The Art of Genes How Organisms Make Themselves

hardened skin of the embryo to be seen very easily. You may recall that Ed Lewis developed a similar procedure to sort out mutants of the Bithorax Complex that died early (Chapter 4). But whereas Lewis was using this method to study mutations he had already produced, NüssleinVolhard wanted to use it for a primary screen, to identify the mutants in the first place.

NüssleinVolhard and Wieschaus then tried out the method on some existing stocks of flies that had been reported to lay some defective eggs (i.e. eggs that never hatched). When they looked carefully at these eggs, they saw that some of them contained mutant embryos with very specific defects in their cuticle pattern. The embryos were obviously not all scrambled up, as conventional wisdom might have predicted, but had very precise alterations, like particular regions of the body being missing. This was very important because it meant that embryo mutants might reveal genes with specific roles in early development. The mutations were not perturbing development in a non-specific way, they were affecting genes with particular roles that might be deciphered. Encouraged by this, NüssleinVolhard and Wieschaus decided to go ahead and screen a large population of flies for more embryo mutants. Because of the complication that their mutants would die, this was quite a challenge in logistics, involving lots of flies being kept and recorded systematically. NüssleinVolhard, who had a bent for strategic planning, recalls how they set about it:

I organised it more or less because I was the most practical person; I mean I am very untidy, but when I want to do an experiment, I try to be very well organised. Otherwise we did everything together. We had four people working with us: two technicians and two animal caretakers. One collected virgins (unfertilised female flies) all the time and the other one made cuticle preparations and the other technicians scored plates and fixed embryos. When you start to get going the flies come, the flies come!

In the evenings Eric and I looked at the prepared embryos together. It was very necessary that this was done by two people. We had this microscope with a bridge where two people could look at the same preparation at the same time, very very important. And we had our discussions.

Through their observations and discussions, they classified and sorted out many different types of mutants, each with a specific defect in the embryo. This early screen was followed by several others, establishing a systematic collection of mutants with altered embryo patterns. Once these mutants had been identified, it was then possible to isolate some of the genes involved and study them in detail. They were mining a seam of gold, providing an invaluable collection that was to allow the early events in fly development to be unravelled for the first time.

Bicoid makes black

I now want to describe one of the mutants that emerged from these sorts of embryo screening experiments: a mutant called bicoid. Normal flies can be divided into three main regions: head, thorax and abdomen. The bicoid mutant embryos had a rather fundamental defect: they seemed to have almost no head or thorax! The appearance of the bicoid mutant embryo compared to a normal one is shown in Fig. 9.1.

Fig. 9.1 Normal fruit fly embryo compared to bicoid mutant (side view).

To get an idea of what an equivalent mutation might look like in humans, look at the painting in Fig. 9.2 by RenéMagritte— although in this case, it is an adult that lacks the head and chest, rather than an embryo.

Fig. 9.2 The Symmetrical Trick (1928), RenéMagritte. Private collection.

Because the head and thorax are missing in the bicoid mutant, the normal significance of the bicoid gene (i.e. the gene altered by the bicoid mutation) is to promote the formation of a head and thorax. The bicoid gene normally plays a role in establishing the head and thorax regions of the body, so without it, these regions fail to develop. We are talking about a gene that has a very fundamental role in development: it is needed for a major part of the animal to develop, and a pretty important part at that. Without a head or thorax, a fly would not be much to speak of.

The bicoid gene codes for a master protein— a protein that can bind to genes and switch them on or off. Master proteins are equivalent to hidden colours (Chapter 5), so we can think of bicoid as producing a particular type of hidden colour. For convenience, I am going to call this hidden colour, and its corresponding master protein, black. It will help if you can remember that bicoid makes black. The black hidden colour is needed for the region including the head and thorax to form. In bicoid mutants, no black is produced and so this region of the body does not develop.

This black colour will provide a useful starting point for our exercise in internal painting. Before proceeding any further, however, I will need to give a few more details about the starting canvas.

Sizing up the canvas

In the previous chapter I mentioned how the bulk of the fertilised egg, that is, the cell membrane and its internal contents (cytoplasm), is contributed by the mother. Both parents contribute a half-share of DNA to the nucleus but the surrounding cytoplasm is of maternal origin. In terms of the painting analogy, most of the starting canvas comes from the mother picture.

