1900, Massachusetts, U.S., the tale of mouse
In the last year of the 19th century, Abbie Lathrop moved to Granby, Massachusetts. She did not mode the decision of relocation voluntarily. After teaching an elementary school in Illinois for several years, her illness of pernicious anemia forced her to retire at the age of 32, and she needed to find somewhere better for her health. Abbie had the spirit of entrepreneurship and wanted to run a farm that could profit while benefit her health. Initially, she built a poultry farm, but the it did not work out unfortunately. Abbie noticed that “fancy mice”- domesticated mice for exhibition, often exhibiting distinct coat color and pattern- were quite popular as pets among a group of people, who were often called mouse/rat fanciers.
The business of fancy mice went very successful, and Abbie became more ambitious. She built the second farm to breed other fancy and exotic animals, including rodents, guinea pigs, rabbits, and ferrets. The business of both farm was booming, so she recruited two of her close friends to help manage the farm. They employed many neighborhood children to clean cages and feed the animals. At one point, Abbie’s farm housed more than 11,000 mice. Her customers even included the U.S. government: it purchased some of her guinea pigs and sent them to the trenches in battlefields of Europe to detect chemical weapons during World War I.
Abbie’s success was partially attributed to her superb skill in animal breeding. She was able to select traits to breed creamy buffs, white English sables, and other desirable coat variations for mouse fanciers, her major customers. She observed the coat pattern of mice carefully, and kept the record of breeding arrangement and results persistently. Her fancy mice prized for their unusual coat colors: creamy buffs, white English sables, red creams, and ruby-eyed yellows. Like Mendel, these professional qualities made her a natural geneticist. Not too long after her name became well-known among mouse fanciers, she received a big order from a new client who she would never expect: a professor of Harvard University, William E. Castle, who thought to study mammalian genetics by following Mendel’s experiments. The short lifespans and small size and lower maintenance cost made mice the ideal specimen for research. In particular, fancy mice have been inbred for generations to get distinct phenotypes, especially coat color and pattern, and it was relatively easy to control and observe the genetic variables. Abbie had built her reputation in the fancy mouse business; soon she found herself fulfilling orders for mice by the pound for laboratories.
One day Abbie noticed skin lesions on some of her mice, so contacted her customers in the universities to ask if theirs had also developed lesions. Leo Loeb, well-known pathologist in the University of Pennsylvania, did reply, saying that he had found similar lesions in his mice, and they were cancerous. Instinctively, Abbie realized that the cancer in these mice should be associated with their strains, which were “pure” from inbreeding in her farm (though she may not have the idea of genetically homogeneous).
Abbie started to collaborate with Loeb to test how the strain influenced the cancer incidence. When strains of high cancer incidence were bred with strains of low cancer incidence, the offspring would have high cancer incidence. For the first time in the medical history, they proved that the incidence of tumors were associated with genetic background of mice. When strains of high cancer incidence were bred with strains of low cancer incidence, the offspring would have high cancer incidence. In 1915, the duo showed a connection between hormones and breast cancer: mammary tumors decreased in female mice with ovariectomies while the tumors increased in pregnant mice. This led to the development of estrogen blockade for breast cancer treatment 60 years later.
Between 1913 and 1919, Lathrop and Loeb co-authored 10 scientific papers published in prestigious journals, including the Journal of Experimental Medicine and the Journal of Cancer Research (later became AACR’s Cancer Research). Thanks to the digital era; today you can find their papers in the website of Journal of Experimental Medicine (e.g. https://rupress.org/jem/article/28/4/475/8924/FURTHER-INVESTIGATIONS-ON-THE-ORIGIN-OF-TUMORS-IN). At that time, it was highly unusual for a woman to receive full co-authorship. Little only acknowledged Abbie as “a mouse fancier of more than ordinary care and scientific interest.” in one of his papers.
Meanwhile, Castle began to use the mice purchased from Abbie for genetic studies. He became the Director of the Bussey Institute for Biological Research at Harvard University. In the following 30 more years, he trained most of the leaders in the mammalian genetics field, including Nobel Laureate George D. Snell and Clarence Cook (“C.C.”) Little (1888-1971). Little, an undergraduate student in Castle's laboratory, was given the job to take care of mouse colonies. He initiated the mouse breeding program to build the inbred, “pure”, strains, and applied these mice in genetic research. It would be hard to believe that he was never aware of Abbie and Loeb’s experiments, whose results proved the value of inbred mice in research of cancer genetics. However, it is Little who is credited with developing inbred mice and using them in the studies of genetics and cancer. In 1929, Little founded Jackson Laboratory (JAX), the world’s leading supplier for laboratory mice. The all-time most frequently used laboratory mouse strain, C57BL/6J (“Black 6”), is derived from one of Lathrop's mice, No. 57, bred by Little. The truth was that, together and sequentially, they created the inbred strains of laboratory mice and founded the modern mouse genetics.
