Lecture notes for Friday, January 16, 2015

Embryonic Development of Frogs and Salamanders (= Amphibians )


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.

Tissue culture was invented using cells from frog embryos, and clotted frog lymph as the substratum.


    1) Large size of embryos (several millimeters in diameter)
    2) Tolerate surgery very well (although large cells are very fragile)
    (Early salamander embryos repair wounds in minutes or hours.)
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 loose organs and just squeeze them next to another area where you have removed the skin, and they quickly seal into position. 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)

Hans Spemann and Hilde Prösholdt discovered embryonic induction of the neural tube by notochord mesoderm, using tissues from 2 newt species. (Spemann won a Nobel Prize specifically for discovering embryonic induction)

When signals from one part of an embryo stimulate differentiation of nearby cells into some different cell type than they otherwise would have become, this is called "Embryonic Induction"

Examples of embryonic induction:

1) Induction of somatic ectoderm to differentiate instead into a second neural tube, because of contact with notochord mesoderm (and sometimes by contact with other tissues)

2) Induction of extra lenses to differentiate from what otherwise would have been skin, by contact with the optic cup part of the eye.

3) Induction of differentiation of neural retina by signals from somatic ectoderm
(If an optic cup doesn't touch the skin, only pigmented retina differentiates)

4) Induction of salivary glands, lungs, liver etc. to differentiate from endoderm.

(Experimenters dissected out endodermal epithelium from early embryos, and stimulated it to differentiate into lungs, by mixing it with lung mesenchymal cells; Or stimulated that same endoderm to differentiate into salivary glands, liver, or pancreas, depending on the source of the mesoderm cultured with it.)

5) Induction of kidney differentiation by signals from the kidney ducts.

Many humans are born with only one kidney, with both ureters (kidney ducts) connecting to it.
Dozens of other examples of embryonic induction were discovered.

6) 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)

(Plastic beads soaked in fibroblast growth factor (protein) will also induce extra legs).

7) In tooth embryology, the dentine induces stomodeal epithelium to differentiate into enamel-producing cells ("ameloblasts").

8) Conversely, enamel induces neural crest mesenchyme to differentiate into dentine-producing cells ("odontoblasts").

The researchers Fisher and Koller discovered that chicken mouth epithelia can be induced by mouse mouth mesenchyme to form bird teeth. This means that the evolutionary loss of teeth in birds was because bird odontoblasts no longer induced their inductive signal.

But the bird epithelia still had the receptors and all the genes for making teeth.

Two main experimental criteria for discovering an example of embryonic induction:

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

C. H. Waddington had earned a very good scientific reputation by demonstrating that embryonic induction can be produced chicken and mammal embryos. (In other words, it's not just an amphibian phenomenon; proving the truth of some fact that everyone already assumes will make you famous).

In WWII, Waddington became the chief scientific advisor for British anti-submarine warfare, calculating the best size and distribution pattern for depth charges, and analogous questions. He has often been considered the "father" of Operations Research, and much of his military research remained top secret until the 1980s. He also wrote books about the aesthetics of water-color painting.


* 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.

* 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.

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. Their method was to isolate chemicals from embryos and inject them into blastula-stage embryos.

Unfortunately, only non-specific effects were found.
For example, small pieces of salami can induce neural tube formation.

In more recent years, proteins were discovered that stimulate induction of neural tubes, etc.

RNA was isolated from gastrulating embryos.

Reverse transcriptase was used to make cDNAs from these RNAs.

Proteins were synthesized using RNA transcripts of those cDNAs.
A few of these proteins were very effective inducers of second embryos.

Examples of such inducing proteins are named "noggin", chordin, and chordino, goosecoid, frisbee and "cerberus" (guess reasons for the names!)


Salamanders, especially the sub-group called newts, can regenerate their legs, regenerate the retinas of their eyes, the lenses of their eyes,and several other organs. It has never been discovered why they regenerate so many organs that other vertebrates can't! Much is known; but not the answer!

Most people assume that the key process in regeneration is control of location of cell differentiation, rather than active rearrangement of already-determined skeletal and muscle cells


Some other interesting properties of newts are:

    * They have more than 10 times as much DNA per cell as humans;

    * They have VERY BIG CELLS

Cell size is linearly correlated with DNA amount, even if this is "junk DNA"!!

Tetraploid amphibian cells are (exactly!) twice as big (twice the volume per cell) as diploid cells.

Haploid amphibian cells are (exactly) half the size (volume) as diploid cells.

Guess the volume per cell of triploid and pentaploid amphibians. 150% of diploid; 250% of Diploid.


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, and another here.
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.






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