by Amanda Schwartz

The physiological similarities between humans and animals were noted long ago, and the emergence of what we call “animal research” can be traced to ancient times. The earliest documentation can be found in the third and fourth centuries BC among Greek philosophers and physicians. Aristotle, the "first to have made dissections which revealed internal differences among animals,” is just one notable figure who delved into this discipline (Cohen and Loew 2). The field attained its first prominent advancement, however, in the middle of the nineteenth century, when Louis Pasteur successfully isolated the microorganism responsible for anthrax. Through his research on rabbits and guinea pigs, he was able to develop a vaccine for the disease, and ended the spread of anthrax among livestock and woolworkers.

Animal research bridges the divide between experiments conducted in the test tube and Petri dish and those conducted on humans. Particularly in molecular genetics, animal models are needed to understand disease and test hypotheses. In the years since the dawn of developmental biology, we have increased our understanding greatly using animal models. We now know, if development is normal, all of the cells that make up an animal, large or small, have the same genotype—the inheritable set of instructions or blueprint for building and maintaining a living creature—as the zygote, or fertilized egg, from which the animal develops. The remarkable part is, even though all these cells carry the same genes, they are very different in their appearance and functions. It is as if every cell contains a copy of War and Peace but each cell reads only one chapter of it.

Signal Transduction

What directly affects the chapter that a cell chooses to read are the different RNAs and proteins it contains. These important components are manufactured based on instructions received from its neighboring cells. For example, in order for a zygote to develop properly into a mature adult organism, information from the egg’s DNA must be delivered that details a selective use of gene combinations at very specific times and places. The way in which the zygote andthe increasing number of new cells achieve this transmission of chemical information is called signal transduction. The processes of signal transduction involve ordered sequences of biochemical reactions inside the cell, which are carried out by enzymes¹, activated by second messengers² and which result in a signal transduction pathway (“Signal transduction”).

From recent research built on earlier studies, scientists have been able to determine certain truths about these signals. Embryonic³ cells of arthropods⁴, nematodes⁵ and vertebrates—which include humans—make many of their developmental decisions based on which chemical signals they receive from other cells. “[These] cycles of signaling and responding are repeated over and over as development progresses” (National Research Council and Board on Environmental Studies and Toxicology 111). From this observation, scientists surmised that the path of development a cell takes (including the type of RNA and protein it produces) while responding to the signals it receives, relies on its environment of neighboring cells and the developmental decisions it has made in the past. Each developmental decision limits the number of future paths that are available. It is analogous to a person’s college career – each course decision a person makes determines what degree an individual will receive, and it rules out other possibilities. For example, one would not expect coursework restricted to the humanities to lead to a degree in physics. The resulting differences in RNA and protein cause the cells to express different subsets of genes from their genome. “At least 300 cell types are recognized in humans (e.g., red blood cells, nerve cells, and muscle cells). The number of cell subtypes is much larger, perhaps numbering tens of thousands, when further differences are taken into account related to the cell’s stage of development and location in the body” (National Research Council and Board on Environmental Studies and Toxicology 111).

Genetic Mutations

In addition, mistakes made during this process can result in severe or altogether new genotypes. Gene mutation is the permanent alteration of the DNA sequence that makes up a particular gene, either by environmental factors like harmful sunrays, or by mistakes made during the copying or transcription of DNA. Sometimes a gene will be copied incorrectly, with a different base substituted in its place or a portion completely deleted. Many, if not most, diseases have their roots in our genes. More than 4,000 diseases stem from altered genes inherited from one's mother and/or father. Common disorders such as heart disease and most cancers arise from a complex interplay among multiple genes and between genes and factors in the environment. CCMs are an autosomal dominant genetic disorder.

Autosomal dominant diseases are disorders in which you only need to inherit an abnormal gene from one parent in order have the disease. If either parent has an abnormal gene on one of their 22 non-sex (autosomal) chromosomes it can cause autosomal disorder. The dominant part of “autosomal dominant” means that even if the matching gene from the other parent is normal, the abnormal gene will dominate the pair of genes. In the case where only one parent has a dominant-gene defect, their children have a 50 percent chance of inheriting the autosomal dominant disorder.

Conservation from Species to Species

So how can animals help us with finding solutions or treatments for genetic disorders? The answer according to the National Research Council and Board on Environmental Studies and Toxicology is in the similarities they have to us.

