Feb 25, 2005; Biology 2005 Albert Harris   Amphibian Embryo early development Frogs and salamanders are both amphibians, and have similar embryos From 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 salamanders Advantages: 1) Large size of embryos. 2) Tolerate surgery very well. For example, you can graft limbs or heads, or areas of skin, etc. Just keep the embryos is a very dilute salt (saline) solution, and you can tear lose 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! Thousands of research papers have been published on such experiments The core of what we know, or think we know, about mechanisms of development is extrapolated from surgical research on amphibian eggs. The kinds used most have been Xenopus (an African frog), and Amblystoma (includes the spotted salamanders of the eastern US) and also includes Axolotls (a semi-extinct Mexican Amblystoma) and also several kinds of newts (a sub-group of salamanders) Spemann discovered embryonic induction of the neural tube by notochord mesoderm, using tissues from 2 newt species. 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. It has never really been discovered why they regenerate so many organs that other vertebrates can't! Much is known; but not the answer! Still another advantage of newts is that they have VERY big cells. Cell size is linearly correlated with DNA amount, even "junk"!! 95% of biologists don't realize either fact, in my experience. They have more than 10 times as much DNA per cell as us. Nuclear transplantation 'cloning' was first done using Rana frogs. And the first adult animals produced this way were Xenopus A breeding colony of Xenopus is kept at UVA Breeding Axolotl colonies are at Indiana University, and U. of Moscow 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! Spemann's induction experiment works like a charm; I have done 20 or 30 such operations in an afternoon, with every one successful. (bacteria and molds are the only real problem; not the surgery) 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 mechanisms causing that isn't known, either. 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. In that case, if you separate the cells, then gastrulation only occurs in the daughter cells that got the gray crescent area 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) A scientist reported that he could transplant it; and cause formation of two-headed tadpoles. 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 there (but are not in contact anywhere else) 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. And this has turned out to be mostly true, with some changes. 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) How can you hope to find out which is the "real" normal signal? People also assumed that mesoderm, especially notochord, must be the first domino in the line, from which signals cause X, Y & Z The chordamesoderm was called "The Organizer" Experiments that didn't fit this assumption (sequential stimulation of cell differentiation by different signal molecules) were mostly ignored. 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. b) 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 (still living, I think). c) The new approach to identifying signal molecules is to >make c-DNAs from RNA found in different parts of the embryo; >>make proteins from transcripts of c-DNAs, >>> and see whether these proteins can induce second embryos (have the same effect as transplanted chordamesoderm) and of course: >>>> Give these proteins and their genes cutesy names, like "noggin", chordin, and chordino, goosecoid, frisbee and "cerberus" (Remember the 3-headed dog of Greek mythology!) [The word chimera had already been taken, for another meaning] [The worst offenders live in Boston, and are terminally conceited] Dickkopf is a related gene named by Germans, doesn't mean what you might guess, but could refer to some of those Bostonians. 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. Those guys even invent dumb names for their field of study!)

 

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