Bcd-GFP construct: initial characterization
To visualize the spatiotemporal dynamics of Bcd concentration we made transgenic Drosophila
embryos in which endogenous Bcd was replaced with a fluorescent eGFP-Bcd fusion protein (called Bcd-GFP hereafter). Flies were generated utilizing a transcript coding for eGFP (Tsien 1998
) fused to the N-terminus of Bcd. As in previous work with a GFP (rather than eGFP) fusion protein (Hazelrigg et al 1998
), the construct contained endogenous bcd
5′ and 3′ UTRs, which are known to mediate anterior localization and translation of bcd
Embryos expressing Bcd-GFP demonstrated an intricate spatial and temporal pattern of fluorescence concentration dynamics that was captured by time lapse two-photon excitation laser scanning microscopy (Denk et al 1990
; Svoboda et al 1997
). A typical image stack of three focal planes from a Bcd-GFP embryo during nuclear cycle 12 is shown in . The fluorescence consisted of two components: bright nuclei and dispersed cytoplasmic fluorescence of lower intensity. The bright nuclei are consistent with previous antibody stainings of Bcd and the fact that Bcd is a transcription factor that should, at some point, be targeted to nuclei. A gradient in fluorescence intensity from anterior to posterior is observed in both the nuclear and cytoplasmic components, also consistent with previous work (Driever & Nüsslein-Volhard 1988a
). See movie in Supplemental Data
FIG. 1 Time-lapse movie of a Drosophila embryo expressing Bcd-GFP using two-photon microscopy. A Typical image stack during nuclear cycle 12 of three focal planes at 30 μm (top panel), 60 μm (mid panel) and 90 μm (bottom panel) below (more ...)
Fluorescence from Bcd-GFP was first detected in anterior surface nuclei during nuclear cycle 9, approximately 90 min after fertilization. As shown in , during each successive nuclear division 9–14, bright nuclei reappear during interphase in the cortical layer. Qualitatively, these images reveal that in any given perimeter region, the total amount of fluorescence increases with each division. However, the intensity within individual nuclei at a given spatial location, appears, qualitatively, to be the same. To provide initial quantification of these effects, at each time point fluorescence was measured within a rectangular region (containing several nuclei and surrounding cytoplasm) that was slid along the embryo perimeter (Houchmandzadeh et al 2002
). As shown in (), a fluorescence gradient that is approximately an exponential function of distance along the embryo can first be measured about one hour after fertilization. The overall intensity increases progressively with development time. In contrast, as shown in the inset to , the intensity measured in a very small box within individual nuclei maintains a fixed profile as a function of distance along the embryo. Both the shape of the spatial decay and the absolute concentration do not appear to change, within measurement error, as a function of nuclear cycle.
A series of control experiments was performed to test the biological relevance of these measurements from two different perspectives. First, we asked the question: does Bcd-GFP functionally substitute for endogenous Bcd and do flies expressing Bcd-GFP develop normally? Second, we asked: does the GFP fluorescence quantitatively represent the distribution of Bcd-GFP, and how does this distribution compare to that of endogenous Bcd?
First, the P[egfp-bcd
]transgene completely rescues the headless anterior defect normally observed in embryos from bcd
mutant mothers; qualitatively, no developmental defects are observed throughout the entire life cycle. Quantitatively, the position along the AP-axis of the cephalic furrow, a strong indicator of the total amount of active Bcd protein, was measured. In embryos with the Bcd-GFP construct in a bcd
-null mutant background, the furrow location at 32.8±1.3% egg length (mean and s.d. over n
= 10 embryos) was identical to that of wild-type embryos (33.8 ± 0.7%; n
= 12); these results are consistent with previous qualitative estimates of approximately 35% (Driever and Nüsslein-Volhard 1988b
). Thus, by both qualitative and quantitative criteria, fluorescent egfp-bcd
behaves indistinguishably from wild-type bcd
in the early embryo, indicating that its localization and translational regulation are conserved, and that the protein is folded properly despite the attached eGFP epitope.
