3337.5.2.2 The YSL
Early experiments to determine the role of the yolk during teleost development
yielded ambiguous results. The great geneticist Thomas Hunt Morgan (1866–1945)
was the first person to perform a systematic experimental analysis of teleost devel-
opment, working with wrasse, sea bass, and killifish embryos at the Marine
Laboratory in Woods Hole, Massachusetts (Morgan 1893 , 1895 ). He used a needle
to poke a hole in the vegetal pole of the embryo, opposite the blastodisc, gently
squeezed out the yolk, and permitted the sac to backfill with seawater. He concluded
that, “The yolk may be removed from the egg of Fundulus at almost any stage of
development and the embryo still forms.” (p. 809) (Morgan 1893 ). The caveat to this
conclusion was that embryonic development ceased if he removed more than two-
thirds of the yolk content. Morgan viewed the yolk as a passive structure, important
only for the mechanics of embryonic cleavages and epiboly. Oppenheimer reinves-
tigated the role of the yolk in killifish, with a full understanding of embryonic induc-
tion (Oppenheimer 1936a). Her approach was to remove the entire blastoderm from
the yolk at different stages and to grow them as isolates in culture. Blastoderms
isolated after the 32-cell stage produced embryos with structures from all three
germ layers, although they typically lacked posterior structures. Blastoderms iso-
lated before this stage formed a hollow ball of epidermis reminiscent of the atypical
ectoderm formed in Holtfreter’s animal cap explants. Oppenheimer concluded that
the yolk syncytial layer (YSL), which she called the periblast, contains a substance
that is transferred to the blastoderm at or before the 32-cell stage, and is necessary
for formation of the mesoderm and endoderm. Similar results were obtained from
cultures of isolated goldfish, trout, medaka and zebrafish blastoderms (Fig. 7.6a)
(Tung et al. 1954 ; Devillers 1961 ; Hyodo et al. 1996 ; Sagerstrom et al. 1996 ; Xu
et al. 2014b). These transplant experiments were often difficult to interpret, given
differences in staging, and whether or not a bit of the YSL had been included in the
blastoderm isolates. Conclusive proof that molecules produced in the yolk are
essential for embryonic development came from experiments in which RNase was
injected into the YSL of zebrafish embryos (Fig. 7.7a) (Chen and Kimelman 2000 ).
This treatment had dramatic consequences, and the embryos died before gastrula-
tion, failing to express the earliest markers for mesoderm and endoderm. Since
zebrafish embryos lacking mesoderm and endoderm survive perfectly well for days,
it is likely that the RNase treatment affected a number of different processes that are
vital for survival past early embryonic stages.
A complementary set of experiments demonstrated that signals from the yolk can
induce mesoderm and endoderm. In a series of heterochronic transplants, old blas-
toderms with visible shields were replaced with younger blastoderms, lacking
shields. As development proceeded, the transplanted blastoderms always developed
shields in the same location as the original blastoderm (Long 1983 ). In zebrafish,
animal cap cells expressed markers of mesendoderm, including the shield, when
they were directly exposed to the YSL in grafting experiments (Fig. 7.6a) (Mizuno
et al. 1996 ; Ober and Schulte-Merker 1999 ; Rodaway et al. 1999 ). Because the yolk
cell cannot be further physically subdivided, specific regional capabilities of the
YSL have not been addressed by grafting experiments. Targeted knockdown of
7 Establishment of the Vertebrate Germ Layers