5.5.5 Dorso-Ventral Patterning
All higher organisms have definite axes of development. The anterior end is the one where the head grows and the mouth, brain, and major sense organs develop. The posterior end is where the wastes are discharged. The dorsal side is the top of the animal, and ventral side is the bottom. Having these axes is very important in the process of locomotion and separation of bodily functions. By splitting different functions up by body section we can move forward in our environment much more easily while our most important sense organs are pointed in the direction of travel to warn us of danger or potential food. It also allows organisms to create asymmetrical or bilaterally symmetrical structures that would not work very efficiently if they were forced to be in a radial symmetric body.
The purpose of this experiment is to try and reproduce the model for dorso-ventral patterning found in the drosophila embryo with the simulator. The fruit fly, drosophila, is a species that has been studied extensively and the genes involved in formation of these initial patterns have been explicitly mapped out along with the interactions between these genes. This makes it a very good example system to put the simulator through its paces and see if it is powerful enough to reproduce the same behavior found in nature. I believe that anyone who is familiar with this model will be surprised at how closely the simulated example matches the structure and behavior of the natural model. Figure 3 shows the chromosome used to produce the dorso-ventral pattern. We will be referring to it throughout the discussion on how the model works.
2. Creation of the Dorsal Gradient
The dorsal protein is one of the first to form a concentration gradient. It is responsible for patterning the ventral portion of Drosophila, and it induces the formation of another gradient that patterns the dorsal side. Unlike other embryos, insects start out life as a single, multi-nucleated cell called a syncitial blastoderm where a number of the key patterning steps take place in this single cell, and then a little later additional cells form around the nuclei. When the egg is formed dorsal mRNA is laid down evenly along the entire outer edge of the cell, and after fertilization the mRNA is translated and produces the dorsal protein evenly also. Dorsal is a transcription factor and for it to be active it must be transported into the nucleus of the cell. But there is another protein produced in the cell right along with dorsal. This protein is called cactus and it prevents the dorsal protein from entering the nucleus. So the levels of cactus are high enough everywhere throughout the cell to prevent dorsal from entering the nuclei and performing its actions. So if a gradient is going to be formed there needs to be some way to prevent cactus from inhibiting dorsal. The mechanism to do this involves a few more proteins. The Toll receptor is evenly embedded in the membrane of the cell surface, and the ligand for this receptor is called the spatzle fragment. After fertilization spatzle is released only on the ventral side of the cell and it then diffuses up the dorsal side. When spatzle binds to toll it activates an intracellular signaling pathway that leads to the degradation of cactus. Cactus can no longer bind to dorsal and prevent it from entering the nucleus. So the more spatzle that binds to toll, the more cactus that is destroyed, and the more dorsal that enters the nuclei. The level of spatzle is highest where it is released at the ventral side and, due to diffusion, its quantity gradually decreases as we move towards the dorsal side.
It was not possible to directly reproduce this entire natural mechanism in the simulation. The main problem is that the simulator only works with multi-cellular systems and can not reproduce the effects seen in the multi-nucleated blastomere. In that cell, proteins that are normally confined to one cell, like transcription factors, can freely diffuse to effect all of the nuclei. So the system developed here had to be slightly different. The toll receptor is distributed evenly throughout all of the cells, and a gradient of spatzle is manually laid down. This is the only gradient that is manually laid down in this example. All other gradients are derived from this one through the interactions of the genes and proteins. When spatzle activates toll it produces a kinase regulator pelle. Another place where this system differs with the one found in nature is that there is currently no concept of a degradation protein. Creation of such a protein type was contemplated and may be added in the future, but for the moment the complication introduced from having an extra protein type does not seem to be worth the benefits of having it available. In the natural system pelle and tube are involved in the degradation of the protein cactus. But since we can not degrade anything manually another mechanism is needed. Therefore the dorsal protein has a kinase regulation site on it that deactivates the protein by default. The kinase regulator pelle then switches the protein on and activates it. This is essentially equivalent to what happens in the other pathway. The more pelle available the more dorsal that gets activated. Pelle and spatzle degrade very quickly. They perform their job of activating dorsal and then fade away. Figure 6 shows the concentrations of these proteins in a group of cells after the first time slice.
