The road to discovery for foundational research takes time!

Renowned for their incredible regenerative capacity, planarians exist as both sexual and asexual strains and are an excellent research organism for studies on regeneration. On August 31, 1999, Howard Hughes Medical Institute Investigator and Stowers Institute Executive Director and Chief Scientific Officer Alejandro Sánchez Alvarado, PhD, then a staff associate at the Carnegie Institution Department of Embryology, received a much-anticipated package from Sardinia, Italy. A semi-frozen canteen arrived with around 50 hermaphroditic flatworms, a biotype Sánchez Alvarado was hoping to establish as a new research organism to study reproduction and regeneration.

Perhaps these worms were still shivering, or maybe just not in “the mood,” as they struggled to reproduce in the lab. However, a couple years later, when Sánchez Alvarado relocated his lab to the University of Utah School of Medicine, the move to Salt Lake City, Utah, seemed to have done the trick. When Longhua Guo, PhD, arrived as a graduate student in 2009, he began a robust research regimen to generate an extended inbred line of worms, and when Sánchez Alvarado again relocated to the Stowers Institute for Medical Research in 2011, Guo and his research followed.

Now situated at the Stowers Institute in Kansas City, Mo., Guo continued studying the sexual strain of the planarian, S. mediterranea, the fresh-water flatworm indigenous to Tunisia and the Mediterranean islands of Sardinia, Corsica, and Sicily. Greatly aided by the generous resources Stowers provided to establish the Planarian Core Facility, the lab was able to generate, maintain, and perform transcriptomic and genomic sequencing on the 10-generation inbred line of these hermaphrodites.

In a study published in Nature Ecology and Evolution in 2016, Guo and his coauthors found that planarian DNA maintains heterozygosity—having two variations or alleles of a particular gene in a pair of chromosomes that are dissimilar, as opposed to homozygosity, when the alleles are identical. The team was surprised to find that large portions of the DNA retained these heterozygous alleles even after 10 generations of inbreeding.

Just one year prior to the publication, to rule out the possibility that this heterozygosity maintenance may have been an artifact introduced by the artificial inbreeding carried out in a laboratory setting, the team took a trip to Sardinia to collect additional wild worms. And indeed, the same phenomenon they observed in the lab was also occurring in nature!

“We showed that all of the heterozygosity we saw in the lab was actually recapitulated in the wild,” said Sánchez Alvarado.

Prolonged inbreeding typically results in the loss of heterozygosity, or an increase in gene homozygosity, as inheritance in organisms that reproduce sexually generally follows specific rules. Over one third of the island-specific planarian genome violates these “laws” of inheritance. Two groups of the heterozygous DNA could be assembled as haplotypes, the physical clustering of variations in a particular genetic sequence that tend to be inherited together, and may have potentially segregated from a pair of homologous chromosomes, or chromosomes from each parent with similar size, structure, and gene location. In honor of the co-founders of the Stowers Institute, Jim and Virginia Stowers, the team named the male and female haplotypes J and V, respectively.

The culmination of a 23-year journey

Now, new research from the University of California, Los Angeles (UCLA), in collaboration with the Stowers Institute has uncovered compelling evidence to support the theory that sex chromosomes evolved from autosomes—any chromosome that is not a sex chromosome. This autosome not only acquired genes that determine sex but also lost its ability to recombine over time, an important mechanism that maintains sex-determining differentiation. The study, led by Guo in the lab of Howard Hughes Medical Institute Investigator and UCLA Distinguished Professor Leonid Kruglyak, PhD, constructed a genomic map of the four chromosomes for the sexual strain of S. mediterranea. The findings, published online and in print in Nature on June 1 and June 9, 2022, respectively, expound on the previous work from the Stowers Institute and demonstrate that these regions of genomic variants, J and V, are located on a single chromosome, Chromosome 1, that is a prime candidate for evolution to a sex chromosome.

“To Leonid’s credit, he was so mesmerized by the findings that he really wanted to continue this work and see it come to fruition,” said Sánchez Alvarado. “Long and Leonid provided us with a fantastic solution to the problem of why heterozygosity was so stubbornly maintained in these animals. Moreover, their solution also produced linkage maps of all the genes in the planarian genome.”

Why doesn’t genetic recombination occur on Chromosome 1? During a specific type of cell division called meiosis, as the cell prepares to divide following DNA replication and chromosome duplication, pairs of chromosomes align side by side. This sidewise coupling enables portions of DNA to “cross over,” leading to a recombination of genetic information between pairs. Instead, Guo found that pairs of Chromosome 1 form a ring-like conformation during a specific phase of meiosis, suppressing the possibility of recombination. In addition, the researchers discovered that nearly all known regulatory genes involved in reproductive organ development and sexual reproduction maintenance were located on this same chromosome.

Sex determination, or what distinguishes male and female, has been feverishly studied for hundreds of years, and while much of the genetic evolution of sex chromosomes remains unknown, new studies are shedding light on potential mechanisms. The observations in this study serve as novel evidence that the sexual planarians that populate Corsica and Sardinia likely possess an autosome ready to evolve into a sex chromosome.

“This amazingly enjoyable and fruitful collaboration not only provides new insights into potential routes toward sex chromosome evolution, but also establishes planarians as a new genetic system in which to study natural variation in important phenotypes such as regeneration and aging,” said Kruglyak.

And the road continues

Longhua Guo, PhD, will now continue his research as a newly appointed faculty member at the University of Michigan in the department of Molecular and Integrative Physiology. A long road, novel discoveries, and new possibilities for future insights into aging, regeneration, and evolution.

This work was supported by the Howard Hughes Medical Institute and the Helen Hay Whitney Foundation.

Additional authors include Joshua S. Bloom, PhD, Daniel Dols-Serrate, PhD, James Boocock, PhD, Eyal Ben-David, PhD, Olga T. Shubert, PhD, Kaiya Kozuma, Katarina Ho, Emily Warda, Clarice Chui, Yubao Wei, PhD, Daniel Leighton, PhD, Tzitziki Lemus Vergara, PhD, and Marta Riutort, PhD.