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Title: Factors controlling migration of avian primordial germ cells
Author: Pogool, Satian
ISNI:       0000 0004 2697 3070
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2002
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Avian embryos have played an important role in the study of vertebrate development but further understanding of their development may also benefit the commercial poultry industry. Manipulation and use of primordial germ cells (PGCs) as a vehicle for constructed genotypes will be increasingly important for future improvement of commercial poultry including the turkey. However, the manipulation and use of PGCs will depend on a basic understanding of PGC migration. PGCs are known to migrate considerable distances before colonising the gonad. Previous work in the chick and quail have suggested that avian PGCs arise from the ventral surface of the area pellucida before the blastoderm undergoes gastrulation, translocate to the germinal crescent during primitive streak stages and penetrate the extraembryonic blood vessels (stage 10) after they have formed. PGCs are subsequently carried by the blood stream into the embryo proper during stages 12-14 and then leave the blood vessels and migrate to the developing gonads. However, the factors controlling PGC migration are poorly understood. The developmental stages of older turkey embryos are not well documented. Therefore, this study started by classifying them. It was found that turkey embryos at stage 4 and younger were slightly different from chick embryos, but from stages 5 onwards, turkey embryos could be staged using criteria described for the chick (Hamburger and Hamilton, 1951). Avian PGCs can be recognized by their distinctive morphology being large cells with large and eccentrically placed nuclei, and a cytoplasm containing refractile granules. These characteristics were confirmed for turkey PGCs. In order to manipulate PGCs, cells need to be identified using appropriate markers. A panel of markers previously used for chick, quail, mouse, rat and rabbit were tested with turkey PGCs at different stages. It was found that turkey PGCs could be detected by the histochemical stains, periodic acid-Schiff (PAS) and alkaline phosphatase (AP) or using antibodies to stage-specific embryonic antigen-1 (SSEA-1), fucosylated polylactosamine carbohydrate groups (EMA-1) and ovomucin-like protein (OLP). At primitive streak stages, turkey PGCs were located in the area pellucida. During stages 11 - 15, they were found in the blood reaching maximal numbers at stage 14. After stage 15, most PGCs were located outside the circulatory system. From stage 18, PGCs began to settle in gonadal ridges and after stage 28, PGCs were found only in the gonads. Turkey PGCs were isolated from the blood with a micropipette, during visual selection under the microscope, and their Identity confirmed using the above markers. These PGCs could be cultured on a gonadal stromal cell layer or on coverslips coated with rat-tail collagen. Isolated PGCs adhered to coated coverslips were prepared and examined with scanning electron microscope (SEM). Such cells had typical features of PGCs seen in situ and some had the morphology of motile cells. PGC movement in vitro, in response to a variety of potential chemoattractants, was then studied. Conventional chemotaxis assays could not be used to study such small numbers of cells. Therefore, the assay was carried out using a Dunn chemotaxis chamber which allows direct observation of cells and detailed analysis of their movement from a timed series of images. A positive chemotactic response was observed with cells exposed to a gradient of medium conditioned by stromal cells or 10 ng/ml transforming growth factor beta-1 (TGFbeta1). Cells exposed to 100 ng/ml stem cell factor (SCF) or control medium showed no such response. These observations indicate that TGF?l can play a role in directed migration of PGCs in vitro, but further experiments are required to determine whether this factor is similarly involved in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available