The fancy mice were pets with interesting outlooks in the eyes of Abbie's customers. What they did not know, or care, was that the coat color and the pattern was the resulted of disrupted melanocyte development. Long before researchers began to investigate where melanoma was from, the tools were already made available. These inbred mouse strains are Abbie's gift for melanoma research.
1942, Berlin, Germany. Eyes to watch.
Although Abbie was famous of skills for breeding special coat color and patterns in mice, she had been fully focusing on cancer and never considered to study the genetics of pigmentation. The first use of mice to investigate pigment cell development took place on the other side of the Atlantic. To explain how this happened, we have to started from a seemingly irrelevant organism: fruit fly.
The modern genetics was born in Thomas Hunt Morgan's legendary "fly room" at Columbia University, where the fruit flies (Drosophila melanogaster) were bred for different phenotypes. Morgan's group had spent two decades mapping genes to chromosomes, proving that hereditary traits were carried by physical structures that could be followed through generations of mating. Muller, restless and ambitious within that tradition, wanted to know whether mutations — the random changes in genes that gave genetics its raw material — could be artificially induced. In 1926 and 1927, he exposed male fruit flies to x-rays, mated them, and counted the mutations that appeared in their offspring. The answer was unambiguous: x-ray exposure elevated mutation rates by a factor of roughly 150 over the spontaneous baseline. He announced the finding at the Fifth International Congress of Genetics, held in Berlin in 1927, and was met with international sensation. The Nobel Prize would follow in 1946. In the modern biomedical jargon, Muller invented a genetic screening method to identify genetic loci associated with specific phenotypes. This approach not only greatly promoted the efficiency of genetic discoveries, but also a new research field, radiation genetics.
In the 1930s, radiation genetics had been the most exciting field in biology. That must influence Paula Hertwig's choice in career path. She was born in Berlin in 1889, the same year Abbie was teaching elementary school in Illinois. In 1919, she became the first woman to habilitate — the highest academic qualification in the German system — at the Friedrich-Wilhelms-Universität Berlin, in the field of zoology. She was the first biologist at any German university to do so. By the 1930s, she had established herself as one of the founders of radiation genetics research in Germany.
Hertwig had been studying the effects of radiation on animal development for years, worried about what the increasingly widespread medical and industrial use of x-rays was doing to human heredity. Now she had both a method and a motivation. She began irradiating mice. It was in one of these colonies, sometime before 1942, that she noticed a small number of mice were conspicuously different: their coats were white, and their eye balls were abnormally small ("microphthalmia", from the Greek for small eye); so small that they were even buried in the eye sockets. Hertwig traced the trait carefully through subsequent breeding experiments, confirmed it behaved as a recessive Mendelian trait, placed it in a linkage group, and published her findings in 1942. She named the gene Microphthalmus, symbol m, after the eye phenotype that had first caught her attention, even though the white coat was far more visible in most of the affected animals.
(There was a twist in the story that Hertwig could not have known at the time. Later analysis would suggest that the mutation she had found was almost certainly not caused by the radiation at all, but had arisen spontaneously in one of the parents of a later breeding — a naturally occurring event that her irradiation screen happened to catch because it put her on heightened alert for aberrant offspring. )
Subsequent investigators over the following decades discovered that microphthalmia mice were also profoundly deaf, deficient in immune mast cells, and afflicted with osteopetrosis — abnormally dense, brittle bones caused by the failure of the cells responsible for breaking bone down. A single mutation, apparently, was devastating across multiple seemingly unrelated biological systems. Although nobody could explain why, gene m must control the development of pigment cells.
1990, Bethesda, Maryland, the United States. The accidental identity.
Up to the 1980s, the advance in molecular biology made it possible to identify genes associated with mouse phenotypes. The genes involved in the melanin synthesis and its control became obvious targets, since the variations in coat color were easily tracked. The very first target for the mouse gene hunters was a very old one: tyrosinase, the enzyme to control the process of melanin synthesis which had been known since the 1940s.