“Signaling pathways are highly conserved across a wide range of animals from chordates to arthropods to roundworms. Many of the kinds of cell responses to signals also are conserved(e.g., responses of selective gene expression, secretion, cell proliferation, or cell migration). The response of developing cells to signals involves activation or repression of the expression of specific genes by transcription factors contained within genetic regulatory circuits. Signaling pathways frequently affect the activity of those factors… Not only individual genes and proteins but also entire pathways of signaling and response and their functions in developing embryos appear highly conserved…This means that, although the embryology of simpler animals might appear superficially very different from that of humans, knowledge gained from those models can often be applied directly to understanding human developmental mechanisms” (112).

The fact that we share the same signaling pathways and regulatory circuits—albeit in different combinations, times and places—with less complex organisms assures us that animal research is applicable to the discovery of genetic solutions for human disease.

Spurred by the discovery of the conservation of signaling pathways between organisms, scientists further discovered other similarities in development between species. Homologous genes, or genes in two different organisms that are believed to share common function or protein codes, have been found to exist between very simple model animals and humans. With this knowledge, experimentation can be done at the simple organism level to find solutions for gene dysfunction at the vertebrate or human level.

The Model Animals for Genetic Research

Now that the need for animals in human development research has been determined, the correct animals for research must be found. Certain factors should be considered when deciding which animals are most suitable for the study of human development. Animals used in research need to be cheap, low maintenance, and easily manipulated. “For study of development, the currently most intensively investigated model animals, in order of increasing complexity, are the free-living soil roundworm (nematode) Caenorhabditis elegans [C. elegans], the fruit fly Drosophila melanogaster, the frog Xenopus laevis, the zebrafish Danio rerio, the chick, and the laboratory mouse…all are relatively small, easy to maintain in large populations in the laboratory, and have short generation times, which allow for rapid analysis of breeding experiments” (National Research Council and Board on Environmental Studies and Toxicology 152-153).

The three model animals currently used for the study of CCMs—chosen primarily based on their convenience for genetic analysis—are C. elegans, zebrafish, and mice.

C. elegans

The nematodes Caenorhabditis elegans have a short life cycle of three days; they are transparent and undeniably simple, having only 959 somatic cells⁶, which make them a great model animal for genetic research. “Because of their genetic tractability, they are a powerful system for understanding gene function and the discovery of new genes that function in distinct biological processes, such as cell death and survival” (Derry).

Much of the research conducted with C. elegans relating to CCMs takes place at the Derry Lab in association with the Program in Developmental and Stem Cell Biology at the Hospital for Sick Children in Toronto.

Part of the research conducted so far has dealt with identifying homologous genes in the C. elegans with those genes in humans that can result in CCMs. So far, scientists have determined that the C. elegans homolog for KRIT1or CCM1 (one of the three genes that can cause CCMs in humans) is kri-1. They have also identified a homolog for PDCD10 or CCM3, which is found to be C14A4.11 in the worms (Ito and Derry).

Through their research, they have determined that kri-1 is required for the programmed cell death (apoptosis⁷) of germ cells⁸ “that have been subjected to various stresses” in C. elegans. They “are actively studying the molecular basis of how this gene controls the core cell death signaling pathway and have identified several novel modifier genes that may be exploited as therapeutic targets for treating this disease clinically. The exact biological function of the C.elegans CCM3 orthologue C14A4.11 in C. elegans is still unknown, but genome-wide studies have shown it has a role in fertility and the survival of germ cells” (Derry).

Zebrafish

Considered an extremely useful model organism, zebrafish have several specific advantages to CCM research. Like C. elegans, they are optically clear, allowing for visual screening. They also develop rapidly—thousands of embryos at once—and are relatively cheap. Unlike the nematodes, however, zebrafish are vertebrates like us, an advantage when studying biological and genetic conservation. “Their embryonic development provides advantages over other vertebrate model organisms. Although the overall generation time of zebrafish is comparable to that of mice, zebrafish embryos develop rapidly, progressing from eggs to larvae in [less than] three days. The embryos are large, robust…transparent and develop externally to the mother, characteristics which all facilitate experimental manipulation and observation” (Dahm 447).