Now, in many organisms the egg cell can grow to be quite large within the mother before it is fertilised. It is as if the new canvas gets a head start, expanding inside the mother to form quite a large expanse. A human egg cell, for example, grows to be about one-tenth of a millimetre in diameter before it is fertilised. This is about five to ten times larger in linear dimensions than a typical human cell. In the case of fruit flies, this expansion of the egg cell is even more dramatic: it ends up being about one-half of a millimetre long. This is an enormous cell, being about one-sixth of the entire length of the female fly. If you were to magnify the fly to human size, the egg cell would be about the size of a rugby ball (or an American football). A female fly may produce more than two hundred of these rugby balls during her life: no mean feat by any standards.

After a large egg cell has been fertilised, it typically starts to divide without much growth in its overall dimensions. The very large cell effectively gets divided up or cleaved into smaller cell portions. Although the total number of cells increases, their average size gets progressively smaller at each division, eventually approaching a size that is more typical for a cell. For example, four days after fertilisation, a human egg will have gone through several rounds of division to give a clump of about one hundred cells, but the overall size of this clump will be roughly the same as that of the initial egg: about one-tenth of a millimetre across. There are more cells but they are each much smaller than the fertilised egg cell. Eventually, this process of cleavage comes to an end and the embryo starts to grow bigger in overall dimensions as its cells grow and divide.

Fruit fly eggs also go through a phase of cleavage without overall growth, but there is an additional complication. After fertilisation, the nucleus in the egg cell divides to give two nuclei, but the membrane around the cell does not divide. In other words, you end up with two identical nuclei in the same cytoplasm. Each nucleus contains the full complement of DNA (derived from both father and mother), but it shares its cytoplasm with the other nucleus. This process repeats itself several times until many nuclei are formed, all immersed in the same cell fluid (Fig. 9.3). At this point the embryo is one large cell with lots of nuclei inside it. The nuclei then migrate to the outer part of the large cell, where a membrane starts to form around each nucleus. The free nuclei have now become enclosed by membranes to form separate cells, giving a ball with an outer layer of about six thousand cells (Fig. 9.3). Throughout this process there is no overall growth, so the final ball of six thousand cells is about the same size as the initial fertilised egg (one-half of a millimetre long).

Fig. 9.3 Early development of the fertilised egg of the fruit fly.

You can think of these cells of the embryo as having arisen in two phases. In the first phase, nuclei divide freely in the same cell fluid, with no cell membrane separating them. In the second phase, the nuclei acquire their own bit of membrane, collectively forming a ball of six thousand cells. All of this happens very rapidly: it takes about three hours from having a single nucleus to becoming a ball of six thousand cells. The various structures of the fly larva, such as the head and body segments, then develop from this ball: the head forming from one end and the tail at the other.

Putting this in terms of painting, the new canvas starts off very large, with one artist (the nucleus) in the middle. The artist then starts to divide to give two artists, which in turn multiply, so that eventually the large initial canvas is populated with thousands of artists. During this process there are no barriers (cell membranes) that clearly demarcate the bit of canvas each artist is painting; the artists just work on the region around them. Later on, some lines of demarcation are drawn, so that the activity of each of the six thousand artists becomes more confined. Throughout this process, the overall size of the canvas does not change.

Whilst this example of development may be slightly atypical, it does simplify our initial painting exercise in two respects. First of all, because there is no overall growth, we do not need to worry about the canvas expanding at the same time as it is being painted. Secondly, the lack of barriers between nuclei early on means that there is relatively free communication between different zones of the developing embryo. This will greatly simplify matters when we have to consider how the hidden colours in nearby regions are coordinated.

A gradient of colour

We can now return to the black master protein, produced by the bicoid gene. The black hidden colour starts to appear in the developing embryo just after the egg has been fertilised. If the fertilised egg is probed to reveal the whereabouts of the black master protein, the intensity of staining appears to change gradually from one end of the cell to the other (Fig. 9.4). There is much more of the black protein in the cytoplasm at the head end of the early embryo (the end where the head will eventually form) than at the tail end of the embryo. In other words there is a gradient in the concentration of the black protein from the head to the tail end of the cell (by concentration I mean the number of black master protein molecules per unit volume, equivalent to the intensity of hidden colour). The concentration gradient is shown graphically next to the embryo in Fig. 9.4.

