The Art of Genes How Organisms Make Themselves

genes for hidden colours being able to interpret themselves, but its meaning should become clearer as we encounter some specific examples.

To show how this allows us to elaborate patterns, I am going to give a simplified story. I will begin by introducing seven different interpreting genes that respond to the black gradient through their regulatory regions. As well as responding to a hidden colour, each of these genes also produces its own hidden colour, a particular type of master protein, from its coding region. We can name the master proteins produced by the seven genes after the colours of the rainbow: red, orange, yellow, green, blue, indigo and violet. Each of the interpreting genes therefore has a coding region that encodes a particular master protein (red or orange or yellow, etc.); and a regulatory region that acts as its own individual molecular antenna, ensuring that it is expressed at particular times and places.

We will assume that the regulatory region for each gene contains a B-site with a particular affinity for the black master protein, allowing the gene to respond in a particular way to the black gradient. As we saw in the previous section, a low affinity B-site will lead to a narrow range of activity towards the head end, whereas a high affinity B-site will lead to activity over a more extended length of the embryo. We will assume that the gene for red has the lowest affinity B-site, so it is expressed only near the head end of the developing embryo. The gene for orange has a slightly higher affinity B-site, so that its region of expression overlaps with red but extends a bit further tailwards. The gene for yellow has a B-site with a slightly higher affinity again, so it extends further back than orange; and so on up to the gene for violet, which has a very high affinity B-site and is expressed all the way from head to tail. The result is a series of overlapping colours, with all seven colours being made at the head end and gradually fewer and fewer being produced towards the rear, ending with mainly violet at the tail end. I now want to show how this overlapping pattern can get converted into a rainbow of separate colours. Before doing this it will help to give an analogy.

Bands around a mountain

Mountains are diverse habitats, rich in many different plant species. The vegetation near ground level is similar to that of the surrounding countryside but as you climb higher up, the temperature gradually drops and the predominant plant species progressively change to those that are better adapted to live in cooler conditions. The gradient in temperature results in bands of particular species around the mountain, with warm-loving species towards the base and cool-loving species towards the top (for our purposes we can ignore other environmental factors that change up the mountain, such as rainfall or soil quality).

In addition to the physical environment, there is another constraint on the distribution of the plant species: they compete with each other. To see this more clearly, we will start with a mountain having no plants growing on it, and then sprinkle some seed of various plant species all over it in the spring and follow their growth over several years. All the plant species will be annuals, living for one year and then setting seed for the next. In addition, we shall assume that the seed they produce is unable to travel very far from the parent plants.

In the simplest case, we could sprinkle seed for one species, let's call it violet, at low density all over the mountain. We will assume that the violet species is able to survive in almost all temperature conditions to some degree. On its own, it would therefore grow to cover the entire mountain, setting seed at the end of the year to give more violet plants next year. Eventually the whole mountain would be covered with a dense population of violet (Fig. 9.9, left).

Fig. 9.9 Distribution of various plant species on a mountain following competition between them. Things get a bit more interesting if we start by sprinkling some seed for two species which compete with each other, violet and indigo. Perhaps indigo does not grow very well on the highest regions, so violet tends to win there. But below this, we will assume that indigo is a more effective competitor and tends to win over violet. At the end of the first year of growth, there is more violet at the top and more indigo lower down. This means that seed for the following year will not be evenly distributed over the mountain: more violet seed will have been shed in upper regions and more indigo seed in lower regions. The advantage to violet in upper regions now becomes twofold: its seed is more abundant, and it grows better than indigo. Similarly, in lower regions indigo seed outnumbers violet and it competes better in growth. You would therefore expect that in the second year of growth, violet predominates even more over indigo in upper regions, and indigo outdoes violet even more in lower ones. Repeating this over several years you would tend to end up with two distinct zones: violet at the top and indigo occupying the remaining mountain (Fig. 9.9,

middle left).

Things would have been slightly more complicated if we had started out by sprinkling seed for three species over the bare mountain: blue, indigo and violet. We will assume that blue is an even better competitor than indigo but doesn't like growing quite as high up. Just as violet and indigo competed in the upper regions of the mountain, so indigo and blue will compete further down. Indigo will tend to do better than blue at higher altitudes but blue will win out lower down. Over several generations of competition we might now end up with violet at the top, then a band of indigo, and finally the rest of the mountain below covered in blue (Fig. 9.9, middle right).

We could keep going in this way, sprinkling more and more species on the initial mountain (green, yellow, orange and red), each new species being slightly more sensitive to the cold but being a better competitor on the lower slopes, until we end up with a rainbow pattern, ranging from a band of red at the bottom through to violet at the top (Fig. 9.9, right).

