Abstract
Developmental biology seeks to study the progression of multicellular organisms from the single cell stage to full adulthood, taking into account the genetic information and chemical signals used by animal cells to mold them into the complex creatures they are at the end of their growth. The main question asked by developmental biologists is: How do genes determine the way an organism develops patterns, or specific structures in its body?
This paper will attempt to provide some insight into what research is currently being done to find an answer to this question, background information as to what the answering of this question entails, and most of all, what, exactly, the question is asking, along with the other questions that are brought up in the course of this exploration. To do this, I have read a variety of research papers, articles, and books; conducted interviews at the lab run by my technical advisor, and, with the aid of my technical advisor, gone through common steps used to find some of the answers needed to fully understand this question. In doing this, I have also related the research currently being done with nematodes to the search for the cure for cancer, and the development of animal life in general.
Head, Shoulders, Knees, and EMS:
Pattern Formation and the Effects of Wnt Signaling on Cell Division in Nematodes and Humans
Introduction
The field of genetics is a rapidly expanding one, and developments in this field will have long term and lasting effects not only on the lives of those living in first world countries, but also on the lives of everyone on Earth. Genetics has and will continue to provide ways to cure disease, increase agricultural output, and decrease waste. As a society, we will see changes in the way we are born, how we live, and when we die. We will see changes in our mortality, and we will see changes in our societal structure. More than any of these, however, will be the changes we will see in the way we see ourselves and our humanity.
Part of the process of understanding ourselves is the understanding of how we develop from fishlike fetuses to walking, sighted, limbed adults. In this search, biologists seek not only to understand the development of humans, but the development of organisms in general. This area of science is called pattern formation, or developmental biology. Developmental biology studies the role genes play in the maturation of organisms. Specifically, a developmental biologist studies how arms, legs, fingers, and toes are separated and specialized between conception and maturity, as well as how DNA determines where the brain is put, and whether you have six legs or two. By breeding organisms with a very short life span, and looking for fatal defects, the effects certain genes have on the development of a given organism can be deduced. A great deal of work has recently been done in this area, and an important breakthrough was made in October of last year, when a pair of headless tadpoles were cloned, revealing the nature of a protein that is vital to the development of a head during embryo growth (John Travis 55).
In order to study the development of organisms in general, one must select an organism that is both easy to observe, and which develops, and is able to mate, rapidly, giving many generations of data to work with. An organism that does a very good job of meeting these requirements is the nematode Caenorhabditis elegans, a worm which reaches maturity in a matter of a few days. Some types of flies are also used, but the worms allow easier containment and selection in breeding, and are therefore more popular. These, in fact, are the organisms which Bruce and his lab aides use in their research at the U of O.
Another popular, though unlikely, organism for developmental research is the small, striped zebra fish. These are bred normally, but then are subjected to radiation, viruses, and other harmful effects, to see who survives, and therefore, who has genetic defects (John Travis 360).
In order to study the development of the C. elegans, it is necessary to breed two of them, both of which have only one of two genes necessary to development, and examine the offspring. One quarter of these should have neither necessary gene, and therefore be unable to develop, usually resulting in death.
This is much more difficult in the lab, however, because one needs to breed nematodes until a quarter of the offspring die, and then determine which gene both the parents lacked, and therefore which gene, of many, is vital to development. Eventually one can get a picture of all the genes that have a part in development, and what each one�s role is, and from there an idea of how development is orchestrated.
Therefore, the goal of Bruce Bowerman and the people he works with has been to search for the genes that are central in the development of patterns in organisms, and thereby figure out exactly how genes affect development.
Research Methods
at the University of Oregon
My research into this subject began in fall of last year, as I read through articles and papers and e-mailed professors at the University of Oregon about their involvement in genetics, the area of my interest. Specifically, that realm of genetics concerned with what makes your foot a foot and not a fin, as opposed to that concerned with what makes your eyes blue and not green. In other words, I was interested not in hereditary, or classical genetics, but in the more fundamental aspects of genetics.
