BIOL 390 (modified 2/24/98)

2/3/98

Review of concepts: epigenesis, cell theory, central dogma.

More on central dogma. What is involved in transcription – RNA polymerase, RNA primer, topoisomerase/helicase, promoters, inducers - in front of introns/exons. The RNA polymerase binds to a promoter site to start the transcription. There are other factors – they put DNA and RNA on the track to start producing RNA -> protein. As we understand the number of different factors influencing the path DNA -> protein, we see how the development could be changed. From RNA to protein – ribosomes, t-RNA, aminoacids, and all others. And, even when you get to the protein level, there is still more – post-transcriptional modifications. It’s extremely important – we are not only maintaining, but constructing, what takes much more precision.

Paper which was on the exam (Mediation of Sonic Hedgehog-Induced Expression of COUP-TFII by a Protein Phosphatase).

Axis formation – at the point of blastula – a ball of cells without a lot of structure. But we do know that the positional information is basically have been laid down. It tells us where the axes should be. At this point we get a little "fuzzy", we are missing the details. And these details are different for different organisms. That’s why we are starting to look at different organisms separately.

The maternal genes – have a lot of impact on the development. These maternal factors (RNA and proteins) are put in the egg, and they help to determine the body axes.

At gastrulation, we see the axes. One side is dorsal, another is ventral. If you’re going through gastrulation, you are going through axes – you will always have axes formation. On the point of gastrula, we get the expression of the information which we couldn’t see in the previous stages. The humans develop along the same pathway. But depending on organism, the blastopore can be anterior or posterior.

Once we have the blastopore, we have the gut going through the whole organism, the inner lining of the tube having the endoderm origin, Mesoderm and ectoderm are defined.

Overhead ecto-, meso-, and endoderms are going to be consistent in all the organisms. Mesoderm will form, clump up in a central rod – notochord. How? – all these little things are the story of the development. On the side on notochord, there are other parts which form. After we understand these general notions, we can take this concept and explain the organogenesis. NS is an organ – so it’s an example of organogenesis.

Chapter 5 (continued) – now it is easier to understand the development of fruitflies.

Fruitfly has a centrolecital egg. The yolk material is concentrated in the center of the embryo. The division process is incomplete – we are getting a syncytium. Layout: the tissues are arranged in ant-post and ventr-dors ways. In the various segments, blocks of information are put. The basic question is ho the genes are turned on and off. Textbook – there is maternal information linked to mother. Then the zygotic genes are starting to come in and take place in controlling the process. The environment then becomes a significant factor in regulating these genes. Polytene chromosome (large chromosome in fruitfly) genes can readily be turned on and off at any time. When they’re on, the chromosomes start to puff, and a product comes later on. A puff is a large area on a chromosome in which a lot of copies made, and then it starts to control transcription. If you have a lot of copies, you can transcribe a lot of it. This is an example of how the chromosomes are turned on and off.

Look at the textbook topics… Nanos – a gene product which controls other genes – what are the ways it works? Now we can talk with a high degree of accuracy after understanding the basic concept (of central dogma). Pages 134-135 (and so forth) – specific mechanisms used to control the development. Page 139 – zygotic genes.

2/5/1998

The control mechanism for the transformation of one cell into syncytium is environment. Temperature, pH, whatever, but when we start to focus on some hardcore control mechanisms, we are start talking about the maternal genes (maternal factors). They will be mRNAs and proteins. Those mRNAs and proteins will take this organism and guide its development. In terms of the function, what do they do specifically? Basically, to run around and agitate others – they activate other genes.

In the whole protein activity, if we have a structural gene, we’ll have promoter regions, activators and suppressors – there is a whole set of mechanisms to activate the expression of this structural genes. But from the paper we learned that there are other genes that activate a different type of system (receptors). These factors are going to regulate the transcriptional process.

• Understand the process of regulating a gene expression.

A -> B -> C (almost like an enzyme) – phosphatase stimulates the material to do something else. Some of them can be transcriptional elements, some – receptors, processors of the signals, etc. This process is going to stimulate/suppress (compound C, in molecular terms) the production of some compound D (and we can possibly have a gradient). D, then, is going to influence E, etc. Therefore, the maternal genes start the fall of the dominoes. How do we get to the developmental changes? And, does it stop after the developmental process is finished? But the process is not linear – there are C’, D’, etc… Homeotic transitions are driven out from this notion. If all events are a big tree, the lower to the root we cut the branch(es), the more damage we are going to do.

Then, textbook goes into explaining what are those genes are. One of them is the maternal protein, or the dorsal protein. They were identified and shown what they exactly do. Gradient effect.

P. 136 – orientation of the egg in its development. The process of maturation is shown. The three types of cells involved are the follicle cell, nurse cells, oocyte. The nurse cells are positioned, and think from a physical point view. The concentration gradient goes from the site of the production. The materials are starting to concentrate in a particular area. As the cells are developing, these compounds are being concentrated in a particular area, and they develop in a different way.