The important message is that the final pattern on the mountain does not just depend on how the species respond to the temperature gradient but also on how they respond to each other over several generations. On its own, each species might occupy a large territory extending from the base of the mountain upwards to various extents. But put together, the species compete and restrict

each other to narrower zones.

A conflict of colour

We can think of the seven interpreting genes responding to the black gradient in the embryo as analogous to the plant species responding to the temperature gradient on the mountainside. With the mountain, the initial response was further refined through competition between species for survival. We shall see that something analogous occurs in the case of the embryo, involving competition between interpreting genes over which one gets expressed.

To explain how this molecular competition works, I want to consider two master proteins: indigo and violet. As before we will name the binding sites recognised by each master protein after its colour: violet binds to a V-site and indigo binds to an I-site. Now each of these master proteins is encoded by a gene with its own regulatory region attached. Suppose the gene for violet has an I-site in its regulatory region, as shown on the left part of Fig. 9.10. This would mean that the indigo protein could bind to the violet gene and influence its activity. We will assume that when the indigo master protein binds to the violet gene, it switches the gene off (i.e. the binding of the indigo protein interferes with or blocks the production of RNA from the violet gene). This means that wherever indigo hidden colour is produced in large quantities, the gene for violet will be off. In other words, if the indigo gene is on, producing lots of its protein, the violet gene will be off.

Fig. 9.10 Interactions between indigo and violet genes. On the left, the indigo gene is on and switches the violet gene off. On the right, the violet gene is on and switches indigo off.

The right part of Fig. 9.10 shows the converse situation. In this case, the gene for indigo has a V-site in its regulatory region, allowing the violet protein to bind. Whenever violet binds there, indigo is switched off. This means that if the violet gene happens to be on, producing a lot of its protein, the indigo gene will be off.

We can now imagine a situation in which there are two competing outcomes. If we combine both the left and right parts of Fig. 9.10, such that the violet gene carries an I-site, and the indigo gene carries a V-site, there will be two conflicting interactions: indigo tends to switch the violet gene off, whilst violet tends to switch the indigo gene off. Indigo and violet master proteins tend to inhibit each other's expression, much as two different species might compete for a place on the mountainside. Which colour wins? Will indigo eliminate violet or will violet supersede indigo? We shall see that the outcome depends on two factors: the initial activity of each gene and the

affinity of the master proteins for their binding sites.

Let us first take the effect of initial activity (i.e. the degree to which the genes are on or off to begin with). Suppose that we start with the gene for violet having a slightly higher activity than the gene for indigo (i.e. the violet gene is on more often than the indigo gene). This will mean that more violet protein is produced than indigo, raising the relative concentration of violet. Because the violet protein is more abundant, it will be able to inhibit the gene for indigo more effectively than indigo can inhibit violet (assuming that each master protein binds with a similar affinity to its sites). The result will be that the gene for indigo will become even less active, producing less protein, than the gene for violet. Effectively the violet gene now has two advantages over indigo: its initial activity is higher and it inhibits indigo more effectively than the other way round. The effect will be that the initial difference in activity between violet and indigo becomes even more biased towards violet than it was before. An initial slight advantage for violet has been amplified to produce an even greater advantage. The process can repeat itself again and again until, eventually, violet comes to predominate, with indigo making only a minor contribution.

In this example, the bias towards one colour came from an initial difference in activity, but we could also create a bias by changing the affinity of the binding sites in the regulatory regions. For instance, suppose that the I-site has a higher affinity for the indigo master protein than the V-site has for the violet protein. This would mean that at equal concentrations, indigo will switch off violet more effectively than the other way round. So even if we start with each gene at the same level of activity, producing each protein in equal abundance, violet will be inhibited more than indigo. This will in turn affect the overall concentration of the proteins, leading to a higher level of indigo than violet. Now indigo has two advantages over violet: a higher concentration as well as being a more effective inhibitor. The result will be that even more indigo is made than violet, further increasing indigos preponderance. Eventually, indigo will take over and violet will be a very minor colour.

To summarise, the final outcome of this molecular competition between indigo and violet will depend on both the initial activity of the genes and the affinity of the master proteins for their binding sites. The colour that wins will be the one with the higher initia l activity or affinity.

To avoid any possible misunderstandings, let me be quite clear that the sort of competition I am talking about is purely at the molecular level. It is not about one gene trying to outdo another in terms of survival. Both violet and indigo genes are in the DNA of the same organism, and I am using competition simply to refer to the sort of molecular interaction that has evolved between them. In terms of the organisms survival, the genes are actually cooperating, helping to refine each other's expression pattern, as we shall now see.