The classical albino allele carried by BALB/c, FVB, AKR, and many other inbred strains had long been presumed to represent a loss-of-function mutation in tyrosinase, but what kind of loss? The answer came in 1990, when Shibahara and colleagues sequenced the tyrosinase gene from BALB/c melanocytes and found a single G-to-C transversion at nucleotide 387, resulting in the substitution of serine for cysteine at amino acid position 85, within a region critical for the enzyme's copper-binding and folding. The mutation abolished enzymatic activity completely, rendering BALB/c mice fully albino with pink eyes, a total absence of melanin in skin, hair, and retina. Tyrosinase did not work alone; the melanosome contained at least two structurally related enzymes. When the sequences of the brown-locus protein and the slaty-locus protein were determined by Ian Jackson and colleagues in the early 1990s, they turned out to be close structural relatives of tyrosinase: tyrosinase-related protein 1 (TYRP1) and dopachrome tautomerase (TYRP2/DCT), each catalyzing a different step in the pathway that converts tyrosine to the various forms of melanin. Their relation with different types of human albinism have also been clarified. From the late 1980s to early 1990s, many genes involved in the melanosome functions (e.g. Oca2, Slc45a2, Rab38, Pmel17) and the control of melanin synthesis (Mc1r, Asip) have been either identified or explored in different inbred mouse strains. A few growth factors and their receptors discovered in other tissues or cell types (e.g. Kit, Kitl, Pdgfra) have been found controlling survival and migration of melanocytes during their development and maturation in mice. However, the true identify of melanocytes was still a mystery; in other words, how a melanocyte was generated in embryo? The early clue to answer this question, however, was not provided by pigment cell researcher, but by a virologist, in an accident.
In 1970s, a medical student in Switzerland, Heinz Arnheiter, was thinking a serious question. He had learned the myriad possibilities of becoming sick, so why there are still healthy people? He was fascinated by the mouse model of Prof. Jean Lindenmann, co-discoverer of interferon, which exhibited natural resistance to influenza. That would be the key to break the code of health and diseases, he thought. Therefore he joined Lindernmann's laboratory at the University of Zurich to study genetic determinants of the protective effects of interferons against viral infection.
1981, primarily because of the necessity for any basic researcher to be exposed to research in an English-speaking country, Arnheiter decided to do a short-term research in the U.S. He chose the National Institutes of Health (NIH) at Bethesda, Maryland, for two reasons. First, he liked that it is called the Institutes of HEALTH, reminding him that he wanted to study how to keep healthy, not individual diseases. Second, he communicated with Bob Lazzarini, section chief at National Institute of Neurological and Communicative Disorders and Stroke (NINCDS). Lazzarini is not only a virologist, but also a molecular biologist. Arnheiter was intrigued to introduce the burgeoning molecular biology methods into his own research. Finally, in those years, NIH accepted only visiting scientists supported by their own affiliated institutes. It would be much easier to join NIH with his own Swiss stipend. So he got accepted by NINCDS.
Arnheiter kept working on the same studies initiated in Lindernmann's laboratory. He published steadily and was soon promoted to be a faculty (investigator) in NINCDS. If nothing unexpected happened, he would keep working on virological studies until retire. However, an accidental finding totally changed his scientific track.
In the late 1980s, two investigators at NINDS decided to explore in what tissues the genes of arginine vasopressin (Avp) and oxytocin (Oxt) were activated by using transgenic mice. For that purpose, one of their postdoctoral fellows, Yoshinobu Hara, constructed transgene vectors by connecting the regulatory sequences of the studied genes with β-galactosidase, a reporter gene. The regulatory sequences are like a genetic switch of the reporter gene. Through the pronuclear injection into the embryo, the mice born to carry the transgene were the "founder line" of transgenic mice. In theory, the transgene is expected to insert into the genome of every cellthe reporter gene will be turned on in the tissues where Avp or Oxt are activated; therefore the reporter signal can be located. The researchers could also use such transgenic mice to study what environmental or diet factors can turn on the genes.
In 1988, Arnheiter performed pronuclear injection of the into embryo of the mice with dark coat color. Unfortunately, none of the transgenic offspring showed the reporter signal, because the its sequence contained a mutation that was not recognized before the mice were made. Therefore, he and Yoshinobu tried to label the RNA generated by the transgene in the mouse tissues (so-called in situ hybridization). However, none of the offspring expressed transgenic RNA in brain nuclei where Avp and Oxt were known to be expressed. At that moment, each offspring mouse carried only one copy of the transgene ("heterozygous"). Yoshinobu tried to get each mouse two copies of transgenes ("homozygous") by intercrossing the offspring of each mouse line, thinking that increasing the gene amount may allow detection of its RNA product. Still, the transgene RNA was still not detected in any offspring from the breeding.
