In mammals, germ cells migrate to the gonad from the base of the allantois. From the time germ cells leave the allantois, they travel as individual cells often connected by long cytoplasmic filaments (Gomperts et al., 1994
). However, when germ cells colonize the gonad, they form large clusters of eight or more cells. During this period, germ cells are also undergoing cell divisions (Pepling and Spradling, 1998
). Whether the clusters of germ cells in the gonad arise through aggregation or cell division is unclear.
Imaging analyses suggested that filopodial connections between migrating germ cells might lead to their aggregation once they reach the genital ridge (Gomperts et al., 1994
; Bendel-Stenzel et al., 2000
). When cells from the hindgut mesentery and associated mesonephroi were disaggregated and plated on STO feeder cells, germ cells formed clusters. Furthermore, when half of the germ cells in the cultures were labeled with rhodamine-conjugated dye, clusters were composed of labeled and unlabeled cells (Gomperts et al., 1994
), compatible with the idea that clusters can arise through aggregation, rather than solely through cell division. Further investigations showed that blocking E-cadherin, which is expressed by germ cells upon arrival in the gonad (Di Carlo and De Felici, 2000
), led to a disruption in germ cell aggregation in slice cultures and in dissociated reaggregation assays (Bendel-Stenzel et al., 2000
). However, it was not clear that these in vitro assays reflected aggregation properties in vivo.
Other investigators have noticed the similarity between premeiotic germ cell clusters in vertebrates and those found in invertebrates such as Drosophila
. In Drosophila,
germ cell cysts contain 16 cells that arise through stereotypic divisions. Divisions in the cyst are incomplete and result in ring canals that form cytoplasmic bridges between all 16 cells in the cluster (reviewed by Pepling et al., 1999
). Similar intercellular bridges have been seen in electron microscopy studies of mice, rabbits, rats, hamsters, and humans (Fawcett, 1961
; Gondos, 1973
; Gondos and Conner, 1973
; Pepling and Spradling, 1998
). Pepling and Spradling further analyzed these structures in the mouse ovary and showed that intercellular bridges resembling Drosophila
ring canals are present between germ cells in the ovary between embryonic day E11.5 and E17.5. Germ cells in clusters also show synchronous cell divisions, which are characteristic of germ cell cysts in Drosophila
and medaka (Pepling et al., 1999
; Nakamura et al., 2010
). In addition, clusters tend to be comprised of an even number of cells, consistent with the idea that clusters arise through cell division rather than random aggregation (Pepling and Spradling, 1998
, only one of the germ cells in the cluster becomes an oocyte, while the others develop into nurse cells that nourish the oocyte by a directional transport of mitochondria and specific mRNAs through the cytoplasmic bridges (de Cuevas et al., 1997
). In mice, there is at least a 3-fold reduction in the number of germ cells in the ovary between the time that their numbers reach a maxima and the time of follicle formation just after birth (Pepling and Spradling, 2001
; McClellan et al., 2003
). There is currently no clear explanation for this high level of germ cell atresia during fetal ovary development. However, the identification of endoplasmic reticulum and mitochondria within intercytoplasmic bridges between adjacent germ cells in mouse ovaries led to the idea that germ cell atresia could be explained by a selective mechanism similar to the one that operates in Drosophila
(Pepling and Spradling, 2001
). The idea that processes underlying oocyte selection are conserved is appealing.
More recently, molecular components of intercellular bridges in mammalian cells have been identified (reviewed by Greenbaum et al., 2011
). The first of these was TEX14, a protein that is required for the stabilization of intercellular bridges and essential for spermatogenesis in males (Greenbaum et al., 2006
) but not oogenesis in female mice (Greenbaum et al., 2009
). Using a proteomics approach on testis fractions enriched for cytoplasmic bridges, several components of the cytokinesis complex were identified and shown to colocalize with TEX14 to cytoplasmic bridges during early spermatogenesis, including three components of the midbody, the mitotic kinesin-like protein 1 (MKLP1/KIF23), RACGAP1 (MgcRacGap), and centrosomal protein 55 (CEP55), as well as several septin proteins (Greenbaum et al., 2007
; Iwamori et al., 2010
). These elegant studies provided clear evidence that cytoplasmic bridges can form as the result of incomplete cell divisions during germ cell development.
To investigate whether germ cell aggregates arise only through clonal divisions in mice, we produced chimeras between GFP-positive and GFP-negative embryos. Our results indicate that while fetal germ cell clusters arise through both aggregation and clonal divisions, we detected clear evidence of bridges only between germ cells of the same genotype.