Embryology Biology 441 Spring 2009 Albert Harris
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Lecture notes for Monday, Feb 16, 2009Amphibian Embryo early developmentFrogs and salamanders are both amphibians, and have similar embryosFrom the late 1800s until after the mid-1900s, most of the best embryological research was done using salamander & frog eggs Even tissue culture was invented using cells from frog embryos, and frog lymph
Advantages of amphibian embryos for embryological research::
2) Tolerate surgery very well (although large cells are very fragile) (Early salamander embryos repair wounds in minutes or hours.) Just keep the embryos is a very dilute salt (saline) solution, and you can tear loose organs and just squeeze them next to another area where you have removed the skin, and they quickly seal into position. It takes a few weeks to learn the art, but then you are Dr. Frankenstein!BR> Always remember to build only very small monsters!
Thousands of research papers have been published on such experiments
The kinds used most have been Xenopus (an African frog), and Amblystoma (includes the Spotted Salamanders of the eastern US) Hans Spemann discovered embryonic induction of the neural tube by notochord mesoderm, using tissues from 2 newt species. (Won Nobel Prize)
Holtfreter used salamander & frog cells to study cell sorting.
Another advantage of newts is that they can regenerate their legs, regenerate the retinas of their eyes, the lenses of their eyes, etc.
Still another advantage of newts is that they have VERY big cells.
Nuclear transplantation 'cloning' was first done using Rana frogs.
A breeding colony of Xenopus is kept at UVA, and another here. Slow life-cycles (and also very large genomes) are big disadvantages for doing genetic embryology. But it's hard to do surgery on flies! Or even mice, or fish, or birds! With salamander gastrulas, you just cut out the dorsal lip of the blastopore with a sharp needle; then slit open the blastocoel of another embryo, push this dorsal lip in through the hole, watch the hole heal in a few minutes, and next day a Siamese twin salamander will form! Call the National Inquirer
Spemann's induction experiment works like a charm;
I have done 20 or 30 such operations in an afternoon,
with every one successful. Hildegard Pröscholdt & Hans Spemann discovered (and named) the phenomenon of "Primary Induction") This was the ability of grafted cells from the dorsal lip of the blastopore (put more or less randomly into the blastocoel of another newt embryo) tostimulate changes in the differentiation of the "host" embryo cells so that host cells near the graft get recruited to form an entire new body. For example, the somatic ectoderm next to the graft is induced to form an extra neural plate, which then neurulates and forms a neural tube, which forms a new, extra brain and spinal cord!
Please understand that this is not just a nifty parlor trick. Furthermore, this example of "re-programming" cell differentiation by cell-cell signals from the future notochord cells led to beliefs that many (all? Most?) cases of cell differentiation might be caused by (chemical!) signals from nearby cells. Inductive signals would activate the switches of the branching train tracks that I used above as diagrams of successive determinations of cell fate. Many more examples of embryonic induction were then discovered: a) The optic cup (future retina) induces epidermis to differentiate into lens. b) Epidermis induces the part of the retina that touches it to be neural retina. c) In tooth embryology, each of the two layers induces differentiation of the other layer (Future dentine-forming cells induce enamel-forming cells) d) Endoderm can be induced to differentiate into either lungs, or pancreas, or liver, or salivary glands e) Kidneys are induced to differentiate by contact with kidney ducts (and if both an embryo's kidney ducts "grow" (crawl) to the same side of the body, then kidney will differentiate only there. (So you might have two left kidneys, with 2 ducts; & no right kidney) f) An extra leg can be induced to form along the flank, by grafting an inner ear rudiment under the flank skin. (you can easily make 6-legged salamanders, or other animals)
Embryological facts are more strange than Mary Shelley could imagine!
Many further examples of induction were discovered: Two main experimental criteria: 1) Grafting some tissue to an abnormal location results in either the tissue next to the graft and/or the tissue of the graft itself differentiating into some different cell type than they would have, normally. 2) Inserting impermeable barriers between where two embryonic tissues would have contacted each other results in one or both failing to differentiate into the normal cell type they would have become.
An important related concept: "Competence" =sensitivity to be induced This special meaning for the ordinary word competence was proposed by Conrad Waddington Waddington had earned a very good scientific reputation by discovering/demonstrating that grafting Hensen's node to peripheral parts of bird & mammal embryos will induce formation of entire second bodies. In other words, Hensen's node has the same inductive power as the dorsal lip of the blastopore (which makes sense! It does form the notochord).
Some history that you don't need to learn:
Hilde Pröscholdt married Otto Mangold (a post-doc in Spemann's lab). Otto Mangold became a leading Nazi; Spemann was a Nazi supporter. Both died during the Second World War. Spemann's other student, Hans Holtfreter, opposed Nazis, was attacked by them, & escaped to Canada. The first Nobel Prize for any embryological discovery was awarded to Spemann. Another embryologist (Morgan) had previously won a Nobel Prize, but for discoveries in genetics, which he said was much easier! Waddington was Chief Scientist in optimizing depth charges and other anti-U-boat weapons and strategy in the Bay of Biscay, west of France, is generally credited with inventing "Operations Research" as a subject, and made several very large contributions to the defeat of Nazi Germany. He then headed a center for genetics research in Edinburgh, Scotland, discovered the controversial phenomenon of "Genetic Assimilation" explaining examples of what had seemed to be Lamarckian evolution, and wrote many good books about embryology, and a weak one about art. He lived until the mid-1970s, and wrote for the New York Review of Books, which was then, and still is, the most intelligent periodical in the world.