Before giving up, Yoshinobu decided to do more intercrossing within each mouse line; maybe one of the offspring could have higher expression of the transgene if lucky enough. One day, to his surprise, Yoshinobu found white mice with small red eyes among the offspring of the one founder line. He soon realized that these mice looked like the microphthalmia mice derived from Hertwig's discovery, and called them vga-9 (the ninth mouse inspected from the first injection of the vasopressin/β-galactosidase/human AVP transgene). These mice attracted attentions from experts in different fields. Heinz and his collaborators found that Vga-9 mice had defect mast cells and no melanocytes, exactly like Hertwig's mice. All these results confirmed that vga-9 was indeed a mutation at the microphthalmia locus.
Arnheiter told Nancy Jenkins and Neal Copland, wife-and-husband investigators at the ABL-Basic Research Program, NCI-Frederick, about these mice. The couple were renown experts in mouse genetics and mouse cancer models; they immediately expressed the interest in collaboration to clone the gene that caused these phenotypes. Colin Hodgkinson, the visiting fellow in Arnheiter's lab, with persistence, skill, and a blessing of luck, cloned the flanking regions of the transgene. In these clones, Jenkins and Copland's group identified the entire open reading frame of a novel member of the E-box binding basic-helix-loop-helix-leucine zipper family of transcription factors. Arnheiter and the couple's groups published it in 1993, termed this factor Microphthalamia-associated Transcription Factor, MITF. Years later, Arnheiter commented on this discovery: "Had the experiment not been such a failure, the idea of systematically generating transgene-homozygotes might never have come up, and given the high costs of keeping laboratory mice, the vga-9 line might have been terminated long before the appearance of any white and small-eyed mouse." Three years later, in 1996, a study showed that forced expression of MITF could convert a fibroblast to a melanocyte-like cells, proving the dominant role of MITF to determine the melanocytic lineage.
The Mitf gene also changed Arnheiter's career. He was fascinated with this new discovery after two decades of research in virology, and thus decided to turn his lab to study the function of Mitf in the sensory organ development, as he tried to fit into the NINDS’s principle focus on neuroscience. Years later, he stated, "Little did I know at the moment that this observation was the beginning of a new field of research and a switch of my career from 20 yr of virology to now nearly 20 yr of developmental biology... It was by no means a totally smooth process as I had to learn a lot of new things and slowly change the character of my lab. But with some excellent collaborators we eventually succeeded." Meanwhile, he also formed extensive collaboration with pigment cell researchers and melanoma researchers. An amazing fact is that intramural investigators in NIH could change their research focus any time they want; they are not bounded by the research proposals written in any document. This is still the case today.
However, the discovery changed not only Arnheiter's research career.
Away from eight-hour driving on I-95, Philip A. Sharp was discussing data with postdoctoral fellows in his laboratory at Massachusetts Institute of Technology (MIT). He was well recognized as the co-discover of RNA splicing in molecular biology field, and now he was focusing on the MYC oncogene and its family member genes, which all are a transcription factor family called bHLHzip (basic-helix/loop/helix-leucineZipper, named for their DNA binding and dimerization domains). In the room next to Sharp's office, a MD/PhD postdoc, David E. Fisher, was leading the project for the analysis of TFEB, a MYC family gene that was a major regulator of lysosome biogenesis and autophagy. He just accepted the offer as an investigator from Dana Farber Cancer Institute. One day Sharp received a phone call from Nancy Jenkins, because they had just cloned the gene of Mi trait, which had not been officially named Mitf yet. She noticed strong homology between the sequence of the gene and TFEB. Sharp told Nancy to call Fisher in the next room, because he was leading the work on this family of proteins. At that time, Fisher was about to start his own lab at Dana Farber Cancer Institute as young PI, so the study of MITF became the major focus in his own research.
Fisher started studying MITF virtually as soon as Jenkins called him. When Jenkin's postdoctoral fellow, Eirikur Steingrimssen, identified the mutations responsible for multiple interesting mouse mutants of MITF, he sent Fisher the DNA vectors of those mutant sequences. In turn, Fisher generated proteins from those mutant sequences and then tested their functions. He would made phone calls to tell the new results to Steingrimssen, who connected the biochemical results with the remarkable genetic behaviors of the mutants he had sequenced. As Fisher recalled, "While this was certainly fascinating, and collaborating with Nancy Jenkins, Neal Copeland, Heinz Arnheiter, and Eirikur Steingrimmsen, was super fun."
In 1993, Arnheiter, Jenkins, and Steingrimssen published their discovery of Mitf gene. Philip Sharp received Nobel Prize for co-discovering RNA splicing. Fisher got the offer from and moved to Dana Farber. It must be the most memorable year from them.