2. Creation of the Decapentaplegic (Dpp) Gradient
The dorsal protein gradient is directly responsible for pattern formation in the ventral portion of the developing embryo. But there is virtually no dorsal found in the nuclei above the equator. So how is the pattern on the dorsal side formed if all of the chemicals have a constant concentration? The answer is that dorsal induces the formation of a new gradient in the dorsal side that is responsible for forming that pattern. Just as dorsal mRNA is distributed evenly throughout the cell and then translated after fertilization to produce an even quantity of deactivated dorsal protein, there is another evenly distributed mRNA that produces the transcription factor decapentaplegic (dpp). Dpp is created active and it is responsible for forming the dorsal pattern. But if it is evenly distributed then how can it form this pattern? When dorsal is activated it begins producing the short gastrulation (sog) protein and represses the transcription of dpp. Sog is an allosteric regulator that diffuses away from where it is created and into the dorsal portion of the cell. The more dorsal protein that is located in the nuclei the more sog that is produced. As it diffuses into the dorsal side it inhibits dpp and forms a new gradient of active dpp that is maximum at the dorsal end and decreases the closer you get to the center.
Once again the multi-nucleated blastomere is giving us trouble by allowing sog to directly diffuse to other cells. In the simulator system only diffusible ligands are capable of diffusing between cells in this manner. So we have to make things a little more complicated in order for this to work. You can see in figure 8 that dorsal represses the transcription of dpp since there is no raw dpp found below cell 25. Dorsal also produces the sog protein in large quantities directly related to the amount of active dorsal found in the cell. However, in this instance sog is really a diffusible ligand. Each cell also produces a constant quantity of the receptor sogr that binds to the ligand and produces the allosteric regulator sogar. Sogar is what actually inhibits dpp activity and forms the gradient of active dpp seen in the figure.
2. Dorsal Pattern Formation
The dpp gradient does not form instantly. It takes a little time for the sog protein to be produced and diffuse into the dorsal section to inhibit activity of dpp. Dpp is responsible for transcribing several other genes that make up the different sections seen in the dorsal portion of the embryo. However, since it takes a little while for sog to diffuse and inhibit dpp, some of these proteins will be transcribed in areas where they will not be found in the finished pattern. This is not really a problem though because dpp does not have enough time to produce these other transcription factors in quantities sufficient to determine the fate of those cells. Only prolonged exposure to the build up of those proteins in the cell over several seconds will have a long term effect on them. A transient production of those proteins have little effect.
The patterning found in the dorsal side is pretty straightforward compared to the patterning found in the ventral side. Dpp transcribes the two genes zerknullt and tolloid. Tolloid is transcribed at a constant level everywhere that dpp is active. Zerknullt is transcribed only when dpp is active in sufficient quantity. So this way tolloid protein will be expressed evenly throughout the dorsal side of the embryo, and zerkullt protein is only expressed at the extreme dorsal side.
2. Ventral Pattern Formation
Ventral patterning is slightly more complicated than dorsal patterning. Dorsal inhibits transcription of the genes that are responsible for pattern formation in the dorsal portion: zerknullt, tolloid, and dpp. It is also responsible for the transcription of the genes responsible for the ventral pattern. These genes are rhomboid, twist, and snail. Figure 3 shows the model of how these genes interact. The transcription factor twist autoregulates itself. You can see this by viewing the analysis of figure 3 and looking at enhancer 2 of the G_Twist gene. The expression function is linear with a negative slope. This is what regulates the level of twist so that it remains at a set point. If the level of twist is less than the set point then more of it is produced and if the level is higher than the set point then expression of twist is repressed until it falls back to the set point. Enhancer 1 for twist only expresses the protein when dorsal is above a certain threshold. this limits production of twist to the lower ventral region. The transcription factor twist also leads to the expression of snail and the production of the snail protein. Snail is a transcription factor that represses the activity of the rhomboid gene. So what eventually develops is that the protein rhomboid is only expressed between the center line and the beginning of twist, and twist and snail are only expressed in the most ventral section.
Video 1 shows the formation of the dorso-ventral pattern. The last two charts on the bottom are the most important. They demonstrate the final pattern like that one shown in figure 9. Since only three colors can be used for each chart it was not possible to superimpose them all together so the first chart shows the dorsal pattern and the next chart shows the ventral pattern. You can clearly see how tolloid is expressed throughout the top portion of the embryo, and zerknullt is only expressed in the most dorsal section. In the next chart you can see that rhomboid is expressed in a portion of the ventral section between tolloid and twist. Twist and snail are only expressed in the most ventral section. The levels of these transcription factors will stay constant for as long as the dorsal and dpp gradients remain constant. However, in nature this would just be a first step. Once these patterns were formed and stable for long enough they would lead to the switching of certain genes on or off, and ultimately seal the fate of those cells. Once these genes had been switched the dorsal and dpp gradients would no longer be important. They would have served their purpose and moved the developmental process on to the next phase.
The formation of the dorso-ventral pattern of drosophila is a fairly complex process and provided a good test of the capabilities of the developmental simulator. In fact, in the process of building this example I realized that the way I was modeling transcription was not correct and I had to go back and fix it. But the new version is more than capable of reproducing the same kinds of pattern formation found in the natural model. In fact, the graphs of the concentration levels look very similar to those found in the real fly.