The farm of cloned animals (Instead of prologue)

The history of cloning of complex organisms has it all...Innovation, competition, turnovers, deceits, love and hate....

And it is so long that for the first time in the history of this blog, I will dedicate two posts to it.




Page from Nicolaas Hartsoeker's book where

he described the homunculus -little child- that

he observed in the sperm. Taken from

Nicolaas Hartsoeker, Essay de dioptrique

Paris: Jean Anisson, 1694, p. 230.

For over two centuries, from the 17th till the 20th, a very heated argument over how life is created had been taking place. There were two dominating theories; Preformationism and Epigenesis. Preformationism supported that organisms develop from miniature versions of themselves while the followers of Epigenesis believed that "each embryo or organism is gradually produced from an undifferentiated mass by a series of steps and stages during which new parts are added." (Magner 2002, p. 154)

In 1883, August Weismann, professor of zoology and comparative anatomy at the University of Freiberg and one of the most influencial biologists of his era proposed the first testable model of cell specification, the germ plasm theory. Of all the hypothetical theories on morphogenesis of that era, the germ plasm theory can claim to be at the same time the most logical and the most elaborated and it have had the greatest influence on his peers.

In his theory, Weismann supported that the chromosomes carried the inherited potentials, determinants, of a new organism. However, not all the determinants on the chromosomes were thought to enter every cell of the embryo; the chromosomes of a fertilized egg wouldn't be divided equally in the cells of the embryo but different chromosomal determinants enter different cells. Only the nuclei of those cells destined to become gametes (the germ cells) would retain all types of determinants while the nuclei of all other cells would have only a subset of them. Hence, although the fertilized egg would carry the full complement of determinants, certain somatic cells would retain the “blood-forming” determinants while others would retain the “muscle-forming” determinants, etc. 

Illustration showing how according to Weismann the heritable substance combined in the cell’s nucleus during sexual reproduction. His view that this heritable substance was distinct from, and unaffected by, the rest of the organism’s body, would later underlie the genetic theory of inheritance. Taken from August Weismann's The Germ-Plasm: A Theory of Heredity translated by W. N. Parker in 1893.

At the same time his student Wilhelm Roux, who also studied under Ernst Haeckel, proposed a hypothesis that could explain the embryo differentiation by a "mechanistic" process that was similar to Weismann's theory and would later be known as the Roux-Weismann hypothesis of mosaic development.  

In 1888, while working in the Anatomical Institute in Wroclaw -part of Germany at the time-, Roux published a series of experiments that corroborated  this theory and became the starting point of experimental embryology. He took 2- and 4- cell frog embryos  and destroyed half of the cells of each embryo with a hot needle. If the mosaic development theory was correct then only half embryos would be formed because the remaing cells would only contain the determinants for the characteristics of the cells of that type. Indeed the embryos that survived till the stage of gastrulation produced half-embryos which was a victory for the mosaic theory. 

Three years later Hans Dreisch, another of Haeckel's students, started experimenting with sea urchins trying to confirm Roux's findings while working at the Marine Biological Station in Naples. He chose sea urchins as his animal model because they have large embryo cells, and grow independently of their mothers.  Dreich took a 2-cell embryo of a sea urchin and shook it in a beaker full of sea water until the two cells separated. He expected each cell to develop into the corresponding half of the animal to which it has been destined or preprogrammed, but instead found that each developed into a complete sea urchin. This also happened at the 4-cell stage: physiological sea urchins were formed although they were smaller than usual. 

 a₁ and b_1: Normal gastrula and normal pluteus. a₂ and b_2: “Half”—gastrula and “half”—pluteus, that ought to result from one of the two first blastomeres, when isolated, according to the theory of “evolutio”. a₃ and b_3: The small but whole gastrula and pluteus, that actually do result. Driesch's drawing taken from his Gilfford Lecture.

His experiments contradicted the mosaic theory but also resulted in him abandoning science. Driesch renounced the study of developmental physiology and became a philosophy professor few years later, in 1909, proclaiming vitalism (the doctrine that living things cannot be explained by physical forces alone) until his death in 1941.

In 1897, Hans Spemann started experimenting with salamander embryos at the University of Würzburg in Germany. He had been inspired by Weismann's book The Germ Plasm: A Theory of Heredity which he had read one year earlier while guaranteed in the Alpes trying to recover from Tuberculosis. In 1902, trying to give an answer to the Roux-Driesch dispute he used a revolutionary, but yet rather simple method, to divide embryo cells by constructing a nose with his baby daughter's hair. Spemann tied this loop around the middle of the jelly-like membrane that salamander embryos have and squeezed it so that the two divided cells were formed into opposite halves of the egg's membrane. From these two newly formed cells two identical salamander embryos were developed both of them whole and normal. 

