier studies [12]. Nikon A1-RS scanning confocal microscope with a 60× Plan-Apo oil objective was used in imaging. Additional details are provided in the Supplemental Data.

Supplementary Material

1
2

Highlights.

  • High-resolution analysis of ERK dynamics in the early fly embryo

  • A pulse of ERK activation is controlled by short-ranged ligand

  • Switch-like control of neurogenic gene by transient ERK signal

ACKNOWLEDGMENTS

B.L. and S.Y.S. were supported by the National Institutes of Health Grant R01GM086537. C.J.D. was supported by the Department of Energy Computational Science Graduate Fellowship (CSGF), Grant No. DEFG0297ER25308, and the National Science Foundation Graduate Research Fellowship, Grant No. DGE 1148900. S.Y.S. thanks Norbert Perrimon for suggesting the csw RNAi experiment. T.J.L. and H.L. were supported by the National Science Foundation Grant Emerging Frontiers in Research and Innovation (EFRI) 1136913. T.S. was supported by the National Institutes of Health Grant R01GM077620. I.G.K. was supported by the National Science Foundation (CS&E program).

Footnotes

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AUTHOR CONTRIBUTIONS: B.L., C.J.D., T.S., I.G.K., and S.Y.S designed the study. B.L. performed experiments and analyzed data. C.J.D. developed algorithms for temporal reconstruction. H.L. and T.J.L. developed microfluidic devices for imaging experiments. B.L. and S.Y.S. wrote the paper.

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Supplementary Materials

1
NIHMS705082-supplement-1.docx (106.1KB, docx)
2
NIHMS705082-supplement-2.pdf (2.4MB, pdf)

RESOURCES