With any Revive & Restore project, outcomes must include the development of technologies that can be applied to pressing conservation challenges. It is clear that the bottleneck for all avian genetic rescue projects is the lack of advanced reproductive techniques that are compatible with avian reproductive biology. With Heath Hen de-extinction, Revive & Restore is working on foundational science that is essential both for reviving this extinct species and for developing tools that can have a measurable impact on enhancing genetic diversity, helping to build disease resistance, developing synthetic alternatives, and facilitating adaptation to a changing climate for avian species.
2019: In Vitro Research Continues
The key milestone of Phase Two is successfully culturing the primordial germ cells (PGCs) of the greater prairie chicken, the proximate species to the Heath Hen. Initial phase two lab work was paused in early 2017 after our avian genetics partner Crystal Bioscience was acquired and could no longer work on our project. With bench-work paused, Revive & Restore leveraged the initial Heath Hen de-extinction work into an avian genetic rescue program, secured a new academic partner, and raised the necessary funding to proceed with the first key milestone. Meanwhile alternative protocols for germ-line transmission were explored with international partners (see 2018 below).
Revive & Restore’s recently established Catalyst Fund Program awarded a two-year grant to Dr. Rosemary Walzem of Texas A&M University to develop and optimize techniques to enable germline transmission of primordial germ cells for avian genetic rescue applications, using the greater prairie chicken as the model species. This avian genetic rescue program will not only build upon the work of Crystal Bioscience, but also serve as a model for other avian species. The goal is to jump start additional avian genetic projects around the world that will advance biotechnologies for a wide range of endangered and extinct birds.
2018: An Alternative Pathway for Obtaining Primordial Germ Cells
In 2018, in order to capitalize upon the partial success of Crystal Bioscience, Revive & Restore shipped the late-stage germ-cell cultures established in 2016 to Dr. Benjamin Schusser’s lab at the Technische Universität München, in Munich Germany. Schusser’s lab will conduct germ-line transfer experiments in adult male domestic chickens, as opposed to the standard embryonic implantation method using primordial-stage germ cells. Dr. Schusser’s lab is currently running control experiments with domestic chickens, and experiments using greater prairie chicken germ-cells will follow. If successful, Crystal Biosciences’ cell cultures may yet produce the world’s first greater prairie chickens hatched using biotechnology. The ability to salvage late-stage germ cells and use adult chimeric hosts opens up a wider array of opportunities to apply genetic rescue to rare birds.
2016 – 2017: Phase Two Research with Crystal Bioscience
In order for the revival of the Heath Hen to be possible, gene-edited cells in a petri dish must develop into live chicks. This must be accomplished by using a process known as germ-line transmission, where the primordial germ cells (PGCs) of an early embryo are edited, grown in cultures, and then reproductively transmitted through surrogate host parents to generate live, engineered birds. This technology is routinely used in domestic chickens, but has yet to be developed as a viable reproductive technique for wild species. Using eggs produced by a flock of greater prairie chickens housed at Grouse Park, Crystal Bioscience researchers were able to establish five viable cell lines from a series of experimental culture conditions. Early in vitro analyses of gene expression revealed that the cell lines were germ-cells, the target cell type. Later in vivo experiments, however, revealed the cells to be late-stage germ-cells, not primordial, and therefore incompatible with current germ-line transmission techniques.
Pictured Left: Greater Prairie Chicken germ-cells, fluorescing green thanks to the integration of a GFP marker gene. These are the world’s first genetically engineered Greater Prairie Chicken cells.
Results of Heath Hen Genomic Study
The goal of Phase One was to supply the data necessary to understand the relevant taxonomic and genetic differences between the Heath Hen and its living relatives. In 2014, Revive & Restore sequenced the genomes of the Heath Hen and all three living species in its genus, Tympanuchus: the greater prairie chicken (T. cupido pinnatus) and its subspecies the Attwater’s prairie chicken (T. c. attwateri); the lesser prairie chicken (T. pallidicinctus), and two subspecies of Sharp-tailed Grouse, (T. phasianellus kennicoti) and (T. phasianellus jamesi). A total of 19 genomes were sequenced to decipher the taxonomic relationship of the Heath Hen to its living relatives.
Dovetail Genomics created a reference genome using the greater prairie chicken, which was the basis of subsequent genomic analysis. Our high quality reference genome of the greater prairie chicken is now available on the National Center for Biotechnology Information’s public GenBank database.
What did we learn?
Once thought to be a subspecies of greater prairie chicken, the Heath Hen’s genome reveals that it is distinct from its relatives, meaning that gene-editing is the only way to revive its unique traits. This result gave a green light for Phase 2 of Heath Hen de-extinction to begin.
Where are we now?
Dr. Jeff Johnson analyzed the genomes of the entire grouse family tree using whole genome data, but definitive and reproducible results have remained elusive. While the initial finding that the Heath Hen is a unique species remains unchanged, interbreeding between the other living species obscures the genus’ relationships, making the eco-evolutionary insights necessary for de-extinction difficult to discover. More specimens are currently being sequenced to help overcome the mixed genomic signals. In the meantime, Dr. Giri Athrey of Texas A&M University is using this unique data set to study the evolution of gene families among birds, particularly immune genes, which have shown to be key to conservation success for the critically endangered Attwater’s prairie chicken.
The field of genomics is ever improving and elevating standards of genome assembly. The ultimate gold standard is assembling genomes to their original chromosomal configuration. Dr. Erez Aiden and Dr. Olga Dudchenko of the Center for Genome Architecture worked with Revive & Restore to improve the Dovetail Genomics’ reference assembly to a chromosomal level, an assembly currently available at the Aiden lab’s DNA Zoo project. This chromosomal level reference genome will enable a new level of analysis of the association of genes on different chromosomes, recombination landscapes, and the distribution of diversity across the genome. This tertiary information of the genetic code may reveal key insights into how Heath Hens were able to thrive in fragmented populations that a simple reading the DNA sequence could not reveal, and therefore will provide essential information for revival.