Second, to determine the faithfulness with which eGFP fluorescence reports both Bcd-GFP and endogenous Bcd concentration, fixed embryos were co-labeled with fluorescent antibodies against Bcd and GFP and the fluorescence intensities at locations around the perimeter were compared to each other and to endogenous GFP fluorescence. As summarized in the scatter plots in , the intensity of one probe always is linearly related to the intensity of a different probe. These results demonstrate that, except for differences in background levels, the fluorescence intensity in each case is proportional to the protein concentration. Further implications of these studies, such as the linearity of antibody staining as a method of quantifying relative protein concentration, are discussed in Supplemental Data
FIG. 2 Comparison of Bcd profiles in Drosophila embryos expressing Bcd-GFP. (Embryos were formaldehyde fixed during nuclear cycle 14 and imaged at the mid-sagittal plane via confocal microscopy.) A Embryo stained with GFP antibodies.(Scale bar 100 μ (more ...)
Together, these control experiments reveal that the biological and physical properties of the Bcd-GFP construct reproduce the properties of the endogenous bcd gene. Thus our time lapse observations of dynamics in Bcd-GFP expressing embryos with a bcd-null mutant background quantitatively represent wild-type behavior of the Bcd morphogen gradient.
Constancy of nuclear Bcd concentration
The low magnification time lapse movies in provide initial support for the idea that the gradient in intranuclear Bcd concentration along the embryo develops early and is approximately constant in amplitude and shape over time during nuclear cycles 9–14. This stability is, however, somewhat surprising, since several processes are occurring simultaneously that would be expected to produce local spatial and temporal variations in Bcd concentration: the number of nuclei doubles with each mitotic cycle, the size (diameter) of individual nuclei changes during interphase and between adjacent cycles, Bcd concentrations must rise and fall as nuclear membranes form and dissolve. To further characterize and understand these expected changes in Bcd concentration and what kind of stability may exist at the local level, individual nuclei were tracked over time at higher magnification.
High-resolution time lapse images were taken of nuclei in the anterior half of the embryo, with the field of view reduced to 50 × 50 μm2. depicts two nuclei during interphase 12; the same field of view during mitosis 13 is shown in . The concentration profiles of a thin horizontal rectangle across the center of the nuclei in and the corresponding position in 3B, depicted in , clearly show that the cytoplasmic concentration increases during mitosis at the location between the two nuclei, as expected if Bcd-GFP is freed to diffuse into the cytoplasm as the nuclei enter mitosis.
FIG. 3 Bcd gradient stability. A Close up of two adjacent nuclei expressing Bcd-GFP fluorescence during mid-interphase 12. (Scale bar 10 μm.) B Same field of view as in A during mitosis 13. C Intensity profile of a mean horizontal cross-section through (more ...)
To measure the complete time course of nuclear Bcd-GFP concentration from time lapse movies during cycles 10–14, a fixed size region of interest (ROI) within the nuclear membrane boundary of a single nucleus was determined during interphase. The average intensity within this ROI was determined for a sequence of images starting with the cytoplasm before nuclear membrane formation, through interphase, and continuing in the cytoplasm immediately following nuclear envelope breakdown. To continue the time course into the succeeding cycle, the ROI was switched to an area within the newly forming nucleus that was closest to the position of the first nucleus in order to maintain positional constancy along the AP-axis of the egg. shows two time courses of nuclear concentration, determined by this procedure, from the same embryo.
These data demonstrate a stereotyped response in which peak intranuclear concentrations occur early in each syncytial nuclear cycle. They further reveal that these peaks are nearly constant between nuclear cycles 10 and 14. This is quantified by plotting, in , the concentration in one cycle against that in the preceding cycle. Monitoring 3–4 nuclei in each of 7 different embryos, in most cases the peak is reproducible to better than 10% accuracy. More quantitatively, the ratio of fluorescence intensities from one cycle to the next has a mean of 1.02 and a standard deviation of just 7.9% (N = 77). These results hold over a wide range of absolute concentrations, corresponding to positions from 5% to 35% of the distance from anterior to posterior pole.
also shows the cytoplasmic Bcd-GFP concentration across nuclear cycles 10–14. As for the nuclear concentration, a highly reproducible pattern between interphase and mitosis is observed with cytoplasmic concentration peaks during mitosis representing the equilibration of Bcd-GFP concentration after release from the nuclei, as shown in .
