Monday, 21 December 2015

9th genome of Christmas: Medaka and friends

My ninth genome of Christmas is a bit of an indulgence: the gentlemanly, diminutive Medaka fish, or Japanese rice paddy fish.

When Mendel’s laws were rediscovered in the 1900s, many scientists turned to local species they could keep easily to explore this brave, new world of genetics. In America, Thomas Hunt chose the fruit fly. Scientists in Germany explored the guppy and Ginuea pigs. In England, crop plants were the focus of early genetics. In Japan, researchers turned to the tiny Medaka fish, a common addition to many of the ornamental ponds maintained in Japanese gardens. 

Medaka fish are regular tenants of rice paddies and streams all through east Asia, from Shanghai through the Korean peninsula and the islands of Japan, with the exception of the very northern set of islands in Japanese archipelago. (Naturally, every country has a different name for this fish, but it is most widely used for study in Japan so I am using the Japanese terms.) Fishing for Medaka is as common for Japanese children as fishing for guppies or fry is for European children, and is widely depicted in 19th century Japanese wood blocks.

Medaka also has the honour of being the first organism to show us that cross-over on the sex chromosomes does occur. We now know this to be commonplace, but at the time of its discovery this was a novel observation.

As genetics developed, Japanese researchers continued to inbreed Medaka fish, creating one of the most diverse set of inbred individual invertebrates from a single species in the world. Being fish, they have all the cell types and nearly all the organs that a mammal has: tiny, two-chambered hearts, livers, kidneys, muscles, brains, bones and eyes. Conveniently, one can keep lots and lots of them, far more cheaply than mice, and they reproduce regularly, with a generation time of around three months.

But then a different fish rose to prominence in molecular biology in the 1980s. Zebrafish, native of the Ganges, was chosen by the influential Christiane Nusslein-Volhard as the basis for redoing her Nobel-Prize-winning forward genetic screens in Drosophila, this time in a vertebrate. 

I’ve not yet asked Christiane whether she ever thought about using Medaka rather than Zebrafish, but I am sure that a couple of details to husbandry made Zebrafish very attractive: it lays 1000 eggs at a time, providing for excellent single-female progeny, and is transparent during its embryonic stage, allowing for easy light microscopy of the developing fish. In contrast, Medaka lay only around 30 eggs, and they stick to the female rather than being spurted out, so harvesting them is somewhat complex. Plus, the eggs have an opaque glycoprotein layer, which skilled scientists can remove but again makes it harder to study the embryo

So why am I so interested in Medaka? Well, I was having a beer with my colleague Jochen Wittbrodt, who is one of the rare Medaka specialists outside of Japan, and we were discussing the next stage of experiments. Medaka fish has a neat trick by which one can introduce foreign DNA (e.g. human) coupled to a reporter (green fluorescent protein from jellyfish is a favourite - easy to pick up using a microscope). Even on the first injection, the foreign DNA will often go into every cell. For most other species, you have to get lucky for the foreign DNA to go the germline, and then hope it will breed true. Jochen had done a number of successful reporter experiments based on designs from my group, and we were discussing whether we could draw on the long history of Medaka research with its rich tapestry of inbred lines to explore the impact of natural variation on these reporter experiments. So, I asked him how many inbred Medaka lines there were, and Jochen nonchalantly replied that he had no idea - after all, his colleague, Kiyoshi Naruse, made one or two new lines from the wild every year or so.

My jaw hit the floor. From the wild? I checked. Jochen confirmed. And then I explored some more, and discovered that there was a whole protocol for creating inbred individual Medaka from the wild.

This might sound trivial, but it is not. Keeping vertebrates in a laboratory is hard. Keeping them in a laboratory when they are inbred, such that their diploid genome is identical everywhere, is extremely difficult. Doing this routinely from the wild is basically unheard of (although this 
“self’ing” happens all the time in plant genetics). 

Standard theory holds that every individual, whatever the species, has a number of recessive lethal alleles, which will kill the animal if you make them the same. The trick to making an inbred line that is truly the same everywhere (i.e. homozygous) is regular brother-sister mating and an awful lot of patience, as at some point you have to find the combination of alleles in an individual that does not have a lethal effect. Normal animal husbandry lore would have it that this was such hard work, particular with wild individuals, that it would be best to just continue propagating the hard work carried out by the original founders of whichever organism you are using.

Now, this theory does not hold true for plants, and plant geneticists have enjoyed making inbred lines from the earliest days. And Trudy Mackay, looking at the tricks you can play, created a set of inbreds from wild Drosophila lines. One can study developmental changes by looking at different individuals from the same genetic line, but it has to be at different times. One can study the interaction of genes and environment by raising genetically identical individuals in different environments, but it must be done across a panel of strains that represent a wild population. The model plant Arabidopsis has been used by geneticists to do this for decades; fly geneticists are just starting to. 

This kind of work would have been considered madness in vertebrates. You can’t even keep one or two laboratory zebrafish lines fully inbred - you often need to add back a bit of diversity. There are established inbred laboratory mice, but from a weird multi-species hybrid. Single, wild-derived mice strains have been established, but not at scale - not least because of the complications inherent to keeping mouse facilities pathogen-free, which makes everyone a bit paranoid about wild mice in a laboratory setting. 

But in Medaka, it could be doable. Impressive.

Jochen introduced me to Felix Loosli, the best Medaka breeder outside of Japan, and Kiyoshi Naruse, one of the leading breeders in Japan. The four of us have undertaken to generate and characterise a Medaka inbred panel from a single wild population (unsurprisingly, very close to Kiyoshi’s lab, in Nagoya). 

The Medaka genome has of course been sequenced, in a relatively standard, somewhat quirky way by a Japanese group. This genome is a pretty standard fish genome, around the a third the size of human. Medaka are close to some other evolutionarily interesting fish: the stickleback, beloved of ecologists thanks to the numerous species that form in different river and lake systems; cichlids, with a similarly diverse set of species living around the African lakes and Fugu (and loved by sushi gourmands because of the powerful neurotoxin which, so long as it is only in trace amounts, produces an intriguing taste), and loved by genomicists as the vertebrate with the smallest genome. 

Together, these four funky fish will, I hope, push forward research into vertebrate genetics with evolution, ecology, and environment. Our own contribution is in creating the first ever inbred-from-the-wild panel in vertebrates.

Watch this space.