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Evolutionary developmental biology
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The corn snake yolk sac becomes a solid
tissue filled with blood vessels and
yolk-rich endodermal cells
Richard P. Elinson1,† and James R. Stewart2
Research
1
2
Cite this article: Elinson RP, Stewart JR. 2014
The corn snake yolk sac becomes a solid tissue
filled with blood vessels and yolk-rich
endodermal cells. Biol. Lett. 10: 20130870.
http://dx.doi.org/10.1098/rsbl.2013.0870
Received: 9 October 2013
Accepted: 11 December 2013
Subject Areas:
evolution, developmental biology
Keywords:
amniote egg, yolk sac, corn snake, endoderm
Author for correspondence:
Richard P. Elinson
e-mail: [email protected]
†
Present address: 240 West Neck Road,
Huntington, NY 11743, USA.
Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15282, USA
Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614, USA
The amniote egg was a key innovation in vertebrate evolution because it
supports an independent existence in terrestrial environments. The egg is
provisioned with yolk, and development depends on the yolk sac for
the mobilization of nutrients. We have examined the yolk sac of the corn
snake Pantherophis guttatus by the dissection of living eggs. In contrast to
the familiar fluid-filled sac of birds, the corn snake yolk sac invades the
yolk mass to become a solid tissue. There is extensive proliferation of
yolk-filled endodermal cells, which associate with a meshwork of blood
vessels. These novel attributes of the yolk sac of corn snakes compared
with birds suggest new pathways for the evolution of the amniote egg.
1. Introduction
A defining innovation in the evolution of terrestrial vertebrates is the amniote
egg, characterized by a set of extraembryonic membranes, which support development of the embryo. These membranes are the chorion, amnion, allantois and
yolk sac. One of the major differences between the eggs of extant amphibians and
of birds, snakes and other oviparous amniotes is the large amount of yolk in the
amniote egg [1,2]. This increased yolk allowed the amniote embryo to hatch as a
free-living animal without an aquatic, feeding larva.
The evolutionary origins of the extraembryonic membranes and of the
modifications to the embryo to accommodate the increased yolk are obscure.
Unfortunately, there is unlikely to be a fossil record of these changes to delicate
embryonic tissues. Possible scenarios can be constructed by examining the
physiological and morphological variation in embryos of extant animals [1,2].
Current ideas about the yolk sac and other extraembryonic membranes of
the amniote egg are strongly influenced by their appearance in chickens.
There are however, significant differences in the extraembryonic membranes
of chickens and those of lizards, turtles and snakes [3–5]. Most of these investigations have centred on the chorion and allantois, with little attention paid to
the yolk sac. This neglect is owing in part to difficulties in adequate fixation of
yolk. With this in mind, we have examined the live yolk sac of the corn snake
Pantherophis guttatus by dissection. We show that unlike the yolk of chickens,
the yolk within the corn snake yolk sac becomes cellularized.
2. Material and methods
Oviposited eggs were collected from four captive-reared corn snakes, P. guttatus.
Eggs were incubated in moist vermiculite at 268C. We examined 16 embryos representing Zehr [6] stages 23/24, 25/26, 30, 31, 34, 35 and 36. Oviposition occurs at
stage 22, 50 – 60 days after mating, and hatching occurs at stage 37, 150– 160 days
after mating [7].
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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(a)
(b)
(d)
(g)
(h)
ys
e
(f)
Figure 1. Dissection of a stage 31 embryo. (a) When removed from the shell, the snake embryo (S), surrounded by the amnion, lies deep within a ball of yolky
tissue. The yolk mass was about 27 35 mm. (b) The yolky tissue was cut along the equator, and the top of the ball was flipped up with the embryo in the upper
half. The cut surface of the lower half was covered with a loose mass of white, yolk-rich endodermal cells (e). These have been removed to reveal the yolk
splanchnopleure (ys), rich in blood vessels. (c) The mass of endodermal cells is held together loosely. (d ) The large cells contain many vesicles. (e) Yolk sac endodermal cells of corn snake (stage 33) and (g) chicken are both large and filled with vesicles. The cells are nucleated in (f ) corn snake and (h) chicken as revealed by
DAPI staining. Scale bars: (c) 2 mm, (d – h) 0.2 mm. (Online version in colour.)
(a)
(b)
(c)
Figure 2. Yolk sac blood vessels. (a) In this stage 31 embryo, the yolk sac blood vessels (right) are coated with yolk-rich endodermal cells. Endodermal cells, not
associated with the blood vessels, are on the left. (b) In this stage 34 embryo, yolk sac blood vessels, coated with endodermal cells, form an elaborate mesh. (c) An
enlargement of the stage 34 coated blood vessels. (a,b) Scale bars, 2 mm; (c) scale bar, 0.5 mm. (Online version in colour.)
Eggs were submerged in reptilian Ringer’s (94 mM NaCl,
2.7 mM KCl, 1.8 mM CaCl2, 2.1 mM MgCl2, 24 mM NaHCO3)
[8]. The shell was cut with scissors and the released contents
were kept submerged for dissections. To stain nuclei, cells were
fixed in MEMFA (3.7% formaldehyde, 100 mM MOPS, 1 mM
MgSO4, 2 mM EGTA, pH 7.4) for approximately 20 min and
placed in 1 mg ml21 DAPI in PBS.