You can think of the head end as being black and the colour gradually diminishing through a series of lighter and lighter greys as you go towards the tail end.

Fig. 9.4 Gradient of black protein in the early fruit fly embryo

The gradient arises because the black protein is only made in the cytoplasm at the head end of the cell: it is only at this end that the black protein is produced by translation from RNA. The black protein then diffuses through the rest of the cytoplasm, gradually diminishing in concentration towards the tail end. The head end acts as a source of black hidden colour which gets gradually more dilute as you get further and further away. This means that the fertilised egg already has a basic pattern of hidden colour from one end to the other, a gradient of black protein in its cytoplasm. It would be as if our initial canvas is primed with a graded wash of colour, with say black on the left and gradually merging to white on the right.

You may wonder why the black protein should only be made at one end of the embryo. I shall return to this important question later on in the chapter. For now, I want to look at how this gradient of colour can be interpreted and built upon to establish a more detailed picture.

Interpreting the grey scale

The role of hidden colours (master proteins) is to provide a frame of reference that can be responded to by interpreting genes (Chapter 5). Recall that each interpreting gene is divided into two regions: a coding region that determines the type of protein made by the gene; and a regulatory region that influences when and where the gene is expressed. The regulatory region contains a set of binding sites— short stretches of DNA (locks) recognised by master proteins (keys). In the case of the black master protein, we can call the binding site, or short sequence of DNA it recognises, a B-site. In the simplest scenario, if an interpreting gene has a B-site in its regulatory region, it would be switched on when the black master protein is present (Fig. 9.5, left). On the other hand, if no black protein is present, the interpreting gene will be off (Fig. 9.5, right).

Fig. 9.5 Activity of an interpreting gene with a B-site in its regulatory region in the presence (left) or absence (right) of the black master protein.

I have previously described how an interpreting gene can respond to the presence or absence of hidden colours in particular regions of an organism (Chapter 5). I now want to look at how an interpreting gene might respond to a gradient of hidden colour. Responding to a gradient is more complicated because we now have to deal with the concentration of master protein, not just its presence or absence.

To see how the concentration of black protein might affect matters, you need to appreciate that the binding of black protein to a B-site is a reversible process. That is, when a black protein molecule encounters a B-site it may bind for a period of time and then come away. As long as there are many black protein molecules around, as soon as one comes off the B-site, another will be there to bind in its place, so effectively the B-site will always be occupied.

We can now consider how such an interpreting gene might respond to the concentration of black protein at various positions in the early fly embryo, going from the head end towards the tail. As we have seen, shortly after fertilisation, the embryo comprises a large cell with lots of nuclei dotted all around the cytoplasm (Fig. 9.3). Each nucleus will contain our interpreting gene, so we can look at how the gene will respond in nuclei at various positions in the cell. Starting with nuclei at the head end, where the concentration of black protein is high, the interpreting gene will be on because the B-site is almost always occupied. This might continue to be true as we move towards the tail end, through various dark shades of grey. But there will come a point, say halfway along the egg, at which the concentration of black is no longer enough to ensure the B-site is always occupied. In other words, the concentration of black protein has started to become so low that there is insufficient of it to guarantee that there will always be some bound at the B-site. At this point, the gene might sometimes be on (when black protein happens to be bound), and other times off (when no black protein is bound), giving a reduced overall average activity for the interpreting gene (Fig. 9.6). In other words, rather than producing its protein at a maximum rate, the interpreting gene will be working at a slightly lower level on average. As we go further tailward, the average activity of the gene drops further still as the concentration of black goes down, until eventually the activity may drop down to zero and the interpreting gene will be off at the tail end (Fig. 9.6). The gene has responded to the gradient by being on in the head half, but gradually diminishing in activity in the tail half.

