Overview of germ cell development and gametogenesis: The basic process of germ cell development is conserved in flies and higher vertebrates. In Drosophila (Fig 1), this process begins with formation of primordial germ cells (PGCs) at the posterior pole of the embryo. Once formed, germ cells migrate toward somatic gonadal precursor cells (SGPs) to form two distinct embryonic gonads. Subsequently, gonad morphogenesis ensues, eventually giving rise to ovaries or testes with functional germ line stem cells (GSCs) localized to a GSC niche.
Gametogenesis (Fig 1) begins with the asymmetric division of a GSC to produce one GSC that remains undifferentiated, and a daughter blast cell (termed a gonialblasts or cystoblast in males or female, respectively). Blast cells then undergo 4 rounds of division with incomplete cytokinesis, giving rise to a 16-cell cyst that shares its cytoplasm through ring canals and a specialized organelle called a fusome. In males, spermatogonial cysts then undergo a number of morphological changes and initiate meiosis to produce 64 individual haploid sperm. In females, each 16-cell cyst becomes surrounded by somatic follicle cells to form an egg chamber. Within each egg chamber, the majority of cyst cells then develop into nurse cells that provide energy to a single germ cell that is selected to become an oocyte.
Drosophila gonad morphology and function of the GSC niche: An asymmetrically dividing population of GSCs must be maintained in adult gonads to ensure fertility. This balance between GSC maintenance and differentiation is regulated by the micro-environment surrounding the GSCs (Fig 1). In adult ovaries, this consists of terminal filament cells at the apical tip of each ovariole (approx. 16 ovarioles per ovary), 2 cap cells, and a population of escort stem cells (ESCs) that are intimately associated with 2-3 GSCs. Signaling and cell adhesion between GSCs and cap cells that comprise the GSC niche are critical regulators of GSC maintenance, while ESCs give rise to non-dividing escort cells (ECs) that encapsulate cystoblasts and nurture their development prior to egg chamber formation.
While morphologically distinct (see Fig 1), the micro-environment surrounding GSCs is similar in adult testes. At the apical tip of adult testes, 5-9 GSCs are arrayed around a cluster of somatic “hub” cells that comprise the GSC niche. As in ovaries, adhesion to and signals from the GSC niche are required for GSC maintenance. Furthermore, each GSC is surrounded by (except at the GSC-hub interface) a pair of somatic stem cells, termed cyst progenitor cells (CPCs). These cells divide in concert with GSCs and, like ESCs, produce progeny (termed cyst cells) that encapsulate differentiating gonialblasts. During this process, signaling between germ cells and CPCs/cyst cells regulates germ cell proliferation and differentiation.
Drosophila gonad development and GSC niche formation: The somatic portion of the gonad (SGPs) arises from the mesoderm within abdominal parasegments (PS) 10-13. By ~9 hrs after fertilization, germ cells and SGPs make contact, and gonad morphogenesis ensues. This begins with the process of gonad coalescence, which simultaneously results in ensheathment of PGCs by SGPs, and compaction of gonadal cells into a spherical gonad located in PS10 by ~12 hrs of development (see Fig. 1).
At this time, male and female gonads begin to develop in a sexually dimorphic manner. Indeed, sex-specific apoptosis is observed in posterior germ cells (so called male-specific SGPs or msSGPs; Fig 1) during female gonad coalescence. Additionally, signals from SGPs at this time appear to regulate germline sexual dimorphism. Furthermore, while the female GSC niche does not appear to form until the third larval instar, recent evidence indicates that the male GSC niche has formed by the end of embryogenesis.
Germ cell sex determination: While GSCs are required for continuous gamete production, functional sperm or eggs cannot develop at all without proper assignment of sexual identity. Interestingly, the decision made by germ cells to develop as male or female appears to be regulated differently from all the other cells in the body (somatic cells). Somatic sexual identity (see Fig. 2), is controlled cell autonomously by the ratio of sex (X) chromosomes to autosomal (A) chromosomes, such that an XX individual (X:A=1) develops as female, while an XY individual (X:A=0.5) develops as a male. Differences in sex chromosome karyotype are sensed by an X:A counting system and lead to female-specific Sex-lethal (Sxl) expression. Sxl then functions through transformer (tra) and transformer-2 (tra-2) to regulate splicing of dsx mRNA. In an XX soma, this results in production of female-specific dsx mRNA (dsxF) that promotes female identity, while in males, the male form of dsx (dsxM) is produced by default.
In contrast, germ cell sex is controlled by both germ cell autonomous and somatic factors (Fig 2). Furthermore, many of the genes that promote somatic sexual identity (e.g. tra and dsx) are dispensable for germ cell sex determination. These conclusions have been drawn largely from germ cell transplantation experiments. Indeed, when genetically male (XY) germ cells develop in a female soma (or vice versa), flies are infertile, while transplantation of tra or dsx mutant germ cells into “same sex” embryos results in functional gametes. Closer analyses, however, reveal more specific mechanisms. While infertile, XX germ cells in a male soma initiate spermatogenesis. Furthermore, XY germ cells in adult ovaries form tumors that express genes characteristic of male germ cell development. Thus, both germ cell autonomous mechanisms controlled by the X:A ratio and male somatic signals appear to promote male germline sexual identity.
A number of genes also act germ cell autonomously to promote female identity. The two most extensively studied of these are ovo, which encodes several zinc finger-related transcription factors, and ovarian-tumor (otu). Both of these genes are required for female, but not male, germline development, and specific mutations result in formation of male-like ovarian germ cell tumors. Interestingly, Sxl is also required autonomously for female germ cell development. Because, tra and dsx are not required for germ cell sex determination, it is not yet clear how Sxl promotes female germ cell identity. However, it is thought to act downstream of ovo and otu. Furthermore, otu expression is directly regulated by ovo as well as signals from the female somatic gonad, while ovo appears to be controlled by the X:A ratio. Thus, it appears that germline sexual identity is controlled by both male and female germ cell autonomous factors, as well as signals from the male and female somatic gonad (see Fig 2). While some of these factors have been identified (e.g. ovo, otu and Sxl), many others are yet to be determined. By elucidating these factors and studying their function throughout the process of germ cell development, we hope to gain insights into the process of germ cell sex determination that have implications for our understanding of cancer progression, infertility and regulation of GSC behavior in adult and developing gonads.