Our data show a surprising coexistence of highly dynamic behavior and precise constancy. Over the course of a single nuclear cycle, the concentration of Bcd varies systematically over a factor of four at the location corresponding to a single nucleus. When the next cycle starts, however, the nuclear Bcd concentration is restored to the same value with ~ 10% accuracy. This reproducibility validates a dynamic version of the positional information hypothesis: From one nuclear cycle to the next, Bcd concentration provides information about the spatial location of the nucleus, and the mapping from position to concentration is invariant across cycles. This stability in the nuclear concentration gradient occurs despite the fact that the total concentration of Bcd in a local cortical region containing multiple nuclei is increasing with each cycle.
Nucleo–cytoplasmic Bcd concentration equilibrium
Although peak Bcd concentrations are reproduced from one nuclear cycle to the next, closer inspection of the nuclear concentration time courses reveals a highly dynamic pattern, both in relative and in absolute terms. We identify three distinct behaviors of nuclear Bcd-GFP concentration during each mitotic cycle, marked as intervals I (nuclear envelope breakdown), II (refilling) and III (interphase) during nuclear cycle 13 in . Note that during interphase interval III, a concentration drop of approximately 30% is observed.
FIG. 4 Nuclear Bcd concentration dynamics. A Typical nuclear (blue) and cytoplasmic (green) Bcd-GFP concentration development during nuclear cycle 13. Three intervals correspond to: I) Nuclear envelope breakdown and diffusive nuclear Bcd-GFP release, II) rapid (more ...)
A fundamental question raised by our results is whether the high intranuclear concentration of Bcd results from simple trapping of Bcd or from a dynamic equilibrium of Bcd molecules exchanging between cytoplasm and the nuclei. To address this question, we bleached the fluorescence of Bcd-GFP in single nuclei during interphase. Nuclear fluorescence recovered, demonstrating that Bcd-GFP is being transported across the nuclear membrane. A typical recovery trace of a single bleached nucleus in cycle 14 is shown in . If repetitively bleached during the slow interphase decay (interval III), the recovering concentration asymptotically approaches the intensity level of adjacent control nuclei. These data suggest that there is a dynamic equilibrium between transport into the nucleus and a loss, either via outward transport or intranuclear protein degradation.
To interpret the results of the photobleaching experiments more quantitatively, a simple model is considerd in which molecules are transported into the nucleus at a rate proportional to the concentration outside in the cytoplasm (kinCout) and leave the nucleus either from transport back into the cytoplasm (at rate kout) or by degradation (at rate kd−nuc). The dynamics for the number of molecules within a nucleus n(t) is given by
where τn is the effective lifetime of the molecule in the nucleus. If the cytoplasmic concentration is approximated as being constant during the measurement (consistent with experiment, see ), then if the photobleaching pulse reduces the number of fluorescent molecules by an amount Δn0 the model predicts an exponential recovery
where n∞ = kinτnCout is the steady state number of molecules in the nucleus, and the observable fluorescence is proportional to the number of molecules n(t). Notice that although recovery of fluorescence after photobleaching provides evidence for Bcd transport into the nucleus, the actual time constant for exponential recovery is related to the rate at which molecules leave the nucleus, either by export or degradation.
Following Equation (3)
, exponential functions were fit to 19 fluorescence recovery curves from 4 embryos in nuclear cycle 14. The time constant determined from these fits is τn
= 68.9±17.6 s (mean ± s.d.). Thus, any disequilibrium in Bcd concentration between the nucleus and the surrounding cytoplasm will be corrected within roughly one or two minutes, which is quite fast on the scale of the nuclear cycles. In particular, this time is short enough that at each moment during interphase interval III (cf ), the nuclear concentration should be in steady state. Why, then, is it drifting downward by ~ 30%?
Geometric factors correlate with nuclear Bcd concentration reduction during interphase
The sizes of nuclei during interphase are not constant; their diameters approximately double in size, as shown quantitatively in . Then perhaps the simplest hypothesis, which could explain the drop in intranuclear Bcd concentration that is evident during interphase interval III, is that as the nuclei increase in volume, the intranuclear concentration is reduced by dilution. Indeed, the model above predicts that the steady state number
of molecules inside the nucleus is given by n∞
, and hence the concentration will be given by
, where rn
is the radius of the nucleus, or
; if all other parameters are fixed, the product
should be constant. In fact this prediction is not consistent with the data:
is an increasing function of time during interphase interval III (green curve in ).
