Needs refs

http://dev.biologists.org/content/141/1/124.short#ref-list-1

http://dev.biologists.org/content/develop/136/4/605.full.pdf

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185443

http://science.sciencemag.org/content/360/6388/543

https://www.biorxiv.org/content/early/2018/03/13/280834

https://www.sciencedirect.com/science/article/pii/S1570963917302108

Needs to be broken up

Needs figures

Bodies are scalable

In the Introduction, I brought up the problem of scaling. When we look within a species we see tall and short (or large and small) individuals but they are all scaled more or less the same. For example, we don't generally see tall people with abnormally long necks or short people with abnormally short legs.

Vitruvian Man

Figure 1:

Leonardo DaVinci noted scaling in humans in this famous anatomical study.

Vitruvian Man (Luc Viatour / https://Lucnix.be) illustrates the generalized proportions we see across the vast majority of humans, regardless of height.

As a general concept, we can say that this kind of scaling must be the result of cross-talk between different cells, tissues, and organs. For example, we might expect a growing bone to stretch muscle fibers and induce them to elongate. We might also expect a bone that grows long to also grow wide in proportion. However, the actual cellular and molecular mechanisms that perform this scaling are often somewhat mysterious. Here we are going to begin to examine a case of scaling in a group of super-manipulable animals - the dipterans.

Bicoid in dipterans: Drosophila

Dipterans are a type of fly, and this group includes the flies you are probably most familiar with - houseflies and Drosophila fruit flies. As was mentioned in the introduction, flies undergo extremely rapid development. This is likely selected for by their transient food sources. Moreover, their embryos and larvae lack protection and are a good source of nutrition for insectivorous predators.

One way to speed up development is to pattern the whole animal at the same time instead of patterning one end first, like we and many other animals do. Drosophila and relatives (often called the "higher dipterans") pattern their anterior and posterior ends at the same time. In fact, the oocyte (egg) is patterned before fertilization even occurs, with the "organizer" of anterior patterning (bicoid) at one end and the "organizer" of posterior patterning (nanos) at the other^1,2. There are many more genes involved in this process, but we will mostly focus here on bicoid since it has been the subject of intense study.

Both the nanos and bicoid organizers in Drosophila are proteins that are found in a concentration gradient. Bicoid is highest at the anterior end, while Nanos is highest at the posterior. Towards the middle of the embryo they are both present at only low concentrations (Figure 2)^1,2,3,4 If you deplete Bicoid from a fly egg, you end up with a fly missing anterior (head) structures and an expanded abdomen. If you deplete Nanos, you end up with a fly missing posterior (abdominal) structure and an expanded head. Weird side note that we can get into later if you are interested - the tip of the head and the tail are patterned by two separate developmental mechanisms. We are going to ignore this for now, but it's a pretty spiffy signal transduction story, so lets keep it in the wings if we get interested in it later.

At this point, I am going to make our first foray into light molecular genetics. If you get a bit confused, please read Briefly, Genetics for some clarification.

When Christiane Nusslein-Volhard first made bicoid mutants, she noticed a couple of interesting things. First, you need to make the moms mutant. This makes sense because it is maternal mRNA that gets pumped into the oocyte, so of course it is the mom's genotype that rules the embryo's morphology in this case. Second, even though she didn't have perfect evidence, she noticed that the Bicoid protein gradient looks like a diffusion gradient. A diffusion gradient is the pattern you get when something is diffusing freely from a point source. Imagine you are baking cookies, a visitor knocks on your door. As you open the door, they notice a faint but terrific smell coming from your house. They make small talk with you and slowly walk towards the source of the smell towards the kitchen. As they get closer to the oven the smell intensifies. You open the oven to take out the cookies, and your kitchen is flooded with the smell of cookie goodness. In this case the baking cookies in your oven are the point source and the smell has formed a diffusion gradient in your house, faintest at the front door and strongest in the kitchen. This mechanism for forming a gradient is called the "SSD model" for synthesis, solute, diffusion^1,⁵. Under this "classic" model, Bicoid mRNA is localized to the anterior of the embryo and serves as the point source for Bicoid protein. Bicoid protein then diffuses away from the point source. One early criticism of this model is that over time the gradient disappears - Bicoid protein should eventually diffuse all the way to the posterior of the embryo. The explanation for the finding of a "steady state" gradient is that Bicoid protein degrades at a constant rate. This maintains the concentration gradient over time, since the only new source of Bicoid protein is at the Anterior pole of the embryo. This slight modification to the SSD model is called the "SDD model" for Synthesis, Diffusion, Degradation.