Eventually I was directed, through Diane Hawley, to Bruce Bowerman, who does research in Developmental Biology at the U of O, focusing on the development of the nematode C. Elegans. C. Elegans is used for a variety of reasons. First and foremost, it is small. Too small to bite you or run away, but not so small that you can�t see it with the naked eye, if you look hard enough. They also can be frozen to nearly any temperature, and, for the most part, survive unharmed. Another major advantage of the nematodes used in the lab is the speed at which they reach reproductive age and reproduce. If you pick a nematode at random, chances are it will be an adult with viable eggs. Yet another reason to use nematodes for determination of DNA sequence is that they tend to have a good number of genetic mutations that are definite, in that they don�t occur to varying degrees, that are easily spotted, and that only manifest above a minimum temperature.
Nematodes themselves are small white worms, visible to the naked eye as tiny white dots, and seen easily under the microscope. They can be cut open with an ordinary scalpel, releasing their eggs, which at that point are still viable. The eggs can then be viewed under the microscope as early as the single cell stage, duplicating rapidly, and a four dimensional tape can be made of this, each frame consisting of several pictures taken of the cell at different depths in that instant. A computer program can then be implemented to view the development of a nematode embryo at different depths, or to view that entire cell, from top to bottom, at a particular instant. The nematodes themselves are kept at the lab in dishes with blocks of bacteria, their source of food. Large freezers hold the worms over the long term, and since they have no minimum temperature needs to speak of, a tank of liquid nitrogen keeps worms frozen if the power goes out.
There is also a technique available at the lab for aiding the sequencing of DNA that I found quite interesting. A slab of a substance somewhat like jelly, and derived from seaweed, is placed in a plastic mold, and the DNA of a given nematode is placed in small indentations in the jelly. The slab is then connected at either end to electrodes, and since DNA is charged negatively, it moves toward the positive electrode. The jelly, of course, offers resistance, and what results is the separation of the smaller sections of DNA, those that could travel faster through the jelly, from the longer sections, which were more easily slowed.
The Great Mutato
The Bowerman lab works solely with nematodes, but has several functions dealing with them. One of these functions is the supply of mutant worms to other labs in the circle of which Bowerman�s lab is part. He sends dishes of worm infested bacteria off to labs around the world on a regular basis. A far more complex function of the lab is the determination of the placements of nematode genes, using known mutations. This process is a bit complicated, and is as follows: Chromosomes, of which a nematode has six, can exchange pieces of themselves with each other by crossing one of their tails with the tail of another chromosome, as can be seen in appendix (a) in the back of this paper. The placement of a gene is crucial to it�s expression, so when this happens, a worm that is known to have had a certain mutation might have that mutation nullified, as it is in the wrong place on the wrong chromosome. Now, say that you know the exact location of a couple of mutations, for instance, unc-20 (for �uncoordinated�), a gene which robs its owner of the ability to move at a normal speed, and dpy-2 (for �dumpy�), a gene causing worms to be short and fat instead of long and skinny, and you know that a particular worm has these particular mutations. Say the worm also has a mutation with a known effect, but an unknown location, on the same chromosome as the other two mutations. You can use the probability of the nullification of each known mutation given that the unknown mutation has been nullified to determine the location of the unknown mutation, by finding out if the �unknown� was between two given �knowns�, before the chromosomes overlapped and switched tails, as shown in appendix (b). Random mutations are therefore induced by flooding worms in x-rays or submerging them in EMS, a chemical which causes mutations in worms, and which should be distinguished from EMS the cell. Those mutations that are only lethal above 15-25 degrees Celsius are then singled out, and become the known mutation of unknown location as above. Therefore, those genes integral to the worms� survival can be pinned down and distinguished from the many genes that serve less important purposes.