Fig. 9.6 Response of an interpreting gene to the gradient of black protein

According to this scenario, the point at which the interpreting gene starts to decline in activity depends on where the concentration of black protein falls below a certain level. Above this threshold level the gene is pretty much fully on, whereas below it, gene activity starts to decline. In the example I gave, the threshold level of black protein was reached about halfway along the cell. In principle, the position in the cell where this happens could be shifted by changing the black concentration gradient. The black protein is only made at the head end of the cell, so if more black protein was produced from this source, more would diffuse through the cytoplasm, increasing the concentration to some extent at each point in the cell. This would mean that the point at which the concentration of black falls below the threshold level would no longer be reached halfway along the cell but further towards the rear. In other words, the expression of the interpreting gene would extend further back, say two-thirds of the way along the egg rather than halfway. Conversely, if the source of black protein was reduced at the head end, the concentration would fall below the threshold level sooner, nearer to the head. This would mean that the expression of the interpreting gene was more restricted, say to one-third of the way along the cell.

If the interpreting gene is indeed responding to the concentration of black protein, we would therefore expect that its activity would vary in a predictable way according to the gradient of black. Christiane NüssleinVolhard and her colleague Wolfgang Driever tested this experimentally by looking at the development of eggs with various levels of black master protein. They were able to do this by varying the number of bicoid genes in the fly (bicoid makes black). Typically, a fly has two copies of the bicoid gene, one from its father and one from its mother. By various genetic tricks they produced flies with other copy numbers, such as no copies, or one, three, or four copies. The more copies of the bicoid gene in the fly, the more black master protein will be made at the head end of the egg cell (actually it is the number of bicoid copies in the female laying the egg that counts, for reasons that will become clear later on), and the further towards the rear the activity of an interpreting gene should extend. As shown in Fig. 9.7, this is exactly what NüssleinVolhard and Driever observed. With no copies of the bicoid gene (no black protein) the interpreting gene did not come on anywhere. With one copy of bicoid, the interpreting gene did come on, but only near the head end. As the number of bicoid copies was increased further, so the expression of the interpreting gene extended further and further to the rear. These results confirmed that the interpreting gene was indeed responding to the grey scale, only starting to diminish in activity when the intensity of black fell below a critical level.

Fig. 9.7 Extra copies of the bicoid gene lead to more black protein being made at the head end of the fruit fly embryo, which in turn leads to a steeper gradient. In response, the activity of the interpreting gene extends further towards the tail end.

The idea that gradients might be involved in development was not new; it goes back to the late nineteenth century. At that time Thomas Hunt Morgan was studying the ability of worms with their heads chopped off to regenerate new heads (this was before he started working on fruit flies). He found that the ability to regenerate a head decreased as the cut was made further and further back. It was as if the regenerating powers were gradually diminishing along the worm. As he summarised: 'Perhaps for want of a better expression, we might speak of the cells of the worm as containing a sort of stuff that is more or less abundant in different parts of the body. The head stuff would gradually diminish as we pass posteriorly.' The trouble with this proposed gradient of head 'stuff' was that it was very hypothetical. As Morgan himself noted: 'I do not pretend that this explains anything at all, but the statement covers the results as they stand.'

The importance of the work on bicoid is that for the first time it was possible to identify a particular molecule, a master protein, that varied in concentration from one end to the other and was responded to in a particular way. Instead of a mysterious 'stuff', here was a defined molecule that played a key role in early development whose effects could be studied in detail.

Let me summarise the main points so far. By screening embryos for various types of mutant, a key mutant, bicoid, was found that lacked both head and thorax. This mutant has a defect in a gene (also called bicoid) that normally produces a particular type of master protein, symbolised by a black hidden colour. In normal embryos, the black master protein is produced only at the head end, from where it diffuses to form a gradient of decreasing concentration towards the tail end. This gradient can be responded to by an interpreting gene with a B-site (lock) in its regulatory region, recognised by the black master protein (key). The response may lead to the interpreting gene being very active (switched on for most of the time) at the head end but dropping in activity further towards the rear. The point in the embryo at which this drop happens depends on where the concentration of black master protein falls below a critical threshold level.

Varying the response

I have described how an interpreting gene can make one sort of response to a black gradient. I now want to look at other sorts of response: other ways of interpreting the gradient. Rather than varying the gradient, as I did in the previous section, we shall keep the gradient fixed and vary the response. What I mean by this will soon become dear.

