Precise self-renewal of the germ cell lineage is fundamental to fertility and reproductive success. The early precursors for the germ lineage, primordial germ cells (PGCs), survive and proliferate in several embryonic locations during their migration to the embryonic gonad. By elucidating the active signaling pathways in migratory PGCs in vivo, we were able to create culture conditions that recapitulate this embryonic germ cell environment. In defined medium conditions without feeder cells, the growth factors FGF2, insulin, and Activin A, signaling through their cognate-signaling pathways, were sufficient for self-renewal of germline-competent PGCs. Forced expression of constitutively active MEK1, AKT, and SMAD3 proteins could replace their respective upstream growth factors. Unexpectedly, we found that BMP4 could replace Activin A in non-clonal growth conditions. These defined medium conditions identify the key molecular pathways required for PGC self-renewal and will facilitate efforts in biobanking of chicken genetic resources and genome editing.

Significance: Here, we show that DMRT1 dosage is the key sex determination factor in birds and is essential for testis development. Furthermore, we provide additional evidence that birds, in contrast to mammals, have acquired cell-autonomous sex identity (CASI) and that the sex hormone environment does not significantly influence avian secondary sexual characteristics. This finding highlights an evolutionary divide between mammals and nonmammalian vertebrates. In mammals, the sex chromosomes determine the type of gonad formed, and sex hormones largely define the secondary sexual phenotype. In birds, the sexual phenotype is directly determined by the sex chromosome content of individual cells in different tissues. Our findings help advance our understanding of the evolution of sex determination systems and the nature of sex identity.

The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.

Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape.

In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 (vasa) locus in chicken primordial germ cells. Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germ line competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary leading to adult female sterility. TALEN-mediated gene targeting in avian primordial germ cells is therefore an efficient process.