415et al. 2009 ; Kawamura and Sunanaga 2011 ). Circulatory vasa-positive cells
have been observed in all botryllid species examined to date (Sunanaga et al.
2006 ; Brown and Swalla 2007 ; Kawamura and Sunanaga 2010 , 2011 ; Rosner
et al. 2009 ). These cells are small, round, with large nuclei, and are present in
the vasculature immediately following metamorphosis (Brown et al. 2009 ).
Germ line precursors from juveniles contribute to gamete formation immedi-
ately upon transplantation into adults, demonstrating that these cells are germ
line stem cells that were specified during embryonic development. Other evi-
dence for the presence of mobile, long-lived germ line stem cells comes from
naturally occurring vascular fusion between genetically distinct individuals.
After fusion, germ line chimerism persists even after surgical separation of the
fusion partners (Laird et al. 2005 ), indicating that long-lived, self-renewing
germ line stem cells are actively transferred between the two colonies. These
cells can be prospectively isolated based on expression of Aldehyde
Dehydrogenase, express high levels of vasa, and can reconstitute the germ line
upon transplantation (Laird et al. 2005 ).
Fig. 8.9 Gonad regeneration during asexual reproduction in Botryllus schlosseri. (a) Ventral view
of a colony of Botryllus schlosseri showing individual adult animals, each of which is connected
to asexual propagating primary and secondary buds. Individual animals and buds are connected by
a common extracorporeal vasculature, which ends in terminal projections known as ampullae. (b)
During the asexual budding process, new secondary buds begin as a thickening of the peribranchial
epithelium, which forms a pocket and eventually closes to form a double vesicle. Small, round
germ line stem cells (GSC, 7–10 μm in diameter, green) expressing Vasa are present in the circula-
tion. These Vasa-positive GSCs are also present in primary buds near developing gonads and larger
Vasa-positive germ cell precursors (30–50 μm, green). When a secondary bud develops and closes
to form a double vesicle, Vasa-positive germ cells (green) migrate into the secondary bud. As sec-
ondary bud development proceeds, more and larger germ cells are visible, indicating proliferation
and differentiation. At later stages, Vasa-positive germ cells differentiate into testes and oocytes, as
primary buds develop into the adult form
8 Mechanisms of Vertebrate Germ Cell Determination