Single-cell RNA sequencing reveals heterogeneity in chicken gonadal germ cells

In a discovery that is already circulating widely among developmental biologists and poultry scientists, a research team at Westlake University in Hangzhou, China, has revealed that chicken primordial germ cells—the embryonic ancestors of sperm and eggs—are far more diverse than scientists had long assumed. Using single-cell RNA sequencing, the researchers showed that the germ cells resident in chicken embryonic gonads split into three transcriptionally distinct subtypes, overturning the textbook view of these cells as a uniform population. The work, published as an open-access article in Cellular and Molecular Life Sciences, also demonstrates a striking practical payoff: cultured germ cells, including gene-edited lines that have been grown in the laboratory for extended periods, can be reintroduced into living birds through a simple blood-vessel injection and successfully integrate into recipient gonads.

Primordial germ cells, or PGCs, occupy a special place in developmental biology. They are the earliest committed precursors of the germline, the cell lineage that ultimately gives rise to sperm and eggs and therefore to every generation that follows. In birds, PGCs have an unusual and biologically convenient journey: they arise in the early embryo, circulate through the embryonic bloodstream, and then home in on the developing gonads, where they settle and mature into functional gametes. This migratory behavior has long made chickens an attractive model for germline manipulation, because researchers can potentially access the germline simply by injecting cells into the bloodstream. PGCs are prized tools for breeding programs, germplasm preservation, and the creation of genetically modified birds, with applications ranging from disease-resistant flocks to bioreactor chickens that produce pharmaceutical proteins in their eggs.

Despite their importance, the internal makeup of gonadal PGCs in chickens has remained poorly resolved. Bulk transcriptomic studies, which average gene expression across thousands of cells, inherently flatten any differences between individual cells, and earlier characterization efforts largely treated the gonadal PGC pool as a single entity. The Westlake team, led by Xiaomin Wang and corresponding authors Chengchen Zhao, Duanqing Pei, and Bo Wang, set out to interrogate that assumption using single-cell RNA sequencing, a technique that captures the full messenger RNA profile of individual cells and thereby exposes the heterogeneity that bulk methods conceal. By profiling PGCs isolated from chicken embryonic gonads, the researchers generated a high-resolution transcriptomic atlas of the germline at a critical stage of development.

The central finding is unambiguous: rather than forming a homogeneous population, gonadal PGCs resolve into three distinct subtypes, each defined by its own constellation of expressed genes and regulatory programs. These transcriptional states likely reflect different phases of maturation or functional specialization within the germline, a possibility consistent with what single-cell studies have revealed in other organisms, where germ cells progress through staged molecular states as they prepare for gamete formation. Identifying these subtypes matters because different states may respond differently to culture conditions, transplantation, and gene-editing procedures. A population that looks uniform under a microscope can, at the level of gene regulation, contain cells at very different points along their developmental trajectory, and knowing which subtypes are present—and which are most amenable to manipulation—gives researchers a rational handle on an previously opaque process.

The technical achievement rests on the power and demands of single-cell transcriptomics. Each cell’s RNA must be captured, barcoded, amplified, and sequenced, and the resulting data must be computationally clustered so that cells with similar expression profiles group together while dissimilar ones separate. Distinguishing genuine PGC subtypes from noise requires careful quality control, marker-gene validation, and cross-referencing against known germline markers. The team’s analysis, supported by the High-Performance Computing Center of Westlake University, succeeded in resolving the three subtypes with sufficient confidence to establish them as robust biological entities rather than computational artifacts. The result provides the research community with a reference map of gonadal PGC diversity in an agriculturally central species, one that can now be used to design better culture media, select cells for editing, and interpret how manipulations shift the balance between subpopulations.

Equally consequential is the study’s second pillar: demonstrating that in vitro–cultured PGCs can colonize recipient gonads after blood-vessel injection. This experiment capitalizes on the natural migratory circuitry of avian germ cells. When cultured PGCs are injected into the vasculature of a recipient embryo, they travel with the circulation and exit into the gonads, where they join the resident germline. If the introduced cells participate in gamete production, the recipient can transmit the donor lineage to offspring, effectively converting an injection into a heritable genetic modification. The Westlake team confirmed that cultured PGCs not only integrate into recipient gonads but do so functionally, validating the long-standing premise that laboratory-expanded germ cells can serve as vehicles for germline transmission in poultry.