 Spemann had documented the first in-vitro animal clone! 

He continued working with salamander embryos and with Hilde Mangold they published a series of ingenuous experiments in 1924. In one of them they attempted the transfer of the nucleus of a sixteen-cell embryo to a single salamander embryo cell with no nucleus. The cell took up the nucleus and developed into a normal salamander. With this process, Spemann and Mangold completed one of the first cloning experiments using the nuclear transfer method.

Original pictures from Spemann's experiments. Top: drawing from the brain of a double headed embryo, 1902. Bottom: Double headed embryos after an incomplete constriction along the first cleavage turrow, 1900.

Unfortunately, Hilde Mangold died at the same year from a gas heater explosion in her house. She didn't live to see her thesis on the salamander embryos' work get published neither the Nobel Prize that was awarded to her mentor Hans Spemann in 1935 for their revolutionary work.

In 1938 Spemann published a book with his results entitled  Embryonic Development and Induction. He wondered whether cells that have already been differentiated would be able to form new organisms and speculated about the possibilty of creating a new organism by transferring the nucleus from a differentiated cell into an enucleated egg.  As he explained it: "Decisive information about this question may perhaps be afforded by an experiment which appears, at first sight, to be somewhat fantastical. This experiment might possibly show that even nuclei of differentiated cells can initiate normal development in the egg protoplasms."

......to be continued....

Further reading..
1. August Weismann: The Germ-Plasm: A Theory of Heredity translated by W. N. Parker in 1893
2.Dreisch's Gillford Lecture 
3. Dr. Ann A. Kiessling and Scott C. Anderson: Human Embryonic Stem Cells: An Introduction to the Science and Therapeutic Potential
4. Oscar Hertwig: The Biological Problem of Today

    

Is there finally going to be a drop OR a very emotional story

In 1927 Professor Thomas Parnell of the University of Queensland in Brisbane, Australia, set up an experiment to demonstrate to students that some substances that appear to be solid are in fact very-high-viscosity fluids.

He used bitumen, also known as asphalt, a sticky, black and highly viscous liquid or semi-solid form of petroleum. It may be found in natural deposits or may be a refined product; it is a substance classed as a pitch.

Parnell poured a heated sample of pitch into a sealed funnel and allowed it to settle for three years. In 1930, the seal at the neck of the funnel was cut, allowing the pitch to start flowing. A glass dome covers the funnel and it is placed on display outside a lecture theater. It was named the pitch drop experiment.

The set up of the pitch drop experiment

 And then the experimenters would wait to watch pitch drops fall....

 and wait....

and wait...

and wait.......

 

Only eight drop have emerged from the funnel in the past 83 years. The last one fell on 28 November 2000, allowing experimenters to calculate that the pitch has a viscosity approximately 230 billion (2.3×10¹¹) times that of water. 

The strange thing is that no one has ever seen a drop fall and excitement is rising as a ninth drop looks set to emerge from the pitch block in the very near future. "No one has actually seen a drop emerge, so it is getting quite nervy round here," said Professor Mainstone, custodian of the experiment since 1960, when interviewed last April. "The other eight drops happened while people had their backs turned. For the last drop, in 2000, we had a webcam trained on the experiment, but it broke down … in 1988, when the previous drop was about to emerge, I popped out for a coffee and missed it. This time we have got several cameras trained on the pitch sample to make sure we get a sight of it dropping. It will take only about a tenth of a second, however. On the other hand, I am 78, and the next drop is unlikely to fall for at least another 10 years, so this might be my last chance to see it happen."

 

Unfortunately Professor John Mainstone will not witness the 9th pitch drop as he died on 23rd of August 2013 but his devotion to his cause and his perseverance is an inspiration for me and I hope for many others as well.

Professor John Mainstone with the pitch drop experiment in 1990

The ninth drop hasn't still emerged..

Very exciting update: The ninth drop emerged on the 24th of April 2014. Faithfull to its previous cinematographic tradition it only fell when the new custodian of the experiment Prof Andrew White tried to change the beaker containing the previous eight drops. Apparently, it has been decided for the experiment to go on for another eighty years, so there will be plenty of time for more drops...or not??