P. 137 – gurkin protein/factor – the mechanism actually involved in the production of the material. The concentration gradient has an impact on the development. The nurse cell puts the material in the oocyte, and it creates gradients which control the development. Is this the only way we can get the developmental process? But is there another way this process may have occurred? – No, and that’s where we brought the gurkin gene. This is a structural process, a molecule which stimulates receptors of follicle , which then releases the material going into oocyte. The egg then picks up this signal, which is not causing the expression of certain genes, but causes the microtubules to rearrange and change the shape of cells. The follicle cells are generally smaller cells, general somatic cells providing environment for the oocyte. Graafian follicle has the follicle cells on the periphery, and they are the follicle cells we are talking about. But the nurse cells are very significant in fruitflies.

As we start to see this process occurring, we see the concentration gradient. One is simply by the production of material, another is by the physical process of rearranging the microtubules.

We now can relate this process to the genetics lab on fruitfly.

The zygotic gene pattern is kind of the same way. The difference is that the maternal genes stimulate the zygotic genes, whose products of are becoming the regulators.

It looks like a very complicated discussion. There some stripes on the embryo. How do we get stripes out of a stripeless embryo. Almost like somites. They become organs (thorax, etc) important for the organism. How do we get these seven distinct patterns in a previously indistinct embryo? Remember that we are in the section which deals with zygotic genes. The products of the … cells produce a gene product which causes induction of adjacent segment, They induce each other in a chain-like pattern. It’s not A-B-C-D, however, but more like A-B-A-B-A-B. So, we see a pattern of AA and B going on in this embryo.

Some of the genes are turned on, and the areas around them are deactivated, and the development of the adjacent areas is stopped. The groups appear after the gastrulation. But it’s not the driving force of the segmentation. Picture on p. 149 explains it all. Then, the concentrations from different ends of the embryo cause the stripes to develop into different things.

2/12/98

Ch. 5 - maternal genes, zygotic genes, how they turn on and off… Now we are going into a specific gene and look how it gets turned on, off, etc.

As soon as you create a product, this product becomes a part of the environment, and it starts influencing the DNA. And again stuff on segmentation.

Ch. 6.

P. 173 mosaic regulative development in nematodes. You can distinguish controlling factors at very early stage. The maternal factors are very distinguishable, and after the embryo being many cells, if you take the structure away, the organism is not going to develop. At the 2 cell stage, it is predetermined what they are going to be. The gene products are distributed into different location (cell). But different patterns of cleavage cause factors to be in different parts of a cell, not only in different cells.

Now we are moving more to embryology, morphogenesis, cell differentiation. But before that…

Overhead on frog development. On molecular level, DNA controls it. The 3D structure, transcription stuff. We are attaching the RNA polymerase by means of promoters. TATA box + transcription factor will allow RNA polymerase to bind to right place.

Meristem - embryonic – like tissue in plants. These are like germ layers. The primary difference is that in the development of an animal, these germ layers disappear. One of the other important feature is totipotency - the capacity of a self to develop into any of the cell types found in a particular organism (they are not determined - almost throughout the whole life, but he made a whole argument that totipotent and determined are different things - I still disagree).

Alternation of generations - the tissues of the plants produce a different plant which has a capacity to produce gametes (gametophyte). Gametophyte will then produce gametes.

2/17/98

• similar to animal development. Differences - histodifferentiation instead of gastrulation.

Unique feature - concept of alternating generations, haploid – diploid states, they are distinct organisms. Haploids reproduce via spores.

A diploid organism produces a spore. It grows and produces a haploid organism, which has a primary responsibility of producing gametes. They will then fuse, fertilize, and produce a new diploid organism. Haploids undergo mitosis (no need for meiosis) to produce gametes. These two organisms are quiet distinct.

If we move to other organisms, the two states are basically the same. We create a unique structure - a spore – producing diploid stage, - > haploid organism, which produce male and female gametes. The male and the female components will combine to produce a zygote - > mature plant.

Gemnosperms - similar pattern, but the process of alternating generations is not that distinct. The haploid organism (gametophyte) is housed in a sporophyte. The gametophytes in essence function the same way as the haploid organism.

Gemnosperm is the cone-bearing plants (pine). These structures are reduced to a small set of cells. Pollen grain is a multinucleated multicell, and the sperm will go to the egg (microphyle) to fertilize it. The gametophyte is confined to the plant.

Flowering plants – similar pattern. Produce flowers, act in the same sense. We get diploid/haploid stages, a multinucleated structures develop. Pollen grain grows down into the stigma to take nucleus into the egg. The sporophyte and gametophyte become one organism.

Then, it comes even more like in animals.

Stem, root system, main branch and lateral branches of roots, on tip – meristem (young tissue undergoing mitosis). In stem, we have vascular system, buds, and the apical meristem, responsible for the direction of the growth. The lateral growth is controlled by the lateral meristem.

This meristematic tissue has to transform itself into a number of different tissues. It starts with undifferentiated tissue, receives signals, and transforms into those tissues. Q: where the information comes from? - Can’t come from an egg – the structure is ever present. We have a tree, and the info is there. One way is position. Each location has signals which cause to develop in a certain way. Lateral meristem, for example, has cork on the outside, vascular tissue on the inside. The meristem depending on the signal from the outside will transform into cork (outside of tree, epidermis), where the same tissue on the inside turns into the vascular tissue, which makes growth rings of the tree. The inner core has to maintain its embryonic state. As it divides, it creates outer and inner cells. Outer cells – differentiated; inner sells are totipotent.