However, in the case of bicoid, this model has its critics, as we will soon find out!

both bicoid mRNA and protein show a gradient. Both are higher in the anterior of the pre-cellular embryo

The problem of scaling in dipterans

The SDD model looks good on paper, but there is a funny fact about dipteran embryos that forces us to take a closer look at it. Dipteran embryos vary in size tremendously. Drosophila embryos are teensy-tiny, about a third to half a centimeter long. Housefly embryos, on the other hand, are a whopping 1.5 centimeters long. What does this mean for the SSD model? Imagine your point source in our cookie example: a delicious smell coming from your oven. Only now you are cooking in the UCen dining hall. Someone walks into the UCen, what is the likelihood that they are going to smell those cookies? Pretty low, unless they have a great sense of smell. The door is now way too far for detectable odor to reach by diffusion. Likewise, in a large Musca (housefly) embryo, the diffusion gradient would only reach 1/5 (or less!) as far down the embryo as the same diffusion gradient in a small Drosophila embryo.

How do flies solve this problem? There are several possible solutions. We will discuss some of these in class

Figure

Models for making a gradient

Morphogenic gradients are incredibly important in development for patterning tissues. If a tissue is completely homogenous (every cell doing the same task) it cannot be complex and is often not resistant to environmental fluctuations. In the case of Bicoid, we are considering a morphogenic gradient that patterns the anteroposterior axis - as mentioned above, too much Bicoid gives us a giant head and too little gives us no head at all. There are somewhere around 60 different genes that respond to different levels of Bicoid protein, one of the earliest to be discovered is Hunchback. Hunchback gives positional information to a fly embryo, it sharpens the boundary between the anterior and posterior of the fly. While the Bicoid protein gradient is very broad and resembles a diffusion gradient, the Hunchback gradient is sharp, with significantly different protein levels within 10um, or about 2 nuclei⁶.

FIGURE (after figure 3 in fradin)

So obviously the levels of Bicoid must matter tremendously since the Bicoid gradient and its readout (for example Hunchback levels) are incredibly precise. Several models have been proposed that would allow for fluctuation in embryo size but still lead to a correct Bicoid readout:

More mRNA/protein in larger embryos: This would extend the gradient out further in larger embryos.
Nuclear shuttling: The same as the SDD model, but Bicoid protein gets taken up by nuclei as it diffuses by them. Under this model, the more densely packed the nuclei are (smaller embryo) the more quickly the Bicoid protein gets pulled out of the syncitial cytoplasm. In larger embryos, the protein can diffuse farther.
ARTS model (Active mRNA, Transport, Synthesis): The cytoskeleton actively transports Bicoid mRNA to form an mRNA gradient that gets readout as a protein gradient as it is translated. In larger embryos, the cytoskeleton can transport the mRNA further.
mRNA diffusion and degradation: This is similar to the ARTS model, except that in this model Bicoid mRNA degrades over time to maintain an mRNA gradient. Larger flies could have lower degredation rates of Bicoid mRNA or faster transport.
Facilitated diffusion of protein. This is similar to the ARTS model, only it involves transport of Bicoid protein by the cytoskeleton. In this case, larger flies could have faster transport of Bicoid protein.

FIGURE

There is some evidence for and against each of these models, suggesting that the Bicoid protein gradient is likely maintained by a combination of these models. Additionally, Bicoid protein readout might be more complex than we think - multiple proteins could be affecting how Bicoid interacts with its targets.