Painting a rainbow

With this notion of molecular competition in mind, we are going to look at how the pattern of rainbow colours may be refined in the early fly embryo.

Recall that I mentioned seven interpreting genes, each producing a master protein named after a

colour of the rainbow: red, orange, yellow, etc. We shall be dealing with these genes at the stage when the embryo is a large cell containing many nuclei dotted around in the cytoplasm. All seven interpreting genes will be present in each of these nuclei, but to make things easier I will treat the genes one by one, just as more species were gradually added to the mountain.

Let us first consider the gene for violet. Initially, the activity of this gene depends on how it responds to the gradient of black protein in the embryo. Earlier in this chapter, I proposed that the violet gene has a high affinity B-site in its regulatory region, allowing it to be switched on even where the concentration of black is very low. The violet gene therefore starts off by being active in nuclei throughout the embryo. This means that each nucleus will be producing RNA from this gene which will go into the local cytoplasm around it to make violet protein. Every nucleus is effectively acting as a local source of violet, so together all the nuclei will ensure that the violet protein is present throughout the cell cytoplasm. There are now two hidden colours in the cell, a gradient of black and a ubiquitous violet.

Enter a second gene, the one for indigo. This gene also responds to the gradient of black through the B-site in its regulatory region. But because the affinity of its B-site is slightly lower, the activity of the indigo gene does not extend quite as far to the rear as violet, declining somewhat towards the tail end. Most of the nuclei in the embryo will therefore act as sources for indigo, except those at the rear which will not produce very much.

I now want to look at how the genes for indigo and violet respond to this situation. As before, we will assume that the genes for violet and indigo compete with each other over which one gets expressed (that is, each has a site in its regulatory region recognised by the other master protein, such that the gene will be switched off when it is occupied). We will also assume that the indigo protein has a higher affinity for its binding site, so that other things being equal, indigo will tend to outcompete violet. Now let the competition begin!

The higher affinity of indigo means that for most of the cell, where the initial activity of the two colours is about the same, violet will be eliminated whilst indigo asserts itself. As we get near the tail end, however, the initial activity of violet becomes higher than that of indigo (because of the response to the black gradient). There will now be two conflicting biases: violet is better off in terms of initial activity, but indigo has the higher affinity. The numerical advantage of violet gets better as we go further tailward, so we might expect that there would be a critical point at which violet gets the upper hand and starts to beat indigo. This will in turn mean that violet inhibits indigo even more effectively, and so violet becomes more predominant. The final result will be a zone of violet at the tail end of the embryo. So instead of the violet and indigo colours largely overlapping, violet will now tend to be restricted to just the tail end, and the rest of the cell will be occupied by indigo (Fig. 9.11).

Fig. 9.11 Molecular competition leading to indigo in most of the cell and violet just at the tail end.

There will still be a narrow region of overlap between violet and indigo where their zones meet. Because there are no cell membranes between the nuclei, the colours can slowly diffuse through the cytoplasm. This means that the colour from one zone will leak or diffuse into the other, giving a fuzzy zone in between (Fig. 9.11). The process of diffusion tends to even things out, ensuring that hidden colours change in a gradual and coherent way from one part of the cell to another.

The overall result is that starting with a gradient of black, we have refined the pattern to produce two zones: indigo and violet. I say refined, because the distinction between the zones is sharper than it was when only black was around. In other words, the gradient that separates violet from indigo is steeper than the original gradient of black. This is because of the way the genes for violet and indigo have competed with each other, so that they have become more tightly restricted than at the start. The refinement has arisen because the genes for violet and indigo are both producing and responding to hidden colours.

We can now apply the same principles to genes for the other rainbow colours. For example, what happens when we bring in a third gene, the one for blue? The expression of this gene starts to drop off a bit sooner than indigo (the blue gene has B-sites with slightly less affinity for black than the indigo gene). Blue and indigo now mostly overlap except for a region towards the tail end, where blue starts to decline in intensity. Now suppose that the indigo and blue compete with each other (they each have a site in their regulatory region recognised by the other colour, such that the gene will be switched off when it is occupied). We will also assume that blue binds with a slightly higher affinity to its site than indigo. As with the competition between indigo and violet, blue will predominate over indigo for the most part because of its higher binding affinity (I am assuming that blue can also outcompete violet in this region). But towards the tail end, where the concentration of blue starts to decline, indigo will get the upper hand. Eventually we will end up with three zones: blue occupying most of the length of the cell, then a region of indigo, and finally a zone of violet at the tail end (Fig. 9.12).