For any given gradient, the activity of an interpreting gene will start to decline at a particular distance from the head. Now the point at which the drop starts to occur depends on how well the black protein sticks to the B-site: how much affinity the black master protein has for the sequence of DNA it is binding to. If the black protein sticks very well, having a high affinity for the B-site, then even a low concentration of black protein might be enough to keep the interpreting gene on for quite a while. In this situation, activity of the interpreting gene might only start to drop significantly near the tail end of the embryo, where the level of black becomes very low (Fig. 9.8, left).

Fig. 9.8 Fruit fly embryos showing two different responses to the gradient of black by interpreting genes with high or low affinity B-sites in their regulatory regions.

On the other hand, if the black protein does not stick well, having a low affinity for the B-site, it would take a relatively high concentration of black to keep the interpreting gene on: the black protein would always be coming off the B-site, so many black protein molecules would be needed to ensure that the B-site was occupied for a significant period. This would mean that even in the dark grey areas, there may not be enough black protein to keep the gene mostly on. In other words, the activity of the interpreting gene would start to decline quite near to the head end of the embryo (Fig. 9.8, right). So a low affinity would lead to a narrow range of activity towards the head end, whereas a high affinity would lead to activity over a more extended length of the embryo. This was confirmed experimentally by Driever and NüssleinVolhard, who showed that lowering the affinity of binding sites in an interpreting gene did indeed lead to expression in a more restricted region towards the head.

In molecular terms, the affinity between the black master protein and the B-site depends on how well their shapes match each other. If the master protein fits very precisely into a particular B-site, like a key fitting perfectly into a lock, the affinity will be high. If the fit is not so good, say because the DNA sequence in a particular B-site is not a perfect match for the black master protein, then the affinity will be low. This means that a slight modification of the DNA sequence of the B-site, varying its capacity to match the black master protein, can change its affinity for black.

The importance of all this is that it allows the gradient of black to be interpreted in several different ways, according to the particular DNA sequence of the B-site in the interpreting gene. An interpreting gene with a high affinity B-site in its regulatory region will respond differently from one with a low affinity B-site. In other words there are various ways in which the same gradient can be responded to. The way any particular interpreting gene responds will depend on the affinity of the B-site in its regulatory region. (I have oversimplified things. The response is more complicated than this because an interpreting gene would typically have several B-sites, not just one, and there can be other hidden colours in the background that may also influence how black binds and influences gene expression. These additional factors mean that the activity of the interpreting gene tends to change more rapidly near the threshold level than I have indicated.)

A rainbow of colours

We have seen that a gradient of black master protein in the developing egg can potentially be responded to in several ways by different interpreting genes. This means that any particular interpreting gene may be very active (mainly on) in some regions of the embryo, and inactive (mainly off) in others, according to the way it responds to the black protein through its regulatory region. Now the consequence of an interpreting gene being switched on is that it will produce its own type of protein from its coding region (i.e. the coding region will be transcribed to give RNA which will in turn be translated into protein). The key to the next step in the painting exercise is to look at what the protein produced by the interpreting gene might itself do. In other words, we need to consider the role of the proteins produced by the interpreting genes responding to black. We shall see that the products of many of these interpreting genes are themselves master proteins: these genes produce more hidden colours!

Now I am in danger of contradicting myself here. I previously classified genes into two types: those that produce hidden colours (such as identity genes) and those that respond to them (interpreting genes). I did this in order to avoid confusion between the production and interpretation of hidden colours. This classification of genes has acted as a ladder, helping us to get to this level of understanding. Having got to this level, we can now dispense with the ladder. I am now saying that genes which produce hidden colours are also interpreting genes. This is because the genes for hidden colours also have their own regulatory regions, their own molecular antennas. Like all interpreting genes, each gene for a hidden colour has two regions: a regulatory region and a coding region. But in this case, the type of protein produced from the coding region is itself a master protein, a hidden colour. Whilst it is still true that only a subset of genes produce hidden colours (master proteins), essentially all genes are able to interpret them, including the genes for the hidden colours themselves. This may sound like a circular argument at first, with