Isolation and characterisation of chick embryonic primordial germ cells

摘要

Embryonic stem cells (cESCs) can be isolated from chick embryos, with the ability to contribute to all somatic lineages in chimaeras, but not to the germ line. However, lines of chicken embryonic germ cells (cEGCs), which are able to contribute to the germ line, can be established from chicken primordial germ cells (cPGCs). However very little is known about these cells, or about the changes that accompany the establishment of gonadal cells as self-renewing cell lines. This thesis presents a detailed study of the properties of cPGCs and the parent tissue from which they are derived. Gene expression profiles for 30 genes related to pluripotency and/or differentiation were are compared between gonads at the indifferent stage (stage 26-28HH), in primary gonocytes, established PGCs and cESCs. The results reveal great heterogeneity in the expression of various markers in culture. Several genes associated with pluripotency change dramatically upon culture. The most salient of these changes is that while cSox3 (but not Sox2) is expressed in the gonads, whereas their expression becomes reversed upon culture (becoming more similar to mammalian stem cells). This suggests that these two SoxB1 class genes have swapped functions in chick. In the process of studying the expression pluripotency markers in later (stage HH35) gonads, we made an unexpected discovery: both male and female embryos show left-right asymmetric patterns of expression of some, but not all, of these markers. Expression of pluripotency (cPouV, cNanog, cSox2 and ERNI) in the left gonad is much higher than those in the right gonad of both sexes. The expression of pluripotency markers is irrespective of its colonisation by primordial germ cells, and it appears that this left-right decision is made independently of whether the gonad will regress or be retained. These findings offer a new model system for investigating the roles of pluripotency-related markers during normal development as well as in stem cell lines.