Evidence for and against the models

1. More protein model. An embryo can get more protein by increasing the amount of mRNA, increasing the rate of translation of that mRNA, or decreasing the rate of protein degradation. Supporting this is the finding that putting in extra copies of Bicoid (more mRNA and more protein) into a fruit fly increases the Bicoid gradient and the size of the head. To see if this is what flies with larger embryos do to make a larger gradient, researchers put Bicoid DNA, complete with cis-regulatory sites and UTRs, from large embryo flies in Drosophila. They found that the Bicoid gene from large embryos did not make a longer gradient in Drosophila. Therefore if there are any factors affecting the mRNA stability or protein degradation, they must be specific to the larger flies⁷.

FIGURE for more protein and nuclear shuttling

2. Nuclear shuttling. Early experiments on this suggested that adding a nuclear localization signal to Bicoid protein made a shallower gradient⁷. Despite this, later experiments made Drosophila with a mutant version of Bicoid that did not accumulate in nuclei. If nuclear import made the Bicoid protein gradient steeper, then they would have seen a broader gradient after inhibiting nuclear import. However, they saw a fairly normal looking Bicoid protein gradient, arguing strongly against this model⁸.

3 and 4. mRNA gradient models. In both models 3 and 4, mRNA is actively transported around the cell by the cytoskeleton. This is in direct opposition to the SDD model, where Bicoid protein is translated from an anterior point source of Bicoid mRNA. In these two models, the protein gradient is a read out of the mRNA gradient. Evidence for these two models comes from careful measurement of Bicoid mRNA in developing embryos (see Figure 2). These careful measurements show that Bicoid mRNA itself forms a gradient, and is not simply a point source⁴^,9. This movement is dependent on the cytoskeleton, if the cytoskeleton is disrupted, the mRNA gradient is also disrupted¹⁰. Evidence against this model comes from the finding that Bicoid protein at the posterior of the embryo is older than Bicoid protein at the anterior. If Bicoid mRNA diffuses towards the posterior to make a gradient, then Bicoid protein is made from that mRNA, we would expect to see younger Bicoid protein at the posterior and mixed age Bicoid protein at the anterior end. However, Durrieu et al found the opposite, suggesting that even though Bicoid mRNA may form a gradient, this is not the main determinant of the protein gradient¹¹.

FIGURE

5. Facilitated protein diffusion. Disruption of the cytoskeleton by treating a Drosophila embryo with anti-actin or anti-microtubule drugs leads to disruption of the protein gradient. Since mRNA tethering at the Anterior pole is also dependent on microtubules, just disrupting the cytoskeleton wouldn't tell you whether it is mRNA transport or protein transport that leads to the protein gradient. One study used a funny trick - if you raise Drosophila embryos in low-oxygen (hypoxic) conditions, the Bicoid mRNA will stay at the anterior end even if microtubules are disrupted. This study found that Bicoid protein still forms a gradient even if Bicoid mRNA doesnt, and that this gradient is dependent on the cytoskeleton¹².

A model emerges

Despite all of the seemingly contrary data, a model is beginning to emerge that both explains the robustness of the Bicoid protein gradient and explains how the gradient itself can scale to larger or smaller bodies. First, Bicoid mRNA is mostly sequestered at the anterior end of the embryo, but also travels along the cortex (outer edge) of the embryo via microtubules. Bicoid protein is translated off of the Bicoid mRNA, with a higher amount of translation happening at the anterior end, where the concentration of Bicoid mRNA is higher. Some Bicoid protein is likely also translated off of the lower concentration Bicoid mRNA that has moved posteriorly via microtubules. This leads to a gradient of Bicoid protein that is slightly broader than what we would expect if there was a simple point source of Bicoid protein (i.e. translation from anterior pole Bicoid mRNA).