Another use of this and other labs worldwide is the determination of the effects of various mutations on nematodes. In the lab, nematodes would be dissected, and their eggs would then be removed, put on a slide, and watched as what starts as one egg turns into a worm. Normal, or wildtype, eggs do just that. Mutants, however, usually don�t reach adulthood. Cyk and Zen mutants have trouble in dividing, and never get past the single cell stage. Mutations like Unc and Dpy, however, may allow the mutant to live an almost normal life. Many mutations, and indeed, most of those used in the lab, can be immediately fatal but only become active above 15 degrees Celsius. But what is most fascinating is not how mutations can prevent worms from functioning, but how worms, and indeed, humans, function normally, as this, it seems, is dependent on many events, and is very intricate in its execution. One of the most important recent discoveries has had to do with division at the four cell stage. Normally, while P2 goes on to produce P3 and C, EMS cell splits into the E cell and the MS, which go on to produce intestine in E, and mainly muscle in MS. It may be easiest to explain an excerpt from an article in a U of O publication, �A signal from the sister of EMS, a blastomere called P2, polarizes EMS to induce endoderm [intestine].� (Cell, p.695) EMS and P2 are sister cells, or blastomeres, and P2 sends a chemical signal to EMS, not only turning EMS to have only one end touching P2, but also telling the half of EMS (E) that touches P2 to produce intestine, and the other half (MS) to produce, for the most part, muscle. This is illustrated as appendix (c). The processes of both EMS rotation and the distinction of muscle and intestine is governed largely by a series of genes known as mom, specifically, mom-1 through mom-5. A lack of these genes not only left EMS incorrectly rotated, however, but randomly rotated, as well as unable to create intestine. Mom-1, mom-2, and mom-3 are all needed in P2, while mom-4 is needed only in EMS. The actual effect of the proteins is to diminish the effects of the pop-1 protein in an EMS daughter, thus diverging their fates. (Cell, p.695) There is a similar rotation at the 2 cell stage, when P1 rotates 90 degrees and then divides, while AB, its sister blastomere, divides normally, and therefore at right angles to the division of P1, illustrated in appendix (d). Such rotations in cells are carried out with microtubules, tiny tubes which emanate from two central spindles. The tubules, when they touch each other, push away, and therefore make the spindles they are attached to rotate away from that point. Each microtubule adds on to its base at the spindle, growing longer, and thereby putting a greater distance between the spindle and the place where the microtubules contact each other. It is curious that, though the message to rotate and the message to produce intestine both normally occur at the same time, if that time is delayed, for instance, by removing EMS from P2 and then putting them back together, intestine will still be produced, but the cell itself will not rotate. It stands to reason, therefore, that though the signal for intestine production and cell rotation is the same, Wnt signaling has a different way of carrying out each of them. It has been found, in fact, that whereas Wnt signaling has to target EMS�s genes to trigger intestine production, it directly targets the cytoskeleton in order to induce cell rotation.
This is where developmental biology and molecular biology meet, and is therefore not only more abstract and a little harder to follow, but also more general in its application. At this point the development of worms begins to have an impact not only on our own development, but that of animal, and even carbon-based life, in general. This type of signaling, known as Wnt signaling, not only allows one sister cell to control the fate of the other, but also allows it to subtly control the other�s actual structure. It affects the sister�s use of the genetic code and which end of the sister is affected by this chemical signal.
There are therefore two parts to Wnt signaling. The first part is the actual message to the cell, which basically informs it that it is to follow a set string of genetic code, thereby eventually producing an intestine. The other part is that the signals sent in some way actually alter the cytoskeleton, using microfilaments, the tiny bone-like manipulators in cells, to turn the target cell 45 degrees, so that it will divide with one daughter facing P2, the sender of the Wnt signal, and ready to be further affected by the same signal to create endoderm, and the other facing away from the sister cell, and, not having received the Wnt signal, producing mesoderm. Experiments have actually been done where the target cell was removed from the organism before it could be signaled to, and it not only didn�t turn 45 degrees relative to the surface on which it rested, but it also produced double the normal amount of mesoderm, and no endoderm. This takes chemical signals out of their normal arena, which is the more mundane functions of a cell, such as respiration and hormone identification, and puts them on a level with genetic information as to how they affect not only a cell�s orientation and movement of cytoplasm, but also its fate, changing the biology community�s whole perspective on how external influence can guide development, and, indeed, how chemical signals function to alter their target cells.
Worm Intestines and You
Wnt signaling has more immediate and far reaching importance than as a link between genetics and cell biology, and, indeed, was not originally associated with nematodes. As Elizabeth Pennisi said in How a Growth Control Path Takes a Wrong Turn to Cancer, �Cancer researchers originally discovered the gene for Wnt, a protein that conveys growth and developmental signals between cells, 16 years ago in mouse mammary tumors.� (Science, 1438) The gene for Wnt signaling was first called Int, for �integrated�, as it activates when a certain virus�s DNA is integrated next to it in the same chromosome. 5 years later, Int was shown to be the mouse version of the fly gene Wng, for �wingless�, and has been renamed Wnt, as a cross between Wng and Int. Since then, Wnt pathways have been shown to be integral in formation of many cancers, in that a faulty Wnt pathway can lead directly to colon and liver cancer, melanoma, and possibly prostrate and breast cancer.