Fig. 9.12 Result of molecular competition between three hidden colours.

We can carry on in this vein, considering more and more genes until we get a full rainbow of hidden colours, red through violet, from head to tail. The original gradient of black has been interpreted and refined to give seven regions of colour (Fig. 9.13).

Fig. 9.13 Final pattern of rainbow colours in the embryo.

You can get a feel for what is going on by thinking in terms of artists painting a canvas. Imagine you are one of many artists (nuclei) working on part of a large canvas. Your part of the canvas is not clearly demarcated from that of your neighbours, but overlaps with theirs (there are no cell membranes between nuclei). The canvas has been primed with a gradient of colour ranging from black at one end (head), through various shades of greys, to almost white at the other end (tail). Suppose you are at the tail end and you respond to the very light grey around you by applying a dilute mixture containing mainly violet together with a little bit of indigo (and even less of the other rainbow colours). Being struck by the mainly violet colour now on the canvas, you start to prefer it over the other colours and decide to increase the proportion of violet in your next mix. After painting on this new mix, you find violet even more appealing relative to the other colours, and eventually end up putting only violet on the canvas. Your neighbours do the same, so this whole region becomes painted violet.

Now imagine you are slightly further away from the tail end. Here the canvas is a slightly darker shade of grey, and you initially respond to this by applying a dilute mixture of two colours, violet and indigo, with minor amounts of the other colours. On seeing violet mixed with similar amounts of indigo, the violet looks distinctly less appealing and indigo now stands out as preferable. Whereas indigo had been rather unattractive when it was only a minor part of the initial mix with violet, it looks better than violet when present in equal amounts. So you respond by applying more indigo on the canvas in preference to violet with your next mix. The process repeats itself so that eventually you and your neighbours paint the whole region indigo.

If you were to start further up the canvas again, at an even darker shade of grey, you might begin by placing a dilute mixture of three colours on the canvas, blue, indigo and violet. But after you see them, the blue stands out as preferable, and from then on you and your neighbours start to increase the proportion of blue. In a similar way, green would be made further along, and so on up to red. The final pattern arises because each time you add new colours, you respond to what you see.

Now let us look at what happens in regions of the canvas where different zones of colour meet, say around the junction between indigo and violet. In this area you might be in two minds about

which colour to favour. Subtle differences in the initial shades of grey might lead some artists to prefer violet, others indigo. If each artist was working alone on his or her own region of canvas, you could end up with a higgledy-piggledy pattern of violet and indigo patches. But remember that the area you are painting is not clearly demarcated from that of your neighbours: it is shared with them to some extent. This means that you will be influenced by what they are doing. Say you are in the indigo region, near to where it meets violet, and you happen to deviate slightly from what your neighbours are up to, by starting to apply a bit more violet. Your neighbours, who are applying indigo, would soon smother your bit of canvas as well, raising the amount of indigo to a similar level to theirs. You would be outnumbered by your colleagues and would tend to conform to whatever they were doing. Of course, there will come a point towards the tail end where the artists will gradually switch to applying violet, but they will tend to do this in a collective way, rather than as individuals going off and painting a bit of violet here and there. The overall result is that the transition zones between regions of different colour will tend to be quite straight and narrow, with everyone on one side conforming to one colour, and everyone on the other side applying a different colour.

Throughout this painting process you need have no idea of where you are on the canvas or what the overall picture looks like. You are simply responding to the local colours you see and then modifying them in some way by adding more colour. To someone high above it may look as if all the artists know exactly where they are because each seems to be applying a colour that fits in with the overall pattern. But the artists themselves are oblivious to this; they simply see the canvas immediately around them and respond accordingly. They are no more aware of their position than a plant knows where it is on a mountainside.

Let me summarise some of the essential ingredients that have allowed the gradient of black to be interpreted and refined. First of all, there is a large population of nuclei (multiple artists) distributed throughout the embryo that can respond to the initial gradient. This allows different regions of gene activity to be established (if there was only one nucleus, the system would not work because there could only be one response at any time). Secondly, there are no barriers between the nuclei, so that neighbours tend to coordinate their activities. This makes sure the response is coherent rather than too much of a hotchpotch. Finally, the genes within each nucleus are able to both produce and respond to hidden colours. Each of the seven genes interprets the local colour whilst at the same time being able to modify it by adding a certain amount of its own colour. Taken together, this allows an initial pattern to be interpreted and refined in an interactive manner, with colours continually elaborating on what went before.

Filling in some gaps

I have described the whole process of producing a rainbow as a painting exercise, giving a general sense of how a gradient of hidden colour might be built upon and refined. The significance of this for the organism is not, however, to depict some pretty patterns but to establish a basic frame of reference for development. To see this, we need to look more closely at the biological effects of these hidden colours.