In his own laboratory at Dana Farber Cancer Institute, Fisher became fully committed to study MITF, and continued to collaborate with Arnheiter, Jenkins, Copland, and Steingrimssen. They found that MITF bound and turn on the "switch" (promoter sequences) of many genes involved in melanin production. Fisher also explored environmental signals to induce MITF expression, and the function of MITF in the survival of melanocytes. Later these studies provided foundation to explore how sunlight makes us tanned, and why melanoma becomes tough during progression (These stories will be mentioned in the later chapters).
Following the tradition of academic research, they sent the DNA vectors containing MITF genes to scientists all over the world who requested it after reading their papers. These collective efforts solidified that MITF is the master regulator and melanocyte development. These studies also dramatically impacted melanoma research. It would not be overstated that MITF is the most important gene in pigment cell research.
(Years later, MITF was renamed as "melanocyte inducing transcription factor". Heinz was not happy about it. He told me that the function of MITF is not limited to melanocytes but also many other cell types, such as mast cells as mentioned above. Moreover, traditionally, gene was named after the associated phenotypes or diseases. In that case, "microphthalamia associated" is more appropriate than "melanocyte inducing", as microphthalamia-associated phenotypes include much more than the shape of eyes, but also pigment cell change, neural symptoms, etc. The change of the name symbolized the current trend of disconnection between genetics and physiology.)
Late 1990s, the Cascade of Lineage.
Microphthalmia is not the only trait to impact melanocyte development in mice. Since the Jackson Laboratory began to expand the inbred mouse lines, its researchers kept finding spontaneous mutant mice that give special coat color patterns. For example, the Splotch (Sp) mouse, discovered in 1947, have white patches of fur on their bellies and sometimes their backs. The Dominant megacolon (Dom) mouse, discovered in 1984, features patches of white spotting on their belly and feet, and sometimes lack pigment in their inner ears causing deafness. Moreover, these mice often showed defects in peripheral neural system and pigment cells associated with specific genetic disorders found in human, such as Hirschsprung disease and/or Waardenburg syndrome.
The defects of Dom mice- white-spotted, deaf, dying of intestinal aganglionosis, had been found originated from the failure of neural crest progenitors to differentiate to both melanocytes and enteric ganglia. In the late 1990s, two research groups led by William Pavan at National Human Genome Research Institute, NIH, and Michael Wegner at Center for Molecular Neurobiology at the University of Hamburg, had aimed to clone the gene that gave Dom mice their phenotypes. Pavan and his postdoctoral fellow, Michelle Southard-Smith, used the "carpet bombing" strategy: they bred and genotyped 1,716 mice specifically to catch the rare chromosomal crossovers, detected by genetic markers, allowing them to pinpoint the Dom gene's position with enough precision- we have to remember there was no whole genome sequencing in those old days. Eventually, Southard-Smith and colleagues narrowed down the chromosomal interval, and found it in one of the DNA fragments isolated from the bred mice. Sequencing of the fragment show that it was bearing unmistakable similarity to the HMG box of the Sry-type transcription factors: Sox10. Wegner's group, in contrast, found a surprised short cut. Previously, they had a detailed characterization of Sox10 — its expression pattern, its binding partners, its transcriptional behavior. The protein appeared prominently in the early neural crest, enteric nervous system, and the peripheral and central nervous system glia, exactly the tissues with defect in Dom mice. The Dom gene was very likely to be Sox10. They bred much fewer mice-252- and sequenced 506 DNA fragments isolated from them. In one of the fragment, they found the sequence matched to Sox10, confirming their guess. With the same concept, both groups examine the expression pattern of Sox10 in the embryo of wildtype ("normal") and Dom mice and the structure of mutant proteins. All the evidence pointed Dom gene to Sox10. Their discoveries were published almost simultaneously; January 1998 for Pavan's paper, and April 1998 for Wegner's paper.
The discovery of Sox10 and its function suddenly unlocked the vault of secrets how neural crest differentiates to melanocytes. Working with Arnheiter's group, Pavan established that SOX10 directly activates MITF expression — placing it above MITF in the transcriptional hierarchy that specifies the melanocyte fate from neural crest. PAX3 was also shown to activate MITF, completing a Waardenburg syndrome transcriptional cascade connecting human genetic disease to normal melanocyte development. Separately, Wnt/beta-catenin signaling via LEF1 was shown to induce MITF expression in melanocytes, adding a key extracellular signaling axis to the specification pathway.
At the beginning of the 21st century, how a melanocyte is formed in embryo was pretty well established. The findings in developmental biology prepared the field to ask the next important questions: how does a melanocyte become melanoma cells? Do melanoma cells learn skill from this process?