The researchers then pushed the system further, addressing a question that has constrained the field for years: do PGCs retain their colonizing ability after prolonged culture? Long-term in vitro expansion is essential for practical genome editing, because generating, editing, screening, and validating modified cell lines takes time, and cells that lose their developmental potential during culture are useless for transmission. Remarkably, the team showed that gene-modified PGCs could be re-established as stable lines after long-term culture and then, following a second round of blood-vessel injection, integrate once again into recipient gonads. This re-establishment and reintegration loop means that edited germline lines can be archived, expanded, edited anew, and redeployed repeatedly—a capability that transforms PGC culture from a one-shot procedure into a renewable platform.

The implications for poultry science are substantial. Genetic modification of chickens has historically been slow and inefficient compared with mammalian systems, in part because of the challenges of accessing the avian germline. The ability to culture, edit, and reinject PGCs through the bloodstream offers a comparatively simple and scalable route to transgenic birds. Applications include introducing disease-resistance traits, for example against avian influenza; producing birds with tailored egg proteins for pharmaceutical manufacturing; preserving rare or heritage breeds by banking their germ cells; and studying fundamental questions of germline biology in a model organism that is both evolutionarily distinct from mammals and economically vital. The demonstration that long-term cultured, edited PGCs remain competent for colonization directly removes one of the biggest bottlenecks, since it decouples the editing timeline from the injection timeline.

Beyond the applied dimension, the study adds an important comparative datapoint to the biology of the germline. In mammals, single-cell analyses have revealed staged heterogeneity within fetal germ cell populations, with cells transitioning through states linked to sex determination, meiotic entry, and epigenetic reprogramming. Finding three transcriptional subtypes within chicken gonadal PGCs suggests that germline heterogeneity is an ancient and perhaps universal feature of sexually reproducing animals, even in a lineage where germ cells migrate through the blood rather than settling directly into the gonad. Each subtype may represent a checkpoint along the maturation path, or alternatively a set of functionally specialized cells with distinct roles in gonad colonization. Distinguishing these possibilities will be a natural next step, and the new transcriptomic map provides the marker genes needed to do so.

There are also implications for how PGC lines are established in the first place. The authors note that the demonstrated diversity could be leveraged to establish and expand PGC lines for long-term in vitro culture from differentiated gonads—cells that have already reached their target organ and were previously considered difficult to de-differentiate into expandable lines. If specific subtypes are more permissive for culture adaptation, researchers could enrich for those states and dramatically improve the efficiency of deriving new lines from valuable genetic backgrounds. That would open the door to germplasm preservation strategies for breeds whose genetics exist only in living flocks, a growing concern as agricultural biodiversity contracts worldwide.

The study, which received ethics approval from the Institutional Animal Care and Use Committee of Westlake University under animal protocol 23–132-PDQ, was supported by the Zhejiang Provincial Natural Science Foundation of China, the Yangtze River Delta Sci-Tech Innovation Community Joint Research Project, and the Pioneer and Leading Goose R&D Program of Zhejiang. The work involved collaborators spanning Westlake University, the Westlake Laboratory of Life Sciences and Biomedicine, Zhejiang University of Science and Technology, the Zhejiang Key Laboratory of Biomedical Intelligent Computing Technology, and Fudan University, with assistance from the Guangzhou National Laboratory on PGC separation and culture.

Taken together, the findings redraw the picture of the avian germline at single-cell resolution while delivering a workflow that breeders and geneticists can use immediately. A cell type once treated as a uniform raw material now appears as a structured, multi-state population, and the tools to navigate that structure—markers, culture conditions, and a validated reinjection route—are now on the table. For a field in which each generation of genetically modified chickens has traditionally demanded months of careful animal work, the prospect of renewable, long-term cultured, edited PGC lines delivered by blood-vessel injection marks a genuine inflection point, and the coming years will show how quickly this single-cell insight translates into new birds, new traits, and new biology.