Researchers at the Cardiovascular Research Center at the Massachusetts General Hospital and the Department of Medicine, Harvard Medical School have made several discoveries in their research with zebrafish. Through knockdown experiments—experiments where the goal is to inactivate specific genes—by way of Morpholino injections⁹, they have identified the gene santa (san) to be the zebrafish homolog of the human CCM1, and have also found valentine (vtn) to be the homolog of human CCM2. They have observed that in sanand vtnmutant embryos, the myocardial wall—the muscular wall of the heart—does not develop properly. “During embryogenesis ¹⁰, the myocardial layer of the primitive heart tube grows outward from the endocardial-lined lumen¹¹ with new cells added to generate concentric thickness to the wall.” This “is essential for normal cardiac function. Zebrafish embryos with the recessive lethal mutations santa (san) and valentine (vtn) do not thicken, but do add the proper number of cells to the myocardium. Consequently, the heart chambers are huge” and instead of having multiple layers of myocardium, they have a single layer lined with endocardium ¹² (Mably, et al.).

Michael Malone, Ph.D. and his team of researchers at the Johnson Laboratory and The Department of Pharmacology at the University of North Carolina, Chapel Hill have also conducted research with zebrafish in the hopes of finding answers to CCMs. In their studies, they have concluded that CCM2 morphant zebrafish embryos have decreased cardiovascular circulation, decreased cardiac output and weak ventricular contractions, consistent with a thin myocardium. They have also identified the CCM2 gene product as significant in aortic arch development.

Mice

Perhaps the most relevant model organism being used for CCM research is the laboratory mouse. “Scientists from a wide range of biomedical fields have gravitated to the mouse because of its close genetic and physiological similarities to humans, as well as the ease with which its genome can be manipulated and analyzed” (Spencer). While C. elegansand zebrafish allow the study of CCM genes in simpler organisms and they multiply quickly, they do not form “lesions” like mammals (Awad). Dr. Douglas Marchuk, Ph.D. asserts, while there are thousands of species of mammals on the planet to choose from,

“the mouse is relatively small, and thus costs for cages and food, et cetera, are not as great as the larger mammals. More importantly, the mouse is the only mammal for which the technology exists to specifically mutate a gene at will. Thus when [Marchuk and his team of researchers at Duke University in Durham, NC] discovered the genes causing CCM, [they] immediately attempted to make a mutant mouse for each of the first two genes (CCM1 and CCM2). Once [they] created lines with mutation in the mouse versions of the CCM1 and CCM2 genes, [they] could breed these animals and create a renewable supply of animals with the appropriate genes mutated. No other mammal has this capability.”

Scientists at the Department of Molecular Genetics and Microbiology, Duke University Medical Center, are currently trying to understand what actually causes a CCM lesion to form once a person (or mouse) inherits a mutant copy of the gene. “Clearly, some initiating event causes the CCM lesion to form in some blood vessels in the brain, whereas most vessels are spared” (Marchuk). According to Marchuk, they believe that the initiating event causing the formation of a lesion is a secondary mutation in the patient’s remaining normal copy of the CCM gene. This called the “two hit” theory.

“The two hit theory begins with the fact that we each have two copies of any gene. When one copy mutates, the other is a backup that will perform the same function” (Lee and Awad 3-4). “Since everyone has two copies of each gene (one from mom and one from dad), and CCM shows autosomal dominant inheritance (only one mutant copy inherited from one parent), then all the cells in the patient’s body have one normal and one mutated copy” (Marchuk). Evidence from Judy Gault and Issam Awad first suggested that the two-hit model might be operating in CCMs. The Duke team’s research using their mouse models, along with the study of human patient CCM tissue, suggests that the initiating event causing the lesion to form is a mutation in the normal copy in a cell, which seeds the formation of a CCM lesion. They continue to test this hypothesis using their mice.

The other important quest they have begun thus far is to try to determine which cell type is the key to CCM lesion formation. Dr. Marchuk has narrowed it down to three possibilities: “the endothelial cell (cells lining the blood vessel), the neuron (the nerve cell) or the astrocyte (supporting cell in the brain vasculature). [Their] data with the mice is currently suggesting that the defect is in the endothelial cell”(Marchuk).

Summary

For ages, animals and humans have shared their hearts and homes with one another. We relish in the beauty of the great beasts and the fascinating myriad of diversities that is the Animal Kingdom. And it is in the humblest of these creature’s contributions to medical research that we find hope to cure those afflicted with Cerebral Cavernous Malformations. Through the extensive research of scientists across the globe, clues found from the C. elegans, zebrafish, and mouse have already opened several doors toward understanding this disease. It is only a matter of time before the crucial discovery is made in their investigative journey for a cure.

