HybridMiX started with a simple question. "What if we can cross mice in a petri dish?"
This simple question started an exploration into a world of stem cells and biotechnology. It turns out, if we can breed mice in a dish, we can ask all kinds of exciting questions with medical and evolutionary significance.
In HybridMiX, we decide to push the boundary of what is possible with mouse genetics, by focusing on how species diverge.
Hybrids between animals are usually evolutionary dead-ends. Nonetheless, hybrids are fascinating, not least because they often show surprising features from both parents. Aristotle discussed mules extensively in his treatises. Darwin devoted whole chapters on hybrids. JBS Haldane formulated his famous rule in describing patterns of hybrid sterility. Our interest in such phenomenon has not abated since. This question, fundamental to evolutionary biology, also has great implications in fertility and health.
Fascinating biology aside, hybrids are usually limited, especially in mammals. This is because they tend to be sterile, if not inviable. For geneticists, this is a missed opportunity, because without breeding over many generations and the reshuffling of genomes that come with it, no genetics can be done.
In our ERC-funded projected, we try to break this question wide-open. Rather than tackling hybrid sterility head-on, we will rely on stem cell, transgenic and tissue engineering techniques to generate mice tissues in a petri dish, as if they have been generated through years and generations of laborious crosses. This will allow us to ask (and answer!) some of the most fundamental question in evolutionary biology:
The key to the technique we have developed, called in vitro recombination (IVR), works by molecular sabotage of a key helicase gene called Bloom Syndrome, or Blm. This works because the loss of Blm helicase function leads to highly elevated rates of DNA exchange during cell division, or mitosis. These exchanges occur between newly synthesized chromosome arms (“sister chromatid exchange”), or even between the maternally-inherited and the paternal counterpart (“homologus” or “mitotic recombination”). Human patients with two copies of defective BLM show highly unstable chromosomes, and often develop cancers as early as their 20’s and a host of other defects in regenerated tissues like skin, cornea or gut linings).
Under IVR, we re-direct the otherwise devastating condition towards shuffling of genetic material in the “dead-end” F1 hybrid embryonic stem cells.
In our first HybridMiX paper, Lazzarano et al., PNAS 2018, we demonstrated that we can induce IVR using a small chemical inhibitor against Blm called ML216. In a series of experiments, we showed that we can force genome reshuffling via mitotic recombination. The variable fluorescence seen in the ES cell colony indicates that we can release the latent diversity in the cell through recombination. We also showed that IVR works in multiple strains, including two different F1 hybrid cell lines between the laboratory mouse and their close relatives Mus castaneus and Mus spretus. We also performed cell sorting experiments and showed that we can use the IVR system to map the genetic basis of drug resistance in as few as three weeks!
Most strikingly, we took some of these mosaic recombinant ES cell lines and re-derived whole mouse embryos from them. This was the first time we were able to directly ask if the mixing up of the genome creates novel developmental outcomes. One (un)fortunate outcome is that a high number of the embryos showed developmental defects, including problems in spinal fusion, or even the complete loss of head structures. Even such grotesque outcomes can tell us a lot about the hidden “wiring” in the genomes of these species.
Now that we can create mosaic recombinant genomes, what’s next?
Tissues and organs, of course! We are currently using tissue engineering techniques to assess more complex phenotypes from these "impossible hybrid" material.
This work is generously supported by the Europea Research Council Project HybridMiX and the Max Planck Society