417extirpation of a strip of intermediate mesoderm from early tailbud stage embryos
(Humphrey 1927 , 1929 ). The absence of PGCs on the operated side of the embryo
demonstrated that PGCs do not arise from a medial endodermal origin, as suggested by
others (Bounoure 1925 ), and was thus consistent with their origination from within the
mesodermal germ layer itself. Furthermore, these results provided the first conclusive
evidence arguing against the possibility that PGCs arise de novo from differentiated
somatic cells, which was also a prominent theory at the time. Nevertheless, because
Humphrey’s dissections were executed with embryos of a fairly advanced stage, it
remained a possibility that PGCs migrate to the mesoderm from an earlier alternative
location, such as the endoderm. This hypothesis was favored by workers who presumed
that PGCs must arise from a common origin in urodeles and in anurans.
Nieuwkoop ( 1947 ) later extended investigation into the origins of urodelean
PGCs using embryos from gastrula stages, at which time it is feasible to separate
mesoderm precursors from endoderm experimentally (Nieuwkoop 1947 ). Focusing
primarily on embryos from axolotls, he combined fate mapping with transplant and
deletion studies to precisely map germ cell precursors within the ventral marginal
zone of early gastrulae, where they reside adjacent to the presumptive blood cells.
Specified progenitors of the germ line later pass over the lateral lips of the blasto-
pore prior to taking residence in the dorsal lateral plate. Interestingly, presumptive
germ cells are the last cells to internalize during gastrulation. They pass over the
blastopore during mid to late gastrula stage (at about stage 11), which is after the
somatic cell lineages are established. Moreover, removal of the germ cell domain
during gastrula stages resulted in complete sterility in the majority of operated
embryos. This finding ruled out the possibility that PGCs might originate within the
endoderm and later migrate into the mesodermal layer, unequivocally establishing a
mesodermal origin for the PGCs. Furthermore, these results provided confirmation
that PGCs could not arise secondarily from differentiated somatic cells. However, in
addition to establishing these fundamental principles of germ cell biology,
Nieuwkoop also found that deletion of the caudal endoderm completely eliminated
the germ line from developing embryos. He thus postulated, for the first time, that
PGCs were formed in response to inducing signals. Yet, validation of this hypothesis
would only come decades later (Fig. 8.10 for summary on PGC formation).
8.5.2 Induction of PGCs from “Totipotent” Cells
Weissman ( 1898 ) proposed the Germ Plasm Hypothesis in which the germ line is
maintained across generations by maternally inherited germ cell determinants, and
the subsequent discovery of material fitting this description in the eggs of species as
diverse as frogs and fruit flies fostered the perception that germ plasm was a universal
component of development in the animal kingdom (Blackler 1970 ; Mahowald and
Hennen 1971 ). Germ plasm segregates PGCs from somatic cells in the earliest stages
of development, thus it was conceivable that germ plasm, or an equivalent substance,
segregates with a subpopulation of presumptive mesodermal cells in urodele embryos
8 Mechanisms of Vertebrate Germ Cell Determination