References

Cohen, Bennett J. and Franklin M. Loew. Laboratory Animal Medicine: Historical Perspectives in Laboratory Animal Medicine. 2nd ed. Eds. J.G. Fox, et al. San Diego: Academic Press, 2002.

Biology-Online.org. Dictionary. 24 Dec 2007. Biology-Online.org. 16 Jun. 2008 <http://www.biology-online.org/dictionary>.

Monod, Jacques and François Jacob. “General conclusions: Telonomic mechanisms in cellular metabolism, growth, and differentiation.” Cold Spring Harbor Symposia on Quantitative Biology 26 (1961) : 389-401.

National Research Council and Board on Environmental Studies and Toxicology. Scientific Frontiers in Developmental Toxicology and Risk Assessment Washington, DC: National Academy Press, 2000.

National Research Council. Science, Medicine, and Animals. Washington, DC : National Academy Press, 2004.

"Signal transduction." Wikipedia, The Free Encyclopedia. 15 Jun. 2008, 01:47 UTC. Wikimedia Foundation, Inc. 16 Jun. 2008 <http://en.wikipedia.org/w/index.php?title=Signal_transduction&oldid=219403502>.

Derry, W. Brent. “RE: Angioma Alliance Animal Research. ”E-mail to the author. 7 Jul. 2008.

Ito, Shu and Derry, W. Brent. “Worming our way out of CCM: the role of kri-1 in apoptosis.” PowerPoint Presentation. Doubletree Hotel, Washington, DC. 17 Nov. 2006.

Dahm, Ralf. “The Zebrafish Exposed.” American Scientist Sept.-Oct. 2006: 446-453.

Mably, John D., et al. “santa and valentine pattern concentric growth of cardiac myocardium in the zebrafish.” Development 133 (27 Jul. 2006) : 3139-3146. 6 Jul. 2008 <http://dev.biologists.org/cgi/content/full/133/16/3139>.

Spencer, Geoff. “Background on Mouse as a Model Organism.” 17 Sept 2007. National Human Genome Research Institute. 21 Jul. 2008, 17:59 EST. <http://www.genome.gov/10005834>.

Awad, Issam. “RE: Angioma Alliance Animal Research.” E-mail to the author. 20 Jun. 2008.

Marchuk, Douglas A. “RE: Angioma Alliance Animal Research.” E-mail to the author. 19 Jun. 2008.

Lee, Connie and Issam Awad. “Latest Research.”Angioma Alliance Newsletter. Mar. 2004: 3-5. <http://www.angiomaalliance.org/docs/March_2004_newsletter.pdf>.

All definitions adapted from Biology-Online.org

1 A catalyst or a chemical produced by cells to speed up specific chemical reaction. An enzyme is usually a protein molecule with unique properties.

2Second messengers are molecules that relay signals received at receptors on the cell surface — such as the arrival of protein hormones, growth factors, etc.—to target molecules in the cytosol and/or nucleus. In addition to their job as relay molecules, second messengers serve to greatly amplify the strength of the signal. Binding of a ligand ( a substance that is able to bind to and form a complex with a biomolecule to serve a biological purpose to a single receptor at the cell surface may end up causing massive changes in the biochemical activities within the cell.

3 Related to the embryo—the underdeveloped fertilized egg that eventually becomes offspring.

4The largest phylum of the animal Kingdom that contains insects, crustaceans and arachnids among others.

5 Of the phylum Nemata. Nematodes are structurally simple organisms, more commonly referred to as round worms.

6All body cells of an organism—apart from the sperm and egg cells, or germline cells.

7Programmed cell death as signaled by the nuclei in normally functioning human and animal cells when age or state of cell health and condition dictates. Cancerous cells, however, are unable to experience the normal cell transduction or apoptosis-driven natural cell death process.

8 Reproductive cells in multi-cellular organisms.

9Injections of Morpholinos (Morpholino oligonucleotides—oligos) into a cell, block the base pairing of RNA, resulting in the knock down of gene function. Often used to study the specific functions of a certain protein.

10The process leading to the development of an embryo from egg to completion of the embryonic stage.

11 The cavity or channel within a tube or tubular organ.

12The innermost lining of the heart cavities. Similar to the endothelial cells that line blood vessels.