Hundreds of rather over-ambitious researchers tried to discover THE specific inducing substance that developing notochordal cells were presumed to secrete that stimulates ectoderm to neurulate, or that are the diffusible chemical signal that cause other examples of embryonic induction. They assumed there was just one substance. There was some success, but mechanisms turned out to be more complex. This may be a good place to digress on the general subject of bioassays. Although the word bioassay doesn't seem to be in the textbook's index, this is one of the most important concepts in experimental biology. How was cyclic AMP discovered to be the chemotactic attractant substance used by Dictyostelium? By trying different extracts and comparing which one caused amoebae to re-orient their movement. (actually, in that particular case, someone in the lab already had some c-AMP, because this chemical had already been discovered to be a "second messenger" (equivalent to a hormone, inside other cells). How was the chemical structure of auxin discovered? By comparing amounts of plant shoot elongation stimulated by different extracts. Auxin was named first, and discovered later. It is misleading to say that people "isolated" indole acetic acid. Instead, they used bioassays to compare relative amounts of the (then still unknown) chemical in different extracts from plant tissue; until eventually they narrowed down the possible candidate substances to just one. How was the chemical structure of serotonin discovered? By comparing forces of contraction ("muscle tone") of dissected-out smooth muscle stimulated by different extracts of blood serum. 'sero' - 'tonin' (That's a better choice than "bloodocontractin", you will have to admit!) Chemical analogs of indole acetic acid are the basis of multi-billion dollar herbicide industries, and chemical analogs of 5-hydroxy-tryptamine are the basis of multi-billion dollar antidepressant industries. Almost all of pharmacology is based on synthesizing chemicals that have nearly the same chemical structures as some normal biological signal molecule, but have some differences, like a nitrogen where an oxygen should be or a bromine where a methyl should be, so that they either block or over-stimulate whatever receptors the normal chemicals bind to. Bioassays are how you discover which chemical causes which response. Three principles of pharmacology: Step A) Some basic biologist discovers a phenomenon that seems like probably it could be caused by secretion of some specific chemical. Step B) Other basic biologists design a bioassay method, by which you can compare the amounts of this phenomenon that get stimulated by different sub-fractions of tissue extract (like, if you centrifuged the extracts; or used chromatography.). Step C ) Eventually, one or a few pure chemicals are identified as producing much stronger effects in the bioassay. Many times, no one such chemical is ever found. If your bioassay isn't specific enough, it will react to many chemicals in addition to the 'real' signal. Another reason for failure is that sometimes the signal is more complicated than you assumed. Both these reasons prevented "isolation" of the inducing substance. And the normal signal mechanism wasn't some single, simple chemical. Step D) Large industrial companies keep track of chemicals discovered by bioassays or other methods, nearly always at universities, and published in research journals, like Nature. Step E) Industrial companies synthesize every imaginable structural analog to molecules like auxin and serotonin, and patent them. Step F) These companies persuade the general public, and judges who don't know much biology, that these chemical analogs were great original discoveries, made by the companies themselves (instead of being just the easy and obvious last steps of university research). This last step, bamboozeling the public, the clueless news reporters, and well-meaning judges who just don't understand how science works, is even more important than the patent laws. AZT was developed by federally-funded cancer researchers at the U. of Michigan in 1956, and an NIH researcher developed a T-lymphocyte bioassay by which he discovered that AZT inhibits AIDS virus infection. But using this chemical to treat AIDS was then patented by a certain company, and three Federal Judges ruled this patent valid. Their legal decision is posted on the web.
No newspaper will print such stories. What amount to molecular-genetic bioassays for inducing substances have been developed and used in recent years. One key idea is to compare amounts of inhibition of cell differentiation produced by injection of nucleic acid base sequences complementary to the genes for suspected signal proteins. "anti-sense RNA" and also "RNAi" The results have been a failure, in that lots and lots of genes have been discovered, the inhibition of which interferes with induction & axis formation. But our textbook considers this outcome a big success, just as earlier textbooks were proud of the number and variety of different chemicals that produce the same induction effects as the dorsal lip of the blastopore. Holtfreter one induced a second embryo by implanting a piece of salami. When I was a grad student, I tried this and got much more positive results (induction of secondary embryos) with salami than with bologna.
The "gray crescent" forms below the equator on the side of the oocyte 180 degrees opposite from where the sperm entered. Later, when gastrulation begins, the blastopore forms at the location of the gray crescent. In most species, you can't really see the gray crescent (i.e. it isn't a different color), but the cortex still slides upward on the side opposite from wherever the sperm entered; and the blastopore forms there. In many, or most, embryos the first mitotic cleavage bisects the gray crescent; and the mechanism causing that isn't known, either. The cleavage pattern is often different, but that does NOT produce abnormalitie in anatomical development.