The Wnt pathway consists of several chemicals all competing and interacting to make an embryonic cell divide at a very high rate, while keeping a liver cell in an almost permanent interphase, a state of non-division. The gene that these various chemicals are all focusing around is c-MYC, a gene that, when activated, causes cell division. This is usually regulated by a transcription factor called Lef/Tcf, which is normally attached to two proteins that it needs to keep c-MYC from becoming activated. When a cell needs to divide, however, a protein called beta catenin, which replaces Lef/Tcf�s two �helper� proteins attaches to Lef/Tcf, making it activate, instead of deactivate, the c-MYC gene. Beta catenin normally is created in the cell, but is then either captured by a protein, E-cadherin, which exists in the cell membrane, or is destroyed by a group of proteins which exist mainly for that purpose, namely, APC, GSK-3beta, and axin. Many cancers are a result, therefore, of the gene which creates these proteins being mutated, producing mutated versions of the beta catenin destroying proteins, and therefore allowing a buildup of beta catenin. This, in turn, replaces the �helper� proteins for Lef/Tcf, allowing c-MYC to be active, and causing the cell to divide rapidly, causing a cancerous condition or growth. At the same time, the activation of c-MYC is not normally detrimental. Cells have to divide fairly often, depending on how long a particular cell lasts, and Wnt signaling, which deactivates the beta catenin destroying proteins, inducing cell division, is very important to our development. We, as humans, and indeed, animals, wouldn�t get past the single-cell stage if it weren�t for this complex pathway. It should be noted at this point that the Wnt signaling which distinguishes between intestine and muscle in a worm, and that which makes our cells divide has more in common than Wnt itself. The proteins which help halt the division of cells in humans, as well as the division inducing protein they help halt, exist in the Wnt pathway in nematodes, as does Lef/Tcf, the trasctription factor which regulates c-MYC, the gene that induces cell division.
The discovery of the relation of this pathway to cancer is a major medical breakthrough. At this point, if one could simply introduce a viable version of the mutant protein into cancerous tissues, the cancer could be stopped, and, after a period of adjustment for the body allow the previously cancerous cells to die, most cancers would even reverse, and would disappear altogether. Thus this discovery has given immediate hope to the search for the cure for cancer.
This example also helps illustrate how intricate the steps the cell goes through are, and how easily outside influence, like harmful radiation, chemicals, and just about anything not water soluble, can throw the cell off it�s regular path. Indeed, the fact that disruptions of the cell�s routine are so rare is only attributable to the incredible number of safety checks and fuse boxes in the machinery that is the cell. As Elizabeth Pennisi wrote, �Inappropriate activation of the Wnt pathway is apparently so dangerous that the cell has evolved multiple ways of keeping it in check...the nuclei of developing frog embryos contain factors that repress the transcription of genes activated by the Wnt pathway.� If we can only discover how to recognize malfunctions in the systems of our cells, we can easily catch and correct the mistakes and, more often, external disruptions, that happen to slip through their defense mechanisms.
Conclusion
This paper addresses both nematodes and their mutations, and the effects of the discoveries made in the field of developmental biology with that of human cancer. Thus, it seems to make a distinction between the research being done which affects us as humans, and the research being done on the secret lives of worms. This is not the case, however. Even the most obscure finding about tiny worms is of importance to our day to day lives, as well as countless industries, though especially the agricultural and medical ones. Not only do these discoveries directly affect us, but they also help to answer the question of how our DNA turns us from a single cell into a tremendously complex organism, and through that, the question of what we really are.
As human beings, worms don�t seem to have much in common with us. They are quite a bit smaller, and also have much less genetic material to work with. The similarities, though, far outweigh the differences. Our genes are, after all, both made up of the same basic substances, and the same chemicals are used to convey information in worms as in humans. Any discovery, therefore, of how these materials are used in worms also has a major affect on how human development is viewed, and any discovery having to do with how the materials actually affect their targets, as in the research being done around Wnt signaling having to do with both cancer and the development of the intestines, can completely revise how we see our own development, and what steps are integral to it.
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Acknowledgements
Thanks to Bruce Bowerman and his fellow lab workers for their help in the writing of this paper, and the use of the Bowerman Lab.
If you internet junkie students copy this and pass it off as your own, may you burn in eternal hellfire. Thank you, come again.
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