To understand the concentration decrease during interval III it may be useful to look more carefully at the physical factors which determine the rate of transport into the nucleus, kin
. The largest possible value for kin
can be calculated by assuming that the entire spherical surface of the nuclei is a perfect absorber for Bcd molecules that arrive via diffusion. Consideration of this limit is inspired by ligand binding to receptors on a bacterial cell surface (Berg & Purcell 1977
), and is analogous to the diffusion limited rate constant for an enzymatic reaction (Fersht 1985
). Transport occurs through discrete nuclear pores, but the nuclei have of the order of 2500 nuclear pore complexes on their surface (Kiseleva et al 2001
), or 20–40 pores per square micron. If each pore acts as an absorber with a radius of a few nanometers, then this density of pores may be sufficient to make the entire nucleus act as an almost perfect absorber (Berg & Purcell 1977
). If this is true, the rate of Bcd transport into the nucleus should be given by the maximal, diffusion limited rate kin
, where D
is the diffusion constant of Bcd in the surrounding cytoplasm. Diffusion limited transport means that, as the nuclei increase in size, the flux into the nucleus becomes larger, and this should partially offset the effect of dilution. More quantitatively, with kin
our simple model predicts that
We have seen that the cytoplasmic concentration Cout
and the recovery time τn
both are approximately constant during interphase, so we predict that the product
should be constant, which is consistent with our data, as shows.
The combination of this result with the photobleaching experiment supports a picture in which nuclear Bcd is in rapid dynamic equilibrium with cytoplasmic Bcd, and the transport into the nucleus is as fast as possible, being limited by cytoplasmic diffusion. All of the quantities in Equation (4)
are directly measured except for the diffusion constant D
. Thus this simple model could predict a cytoplasmic diffusion constant in terms of our other data,
. The result, as shown in , is D
= 0.37±0.05 μ
/s (mean ± s.d.). Although our results in are consistent with a simple model, there are several caveats. In particular, the nuclear pores might not act as perfect absorbers, either because the kinetics of transport through the pore itself is too slow [but this is unlikely, see Kubitscheck et al (2005)
], or because competition among many protein species lowers the efficiency for transport of Bcd. We thus view the diffusion constant which emerges from the analysis of not as a measurement, but as a prediction to be tested by more direct experiments.
Bcd-GFP diffusion in the cortical cytoplasm
To directly measure the diffusion constant of Bcd-GFP in the cortical cytoplasm, we measured the dynamics of recovery after pattern photobleaching. A rectangular volume 16 × 16 × 7 μm3 in the anterior cytoplasm of Bcd-GFP expressing embryos was photobleached during mitosis 13, when Bcd-GFP is uniformly distributed (locally) throughout the cortical cytoplasm and the cytoplasmic concentration is relatively high. A typical recovery curve is shown in . Recovery curves were fit to solutions of the diffusion equation to estimate D (see Methods). From a total of 21 recovery curves in 4 embryos, our analysis provided a cytoplasmic diffusion constant D = 0.30±0.09 μm2/s (mean ± s.d.) during mitosis 13, which is very close to our estimate from the transport model.
FIG. 5 Cortical diffusion constant measurements by fluorescence recovery after photobleaching. Recovery curve of bleached wild-type Drosophila embryos expressing Bcd-GFP during mitosis 13. Bleaching was done with a scanning two-photon microscope in a volume (more ...)
This cortical diffusion constant is surprisingly small, especially given earlier measurements (Gregor et al 2005
) which showed significantly higher diffusion constants for biologically inert molecules injected into eggs. As we discuss below, it is very difficult to reconcile the small diffusion constant measured for Bcd-GFP with the fact that the gradient forms in about one hour and remains stable over nuclear cycles 10–14. Within the SDD model, the gradient would never reach steady-state with our measured small diffusion constant for Bcd-GFP, given the obvious developmental time constraints.