This protein gradient is also modified as Bicoid protein gets transported around the embryo by the cytoskeleton (via actin filaments and microtubules). In this way, there is control over the gradient by the cytokeleton and by the proteins that mediate interaction between Bicoid (protein and mRNA) and the cytoskeleton. These mediating proteins may be sensitive to local Bicoid levels and may let Bicoid move more quickly or slowly depending on the anteroposterior position and the gradient level. For example, a large fly might have less Bicoid protein by diffusion at 15% of its length (EL) that a small fly does at 15% of its length. However, all of the Bicoid protein at 15% EL in the large fly can get taken up by the mediating proteins and moved posteriorly to broaden the gradient. On the other hand in a small fly, the larger amount of Bicoid protein at 15% EL could oversaturate the mediating protein and be transported less efficiently.

Additionally, Bicoid proteins from different species of fly are known to have slightly different properties, for example Bicoid from larger fly species such as Calliphora vicinia and Lucilia sericata are not able to completely rescue Drosophila Bicoid mutants⁷. These different properties could also play a role in protein-protein interactions involving the cytoskeleton. Finally, the mediating proteins between the species could also differ, some might bind to Bicoid more efficiently and/or move along the cytoskeleton more efficiently.

[Remove possible solutions section before publishing this page]

Totally different mechanism for A/P patterning

More mRNA - can diffuse further down the embryo (like baking 6 batches of cookies instead of just one).

Maintenance of low posterior concentrations by protein degradation in Drosophila

Different downstream gene responses, making a shallower gradient have the same effect in Musca as a long gradient does in Drosophila.

So bicoid is a paralog of zen, together they are Hox3 genes

Zen patterns the amnioserosa in some insects, it's a novelty since Hox3 normally does AP patterning - but bicoid is a super wierdo: it does oocyte prepatterning of the AP axis

If you look at the RNA or protein for it, it shows a sharp AP gradient, higher at the anterior end

If you deplete it you get two tails

If you add in extra copies you get a bigger head. But here's the first weird part: You add in extra copies and get a big old embryonic head, but the larva and fly it grows into look pretty normal

So that's thing one: developmental robustness despite super early perturbation. There must be waaaaaaaaay downstream developmental checks on this

So Nusslein-Volhard first sees this bicoid gradient and is like

DIFFUSION

Idk if it her or someone just after that finds out bicoid is almost entirely maternally produced by nurse cells. The nurse cells are ONLY at the future anterior end of the developing oocyte.

So voila, mechanism for setting up the first step in AP patterning - a diffusable RNA with a point source at one end of the embryo

They check out the protein pattern, and same thing - exact same gradient, so the protein gradient must be dependent on the mRNA gradient.

boom.

BUT WAIT

THERE"S MORE

Because Drosophila makes teensy tiny embryos

But it has relatives that make ginormous embryos, this includes the housefuly Musca domestica

These embryos are 5X the length of drosophila.

So what's going on? Do they invent a totally new way of making the first assymmetry? That seems totally extraordinary!

Well. . . they don't!

If you scale a Musca embryo to a Drosophila embryo, their gradients of bicoid are ALMOST IDENTICAL

they both reach to about 30% of embryonic length

Yeah - point source diffusion

If that's running the gradient like N-V hypothesizes, it should be the same size gradient no matter the embryonic size.

UNLESS

there is more bicoid mRNA at the point source.

But there isn't.

So you put in extra drosophila bicoid into a drosophila embryo and get a big head

but you put in Musca bicoid into a drosophila Bcd- embryo and get. . . a normal embryo

So there isn't more of it.

BUT you stick in NL-eFP under bicoid regulation (including the 3'UTR which I will get to in a second) and you get a BROADER gradient

So. . . there must be degradation from the posterior end

But it turns out that other big embryos don't rescue Drosophila in the same way

And there's another puzzle piece - the 3'UTR

The 3'UTR makes bicoid mRNA get degraded and also tethers it to a needlessly complex series of proteins that I simply refuse to recall.

