Mike McGrew

Periodic Notch inhibition by Lunatic Fringe underlies the chick segmentation clock

The segmented aspect of the vertebrate body plan first arises through the sequential formation of somites. The periodicity of somitogenesis is thought to be regulated by a molecular oscillator, the segmentation clock, which functions in presomitic mesoderm cells. This oscillator controls the periodic expression of ‘cyclic genes’, which are all related to the Notch pathway. The mechanism underlying this oscillator is not understood. Here we show that the protein product of the cyclic gene lunatic fringe (Lfng), which encodes a glycosyltransferase that can modify Notch activity, oscillates in the chick presomitic mesoderm. Overexpressing Lfng in the paraxial mesoderm abolishes the expression of cyclic genes including endogenous Lfng and leads to defects in segmentation. This effect on cyclic genes phenocopies inhibition of Notch signalling in the presomitic mesoderm. We therefore propose that Lfng establishes a negative feedback loop that implements periodic inhibition of Notch, which in turn controls the rhythmic expression of cyclic genes in the chick presomitic mesoderm. This feedback loop provides a molecular basis for the oscillator underlying the avian segmentation clock.

Oviduct-specific expression of two therapeutic proteins in transgenic hens

Recent advances in avian transgenesis have led to the possibility of utilizing the laying hen as a production platform for the large-scale synthesis of pharmaceutical proteins. Ovalbumin constitutes more than half of the protein in the white of a laid egg, and expression of the ovalbumin gene is restricted to the tubular gland cells of the oviduct. Here we describe the use of lentiviral vectors to deliver transgene constructs comprising regulatory sequences from the ovalbumin gene designed to direct synthesis of associated therapeutic proteins to the oviduct. We report the generation of transgenic hens that synthesize functional recombinant pharmaceutical protein in a tightly regulated tissue-specific manner, without any evidence of transgene silencing after germ-line transmission.

Somatic sex identity is cell autonomous in the chicken.

In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in birds, sexual differentiation is substantively cell autonomous.

Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons

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.

Characterisation and germline transmission of cultured avian primordial germ cells

Background 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. Principal Findings 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. Conclusions 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.

Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity

The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.

The chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites.

BackgroundSomitogenesis is the earliest sign of segmentation in the developing vertebrate embryo. This process starts very early, soon after gastrulation has initiated and proceeds in an anterior-to-posterior direction during body axis elongation. It is widely accepted that somitogenesis is controlled by a molecular oscillator with the same periodicity as somite formation. This periodic mechanism is repeated a specific number of times until the embryo acquires a defined specie-specific final number of somites at the end of the process of axis elongation. This final number of somites varies widely between vertebrate species. How termination of the process of somitogenesis is determined is still unknown.ResultsHere we show that during development there is an imbalance between the speed of somite formation and growth of the presomitic mesoderm (PSM)/tail bud. This decrease in the PSM size of the chick embryo is not due to an acceleration of the speed of somite formation because it remains constant until the last stages of somitogenesis, when it slows down. When the chick embryo reaches its final number of somites at stage HH 24-25 there is still some remaining unsegmented PSM in which expression of components of the somitogenesis oscillator is no longer dynamic. Finally, we identify a change in expression of retinoic acid regulating factors in the tail bud at late stages of somitogenesis, such that in the chick embryo there is a pronounced onset of Raldh2 expression while in the mouse embryo the expression of the RA inhibitor Cyp26A1 is downregulated.ConclusionsOur results show that the chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites. In addition, endogenous retinoic acid is probably also involved in the termination of the process of segmentation, and in tail growth in general.

FGF, Insulin, and SMAD Signaling Cooperate for Avian Primordial Germ Cell Self-Renewal

Summary 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.

The development and maintenance of the mononuclear phagocyte system of the chick is controlled by signals from the macrophage colony-stimulating factor receptor.

BackgroundMacrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model.ResultsBased upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP+ donors. In both cases, the transferred cells gave rise to large numbers of EGFP+ tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP+ cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings.ConclusionsThe data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling.

A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration.

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.