In some embryos, the first plane of mitotic cleavage is perpendicular to that, and therefore one of the daughter cells gets all the gray crescent. People used to assume that there was some special cytoplasmic signal molecules concentrated there, analogous to the "yellow crescent" in sea squirts (and remember that in sea squirts, this isn't always visible either) One very good scientist reported that he could transplant it; and cause formation of two-headed tadpoles.But maybe it was because they were upside down. By holding eggs upside down, especially with centrifugation, the gravitational flow of cytoplasm can create a second gray crescent.
Now, the idea is that it is special because certain (unknown!) combinations of cytoplasmic materials make contact in the grey crecent
After Spemann discovered induction of the neural tube by the mesoderm that will form notochord, and others demonstrated the induction of the lens by the optic cup, and the cornea by the lens,
biologists concluded that locations of cell differentiation must be controlled sort of like falling dominos, with special chemical signals sent from one cell type to the next, stimulating genes to turn on. People wasted lots of time testing different kinds of chemicals, to find out which ones would stimulate skin to form neural tube, etc. (one big problem was non-specific induction: discussed above, in relation to bioassays)
People also assumed that mesoderm, especially notochord, must be
the first domino in the line, from which signals cause X, Y & Z But some changes have been accepted: a) Mesoderm is no longer thought to begin the chain of induction. Separate culture of pieces of tissue in the animal half only differentiate into ectodermal cell types. Cells from nearer the vegetal pole form only endodermal cell types. You DON'T get mesodermal cell types from the cells in between, unless the cells above and below them are left in position. b) Apparently, mesoderm is induced by some combination of signals from the animal and vegetal hemispheres of the embryo. It's the combination! The vegetal cells that induce the "Organizer" are called the "Nieuwkoop center " in honor of a Dutch embryologist who advocated these ideas. c) The new approach to identifying signal molecules is to
>>make proteins from transcripts of cDNAs,
>>> and see whether these proteins can induce second embryos
and of course:
"Dickkopf" is a related gene named by Germans (doesn't mean what you might guess). Many of these vertebrate signalling proteins have amino acid sequences that are very similar ("homologous") to proteins previously discovered serving comparable functions in Drosophila. These similarities surprised many biologists, and stimulated formation of a new sub-field of biology called Evo-Devo.
= "Evolutionary Developmental Biology" Review Questions:* Starred questions are particularly difficult, or require original thinking.What are two advantages of amphibian embryos for surgical experiments on embryological mechanisms? What sorts of animals are newts? What are Amblystoma and Xenopus? For what experiment, actually done by whom, did Hans Spemann win the Nobel Prize? *In what sense was this NOT the first Nobel Prize won by an embryologist? "Cloning" of animals (equivalent to Dolly the sheep) was first done in the 1950s using embryos of what kind of animal? Describe the method by which Spemann had already proved that all the nuclei at the 16 cell stage still contain all the genes needed to make a whole animal? What are two major DIS-advantages of amphibians for molecular genetic research? What is (or was) meant by "primary induction"? What do embryologists mean by "induction" in general? What is meant by the dorsal lip of the blastopore? What effect can cells from that part of an embryo have on other embryonic cells? In bird, mammal, and reptile embryos, what is the equivalent to the dorsal lip of the blastopore? Who discovered this equivalence? In experiments on induction, what is meant by "graft" tissues, versus "host" tissues? Who was Hildegard Pröscholdt? Does induction occur in normal development, or just as a result of surgery on embryos? Would scientists be so interested in induction unless they believed it occurs on normal embryonic development? Hint: No, they wouldn't. Describe at least five more examples of induction, including what tissue induces which other tissue to differentiate into what. What are two kinds of experiment you could do to find out whether induction is responsible for the formation of a certain organ or differentiated cell type? What is meant by "competence" in relation to embryonic induction? What is a bioassay? How were bioassays used to discover serotonin, auxin or the chemotactic substance in Dictyostelium. * Devise a bioassay that should be capable of identifying a chemical signal that causes a particular case of embryonic induction. Briefly tell two different reasons why such a bioassay might fail to be able to find out what causes induction. For what purposes do people use synthetic chemical analogs of biological signal molecules? Do these need to have the exact same chemical structure as the normal signal molecules? Or are they often more useful if they don't. * In order to make human organs from embryonic stem cells, would it help to know the normal mechanisms of embryonic induction? *And will these efforts fail unless the cells can be induced to differentiate? Can cDNAs be used as part of bioassay to prove which genes are necessary for induction, or for other normal embryological processes? What can you find out using the selective inhibition of translation of particular genes by anti-sense RNA or analogous methods. What are the names of 2 or 3 of the genes that have been discovered in frogs by these attempts to understand the molecular genetic basis of embryonic induction? *What do you conclude from the existence of fly genes with very similar base sequences? |