Cortex vs. core
The diffusion constants for Bcd protein we measure on the surface of the embryos would not be representative of the embryo as a whole if the cortex contains structures that trapped the protein or slowed its diffusion. The classic example of this effect is the slowing of calcium ion diffusion in neurons (Hodgkin & Keynes 1956). In the Supplemenal Data
, we show mathematically how the presence of bindings sites would affect diffusion and observed Bcd concentration in the context of the SDD model. The result is that the presence of fixed binding sites should both slow the diffusion and enhance the total (bound + free) concentration of Bcd, both in proportion to the density of sites.
In sectioned cycle 14 embryos (), we do in fact see a significantly higher concentration of Bcd in the cortex relative to the central core of the egg. The intensity ratio between the cortex and the core increases in an approximately exponential fashion with progressing nuclear cycles and the increasing number of nuclei in the cortical cytoplasm (see ). This suggests that structures which bind Bcd in the cortex are associated with the nuclei themselves. The roughly ten fold enhancement of Bcd concentration, presumably as a result of binding to an enhanced density of fixed sites near the cortex, would be consistent with diffusion constants being an order of magnitude larger in the core of the embryo [see Equation (S9) in Supplemental Data
], although we are unable to measure this directly.
FIG. 6 Bcd concentration accumulation at the egg’s cortex. A Confocal images of hand-cut sections of formaldehyde-fixed wild-type Drosophila embryos. (Scale bar 100 μm.) Embryos have been stained with Bcd antibodies prior to cutting. Each row (more ...)
Bcd diffusion and degradation in unfertilized eggs
To evaluate whether the presence of nuclei, or some nuclear related cytoplamic structure, changes the diffusive behavior of Bcd protein in the cortex, we examined the movement of Bcd in the cortex of unfertilized eggs. Although such eggs have only a single female pronucleus and do not undergo the progressive reorganization of the cortical cytoplasm associated with normal development, they still initiate translation of bcd RNA when the egg is laid. In contrast to the syncytial blastoderm stages described above, Bcd protein in unfertilized eggs show no obvious enrichment in the cortex (see ). To determine the Bcd diffusion constant in such eggs, we performed photobleaching experiments identical to those described earlier for fertilized eggs, In 7 measurements on 3 eggs, the diffusion constant was D = 0.35±0.12 μm2/s, nearly identical to that measured in the cortical cytoplasm of embryos. We conclude that the diffusive behavior measured in the cortex is not dependent on the presence of nuclei or associated cytoplasmic structure and may in fact represent the general behavior of all regions of the egg. In particular, to the extent that the unorganized cortex of the unfertilized egg approximates the environment in the central core of the fertilized egg, our results argue against the possibility that the central cytoplasm would provide a faster path for Bcd diffusion (see Discussion).
Although diffusion constants appear to be identical, the fertilized or unfertilized state of the egg may still impact the final distribution of Bcd protein. To assess this possibility, we measured the average Bcd-GFP fluorescence intensity in 400 μm2 regions near the anterior and posterior poles, and compared the results with those from similar measurements in fertilized eggs. (A more detailed comparison of full in vivo gradients in unfertilized and fertilized eggs will be presented elsewhere.) In the fertilized eggs, intensities were 3.8±0.4 times larger at the anterior pole than at the posterior (mean and s.d. over 13 embryos). This ratio is not as large as the full dynamic range of the gradient (c.f. ) because the averaging region is chosen relatively large so as to have a robust signal from the unfertilized eggs.
In the unfertilized eggs, we found that Bcd intensities at the posterior portion of unfertilized eggs are reproducibly greater than the near background levels observed in that region of fertilized eggs (data not shown), which is in agreement with earlier findings by Driever & Nüsslein-Volhard (1988a)
. At the anterior pole, intensities were lower and the overall ratio was 1.5 ± 0.3 (mean and s. d. over 13 embryos). At 5h, when the anterior intensity of Bcd-GFP in unfertilized eggs was more nearly equal to that in fertilized eggs at cycle 14, the the anterior/posterior ratio had not changed (1.3 ± 0.3; n
= 13 embryos).
These observations suggest that gradient formation in unfertilized eggs differs from that of fertilized eggs in two significant respects: The levels achieved at the posterior pole are higher and the overall shape is flatter. Since the level of Bcd at the posterior pole would be expected to be greatly influenced by degradation, this provides support for considering models in which the nuclei present in fertilized eggs contribute to the degradation process itself (see Discussion).