The Seven Stones

Morphogen Paradoxes

Bicoid morphogen gradientA controversy seems to be brewing over some recent theories and quantitative analyses addressing the fundamental question of how the Bicoid morphogen gradient is established and decoded in early Drosophila embryos. The transcription factor Bicoid controls the anterior-posterior patterning of the developing embryo. It is translated from maternal mRNA localized at the anterior pole of the egg and its graded distribution activates, in a concentration-dependent manner, the expression of gap genes, thus determining their spatial domain of expression. Synthesis from a localized source combined with diffusion and uniform degradation of the Bicoid morphogen provides one of the simplest models to explain the approximately exponential shape of its gradient. While, historically, patterning has been thought to rely on the gradient at its steady state – that is when synthesis, transport and degradation processes balance each other – the question arose as to whether steady-state can be reached rapidly enough in the quickly developing embryo (Lander, 2007).

In February last year, Naama Barkai and colleagues published a study (Bergmann et al, 2007) in which they propose that the gradient would in fact be interpreted before it has reached its steady-state, when the gradient is still “moving”. Experimental evidence for a dynamic evolution of Bcd profile between cleavage cycle 11 and 12 is provided using a reporter gene driven by bicoid-binding sites. These authors further show that a pre-steady-state model implies a reduced sensitivity of the gradient readout to variations in the production of morphogen at its source. One biologically relevant example of this robustness is the observation that the domain of expression of hunchback, a Bicoid target gene, shifts much less in embryos from mothers with altered bicoid gene dosage than would be predicted by a steady-state model.

A few months later, Thomas Gregor and colleagues published two papers (Gregor et al, 2007a, 2007b) reporting a detailed analysis of the profile and dynamics of the Bicoid gradient. Quantitative in vivo imaging of a transgenic bicoid-eGFP reporter revealed several paradoxes. While a stable gradient of nuclear Bicoid is quickly established (within 90min, approx. cleavage cycle 9), the (local) diffusion coefficient of Bicoid, as deduced from photobleaching experiments, appears to be far too small (D=0.3 μm2/s, much less than expected from previous estimations made by injecting labeled dextran molecules) to be compatible with such a rapid establishment of the (long-range) gradient by diffusion alone. These experiments further show that nuclear Bicoid is under a highly dynamic nuclocytoplasmic equilibrium, pointing to a fundamental role for the nucleus in gradient establishment and stability. Finally, the precision with which the Bicoid gradient is transformed into Hunchback expression (see illustration, after Gregor et al 2007b) is estimated to be around 10%. This remarkable level of precision would not only be close to the physical limits of the system, but also strikingly matches the accuracy required to detect changes of Bicoid expression between adjacent cells (10%, equivalent to a difference of only 70 Bicoid molecules per nucleus) and the level of reproducibility of the absolute morphogen concentration from embryo to embryo (10% as well).

In a Correspondence published last week, Bergmann and colleagues (2008) dispute these interpretations and claim that a “reanalysis of their [Gregor et al’s] data demonstrates that their findings are consistent with the well-accepted paradigm of diffusion-based patterning and provides further support for the notion that the Bicoid profile is decoded prior to reaching its steady state”. Thus, according to these authors, constant nuclear Bicoid levels are not indicative of steady-state of the gradient itself given that cytoplasmic levels may still be changing. The small diffusion coefficient of Bicoid would then be an additional argument in favor of the necessity of a pre-steady-state decoding mechanism. If this is the case, the differences in Bicoid levels between adjascent cells would be much bigger at cleavage cycle 9 (50% instead of 10% at cycle 14), thus resolving the paradox of the high precision of the hunchback response.

In their response (Bialek et al, 2008), Gregor and colleagues reply that if cells would make a decision by reading Bicoid concentration at cycle 9, the boundary between expression domains would be 5 cells wide at stage 14 (=\sqrt{2^14/2^9}), while in reality it is only a single cell wide. While they agree that the overall gradient might not be at steady-state at these early stages, they argue that the stability of nuclear Bicoid levels is functionally highly relevant given that Bicoid is a transcription factor. Finally, they also point out that the deduced local diffusion constant is so small that it is in fact incompatible with observing any Bicoid in the middle of the embryo in the first place, thus suggesting the existence of additional mechanisms to explain establishment of the gradient at the scale of the entire embryo. These and some additional arguments lead Bialek et al to conclude that “the small values of the diffusion constant for Bcd we reported are superficially consistent with their model, but the model provides no basis for understanding any of our observations.”

Mmmmh… not an easy one. Those who have additional insights into these subtle but fascinating questions, please let us know!


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    Stefan Baumgartner said:

    Well, the solution is that the SDD model is wrong: the mRNA produces a gradient (as described more than 20 years ago: Frigerio et al., Cell 47, 735-746 (1986). This notion was ignored when the SDD model was proposed in 1988. bicoid mRNA is transported along cortical microtubules. This transport is by far fast enough to explain the dilemna of a lack of diffusion of Bicoid by two orders of magnitude. As such, Bicoid protein is simply translated from an existing mRNA gradient. Check the old paper from 1986!!

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    Stefan Baumgartner said:

    check Development 136, 605-614 (2009): A bicoid mRNA gradient dictates the protein gradient.

    These findings solve many problems mentioned above!

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    Thomas said:

    Many thanks for this interesting update! If I understand correctly, the shape of the bcd mRNA gradient would be dictated by the active and random transport process along the non-polar cortical microtubule network and this shape would be ‘frozen’ when the network disassemble.

    With this model, how important is the precision in the timing of microtubule breakdown to ensure proper gradient establishment and accurate positional information?

    Is it possible to determine experimentally the key parameters of the microtubule-dependent (quasi-)random transport process and build a quantitative model?

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