So far I have only mentioned the effects of one of these genes for hidden colour: the bicoid gene that makes black. Recall that mutants without a bicoid gene produce embryos without a head or thorax. In other words, the black colour is needed for the development of about a half of the embryo, towards the head end.

What happens if a mutation inactivates a gene for one of the rainbow colours? It turns out that these mutations give embryos with several adjacent segments missing (normally there are 14 main segments in the fly, Chapter 4). If, say, green is absent from the rainbow, the middle of the embryo (i.e. the region that is normally green) will not develop, giving an embryo with some middle segments missing. Similarly, if violet is absent, an embryo will develop with several segments at the tail end missing. In other words, each of the rainbow colours is needed for the corresponding part of the embryo to develop fully. In fact this is how the genes for the rainbow colours were identified in the first place: they were noted by NüssleinVolhard and Wieschaus in their screens for embryo mutants, as a class of abnormal embryos with chunks of their body missing. Because of this deficit or gap in the embryo, the genes affected by these mutations were christened gap genes. The gap genes are what I have been calling the genes for rainbow colours. Each gap gene codes for a master protein that is expressed in a particular region or band of the embryo, giving it a hidden colour. Together, they divide the early embryo into distinct territories from head to tail, like the rainbow pattern I described, but not quite so simple (there are actually about nine gap genes, rather than seven as given in my account).

According to my simplified scheme, all the gap genes needed for the rainbow colours are activated by responding to the gradient of black. This would imply that a mutant without black should also lack all the rainbow colours. You might expect such a mutant to develop as an embryo that was all gaps, perhaps forming a tiny blob with no segments at all. This is not what is seen in bicoid mutants. As we have seen, the bicoid mutant lacks a head and thorax but it still produces an abdomen. The reason is that the egg is primed with a few other colours (not previously mentioned for the sake of simplicity) in addition to black. These other colours can to some extent compensate for the lack of black in bicoid mutants, particularly in the tail half of the embryo. So rather than the bicoid mutant lacking all the rainbow colours, it still has those in the tail half, say green, blue, indigo and violet, allowing the abdomen to develop. If all the initial or priming colours needed to set up the rainbow are missing (as in mutants that have several genes inactivated in addition to bicoid), the embryo does indeed form a tiny blob without a head, thorax or abdomen.

So in practice, the expression of the gap genes is not established by interpreting just one initial colour, black, but several others as well. By studying how the gap genes respond to these initial colours and to each other, researchers have been able to start working out how the final pattern of rainbow colours is actually produced. The story that has emerged is quite complicated but the general principles are not too different from my simplified scheme of rainbow painting: gap genes respond to their local hidden colours as well as adding to them, eventually establishing territories of gene expression through molecular interactions and competition.

Beyond the rainbow

I have described the painting of a rainbow in detail so as to show the basic principles by which one set of hidden colours can be built upon and refined through interactions between molecules. We can imagine similar principles being applied again and again to elaborate the pattern further. Just as the gradient of black acted as a frame of reference over a large region of the embryo, each rainbow colour provides a steeper gradient that can be further interpreted and elaborated by other genes. The process can continue in this way, gradually refining the colours more and more. Other considerations will of course come into play in these further stages, such as cell membranes forming between nuclei, and the organism growing larger. We shall deal with some of these issues in later chapters. The point I want to make here is that making a rainbow is just one step in an extensive process of internal painting. The rainbow colours are just a prelude to many more hidden colours that will be added, further refining and elaborating this pattern.

One set of genes that respond to the rainbow are those involved in providing distinctions in segment identity. Recall that there are eight of these identity genes, producing apple-green to herb-green in various territories from head to tail (Chapter 6). The green colours appear after the rainbow and have more restrictive effects. Whereas the rainbow colours are needed for the formation of each type of segment, the various greens only affect the identity of each segment: remove the rainbow and no segments develop; remove only the various greens and the segments develop but they all have the same identity.

Now the head-to-tail, layout of the green territories itself depends on the previously established rainbow colours: without the rainbow, the various zones of green would not be established. We can think of the whole process as a series of painting steps: starting with a broad gradient of black, then a rainbow with fuzzy boundaries, then the series of greens with sharper boundaries, as schematised in Fig. 9.14. The internal painting process does not stop here: it continues, based on the greens together with other hidden colours, to produce an even more elaborate and refined picture. There is a changing internal landscape of colour that bec omes more detailed as the organism develops.

Fig. 9.14 Schematic illustration of some hidden colours at various stages of development.