How do we change form/shapes? The basic concept has to do with rearrangement of cells. The plants don’t go through gastrulation, but they go through histodifferentiation. One of the first things that happen is that blastula transforms itself into gastrula. The cells are migrating inward, what causes different layers to be established. Once they are formed, we look at the association of different parts, differentiation, movement, and we start getting the picture.

Concept of the cell migration.

Q: how does a cell know where it’s going? Ex: neural cells, muscle cells, mesenchyme cells… How does that happen? How can a seed be transformed into a tree to keep the continuous flow of cells to get them to leaves, trunk, etc.?

The plants do it by the differential pattern of division. It builds a structure. As cells divide, they push up the structures. Animals are different - no passive growth pattern, but very dynamic. Ex: primordial germs cells of gametes – they migrate through kidneys to gonads – they know exactly where o go. If they go to a wrong location, they don’t form them. One of the mechanisms is what’s known glycocalyx – sugary coat on the outside of cell. Hair – like structures on the outside, like the antigen components. As a cell type comes to another cell type, it starts communicating with that cell. Contact inhibition – cells will just grow & grow, but what tells them to stop? They are all in the contact with other cells, and that stops the growth.

If you go and alter the glycocalyx, the cells can’t find their way, get lost. They also lose their ability to regulate growth by contact inhibition. They keep growing like cancerous cells.

Chemotaxis is another mechanism. Cells follow a basic chemical path as a result of a production of a product by a different cell. The cell will move along that path. Cells will also follow a specific substrate. There are many different proteins to move cells. Connective tissue is a good medium in this sense: it makes a matrix which has ground substance (gel – like material) and fibers (like collagen). This material creates substance for other cells to crawl on. Some specific cell structures, when planted on petri dishes, have to have this matrix, otherwise they will not grow.

Fibroblast is one of the cells that develops from the mesenchimal cell, which then lay out the matrix.

Gradient also allows cells to move along a certain cells. Together with the above mechanisms, this all answers the question how the cells know where to move.

Once these cells actually found their way, how do they transform? Differential growth pattern of cells… Binding pattern/linkages of cells. Epithelial cells get joined with such links. This includes tight junctions, desmosomes. They involve proteins which link these cells.

We are questioning the physical process of organism’s changing shape.

2/24/98

Cleavage, blastulation, zona pellucida & vitiline membrane, cell mobility and adhesion molecules, the induction process, neurulation, notochord formation - we can talk about all these things, how they exactly happen. We understand differential growth patterns. Then, it's time to move into cell differentiation.

Oh, no! Not central dogma again… But it looks like it. Not only it looks like it, it smells and tastes like it : )

Fat cell development is dependent on a protein, but fats are lipids. What's the connection? Enzymes, yeah, enzymes, don't you see it, Kyle? They are produced in lypogenesis, and there are enzymes to carry it out.

Textbook talks about the whole process of cell differentiation. It is a process in which a cell becomes specialized. What we are saying is that it becomes specialized by producing a specific protein (structural - carotene, functional - enzymes, or in-between - transport proteins, hormones). There is something along that process that will turn this cell into such condition. The cell becomes locked in to a specific path. It's a block from going other ways (back or aside), and inhibitors participate in this mechanism.

A factor that allows a cell to be transformed into a fat cell (can happen in a normal organism, but can easily be shown in a lab). They took myoblasts, treated with arachidonic acid, and transformed into fat cell. Both cells are mesenchymal origin. Here, we show that we can change a development even in a predetermined cell. In the case of antibody production, the molecular process involved in the production of these molecules (P. 277). There are variable regions, and they differ according to the type of antigen which they have to fight. Concept of monoclonal antibodies: you built one specific antibody for a one specific antigen. Vaccination is to trigger the process of antibody production.

It's a process of DNA -> RNA (p. 283). RNA polymerase. Repressor stops the developmental process by stopping transcription. Enhancers lead to the greater production of these products, allowing transcription to take place.

Muscle cell system is neat because you take a basic stem cell and you use it to produce myoblasts which are committed cells that continue to divide. They are determined, and we look the kinds of factors that cause the determination of these cells. In case of arachidonic process, there is a two path process: either to go to a fat cell, or to come back into undifferentiated state. Then, we can talk about myotubes in which they mature (terminal differentiation) and produce actin and myosine, fuse together to produce a tube.

Textbook also gets into a concept of blood cell differentiation. You take a hemopoetic stem cell, the one which gives rice to the foreign blood elements, Then it will go in three different paths to produce RBCs, WBCs, and platelets. Q: what factors cause each path to take place? And there are even subpaths to these paths. Each one of these blood cells have distinct proteins they produce.

Once we turn on the genes, translation becomes the final stage. At the determined stage the process of translation haven't occurred. The cell "knows" that it will occur, but translation hasn't occurred yet. There is a factor present in the cell which will turn on the translation, when the terminal differentiation occurs. Before, it's a determined state.