The $$$ one is stauffen

So stauffen is tethering bicoid mRNA to . . . SOMETHING (probably cytoskeletal motility elements) to make an active diffusion (????) gradient

While at the posterior end something else (likely nanos and other crap) is chomping up the mRNA

Ok no problem, so we have a facilitated diffusion of bicoid mRNA from a point source at the anterior end and this results in a gradient of bicoid protein. Bicoid protein gets degraded pretty quickly I guess???

So this is fundementally opposed to the original N-V model (called SSD)

If you tag Bicoid protein with a fluoresncent marker that "ages" (meaning some magic molecule that degrades over time) you see that older bicoid protein is at the posterior most edge of the gradient and younger bicoid protein is at the anterior most edge

that's OPPOSITE what you would expect if the mRNA is setting up the gradient and OPPOSITE what you would expect if the protein is getting chomped at the posterior end

😱

😰

Its a transcriptional activator

but

also a transcriptional repressor

but

also a translational repressor

REFERENCES

1. "The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner" 1988, Cell, Wolfgang Driever and Christiane Nüsslein-Volhard https://doi.org/10.1016/0092-8674(88)90183-3

2. "Nanos is the localized posterior determinant in Drosophila", 1991, Cell, Charlotte Wang and Ruth Lehmann https://doi.org/10.1016/0092-8674(91)90110-K

3. "From gradients to stripes in Drosophila embryogenesis: filling in the gaps", 1996, Trends in Genetics, Rolando Rivera-Pomar and Herbert Jãckle https://doi.org/10.1016/0168-9525(96)10044-5

4. "Bicoid gradient formation and function in the Drosophila pre-syncytial blastoderm". 2016, eLife, Zehra Ali-Murthy and Thomas B Kornberg, 10.7554/eLife.13222

5. "Diffusion and scaling during early embryonic pattern formation", 2005, PNAS, Thomas Gregor, William Bialek, Rob R. de Ruyter van Steveninck, David W. Tank, and Eric F. Wieschaus, https://doi.org/10.1073/pnas.0509483102

6. "The time to measure positional information: maternal Hunchback is required for the synchrony of the Bicoid transcriptional response at the onset of zygotic transcription", Development, 2010, Aude Porcher, Asmahan Abu-Arish, Sébastien Huart, Baptiste Roelens, Cécile Fradin, Nathalie Dostatni, doi: 10.1242/dev.051300

7. "Shape and function of the Bicoid morphogen gradient in dipteran species with different sized embryos", 2008, Developmental Biology,Thomas Gregor, Alistair P. McGregor, and Eric F. Wieschaus, 10.1016/j.ydbio.2008.01.039

8. "The Bicoid gradient is shaped independently of nuclei", 2010, Development, Oliver Grimm, Eric Wieschaus, doi: 10.1242/dev.052589

9. "Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient", 2009, Development, Alexander Spirov, Khalid Fahmy, Martina Schneider, Erich Frei, Markus Noll, and
Stefan Baumgartner doi: 10.1242/dev.031195

10. "αTubulin 67C and Ncd Are Essential for Establishing a Cortical Microtubular Network and Formation of the Bicoid mRNA Gradient in Drosophila", 2014, PLoSOne, Khalid Fahmy , Mira Akber , Xiaoli Cai, Aabid Koul, Awais Hayder, Stefan Baumgartner https://doi.org/10.1371/journal.pone.0112053

11. "Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis", 2018, BioRxiv, Lucía Durrieu, Daniel Kirrmaier, Tatjana Schneidt, Ilia Kats, Sarada Raghavan, Michael Knop, Timothy E Saunders, Lars Hufnagel, https://doi.org/10.1101/280834

12. "Cortical movement of Bicoid in early Drosophila embryos is actin- and microtubule-dependent and disagrees with the SDD diffusion model", 2017, PLoSOne, Xiaoli Cai, Mira Akber, Alexander Spirov, Stefan Baumgartner, https://doi.org/10.1371/journal.pone.0185443