To observe chicken yolk sac endodermal cells, the yolk
splanchnopleure was washed with chick Ringer’s solution
(123 mM NaCl, 4.95 mM KCl, 1.78 mM CaCl2, 2.38 mM
NaHCO3, pH 7.3) to remove yolk. It was cut into pieces and incubated in 1 mM EGTA in Ca2þ-free chick Ringer’s solution for
about 2 h at 378C. Many endodermal cells detached from the
splanchnopleure when the pieces were gently shaken. The cell
suspension was centrifuged at 1500 rcf for 5 min. Red blood
cells and tissue pieces pelleted, and the floating endodermal
cells were collected from the top of the solution.
3. Results
We begin our description of the yolk sac at stage 31, 36 days
after egg laying, because two unusual features were present.
The embryo, which was surrounded by yolk, was about
11 cm long, less than half its length at hatching (figure 1a).
The yolk sac was vascularized, as it is in chickens, but the
yolk mass was coherent and felt solid. When the yolk sac surface was cut, no yolk spilled out. Rather, the inside of the yolk
sac consisted of two components: a mass of large endodermal
cells and a spaghetti-like mesh of blood vessels coated with
endodermal cells (figures 1b and 2).
The endodermal cells were loosely held together and were
filled with vesicles, most probably containing nutrients derived
from yolk (figure 1c–e). Nuclei were detected with DAPI staining (figure 1f ), confirming that the yolky mass was made up
Biol. Lett. 10: 20130870
(e)
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S
(c)
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The yolk sac of the corn snake in the final third of development is a solid tissue rather than a sac. This is owing to
extensive proliferation of yolk-rich endodermal cells followed
by invasion of the endodermal cell mass by blood vessels.
These novel observations open new questions, both with
respect to development and to evolution.
(a) Development of the corn snake yolk sac tissue
Some of the yolk is already cellularized at stage 23/24, 8 days
after oviposition, and the extraembryonic endodermal cells
continue to proliferate. This raises the question whether
these cells express cell-cycle-related genes or whether these
proteins or their RNAs are already present in the yolk.
Regardless, the cellularization of the corn snake yolk
indicates the presence of active cytoplasm.
In addition to the endodermal cells, there is an extensive
growth of blood vessels through the yolk sac. Formation of
blood vessels in the mesoderm depends on signals from the
endoderm in embryos [9,10]. This raises the possibility that
the endodermal cells in the corn snake yolk sac express
genes required for vasculogenesis and these factors induce
growth of blood vessels into the yolk sac.
Invasion of the yolk sac cavity by mesodermal cells is well
documented in squamates, the lizards and snakes. Squamates
develop a yolk cleft in the abembryonic region of the yolk [3].
The yolk cleft forms from a sheet of mesoderm that penetrates
(b) Evolutionary issues raised by the corn snake yolk sac
tissue
A solid, cellular yolk sac tissue, as described here, has never
been reported. Among squamates, development and morphology of yolk splanchnopleure are similar to birds [3], but
the contents of the yolk sac cavity are largely unknown. There
is evidence that the corn snake developmental pattern may
extend to other squamates, and perhaps to other amniote
lineages. Blood vessels and endodermal cells are present
within the yolk sac cavity of two species of scincid lizards
[5,11]. In addition, an 1857 drawing by Agassiz [12] of ‘a
mesh of blood vessels covered by yolk’ in the snapping turtle
yolk sac (p. 631, Plate XVIII) is similar in appearance to that of
the corn snake (figure 2), but the yolk is not depicted as cellularized. One evolutionary question is whether the yolk sac tissue in
the corn snake is a derived or basal character. It would be useful
to dissect living eggs of additional squamates and turtles to
determine the taxonomic distribution of this character.
Holoblastic cleavage is thought to be primitive for terrestrial vertebrates [13]. The second evolutionary question then
is how the transition occurred from the holoblastically cleaving amphibian embryo to the meroblastically cleaving
amniote embryo. One idea is based on the discovery of a
novel tissue, nutritional endoderm, in embryos of Eleutherodactylus coqui, a frog with large eggs [14,15]. These cells
serve a nutritional role only, and once their yolk is used up,
the cells disappear. This state could be the first step towards
the meroblastic condition.
Our current observations on the corn snake yolk sac raise a
new hypothesis on the origin of meroblastic cleavage in
amniotes. The initial event may have been the invasion of a
tissue-like nutritional endoderm by blood vessels. This would
establish a more extensive association between yolk-rich endodermal cells and the blood supply than that is present in
amphibian embryos, and could lead to enhanced movement
of nutrients from the yolk to the embryo. If this occurred, cell
division within the yolky area could be relaxed, as long as
cells eventually formed. Meroblastic cleavage in this scenario
would have arisen secondary to changes in the vasculature surrounding the yolk-rich endodermal cells, so the blood vessel
invasion of the endoderm would be a primitive trait.
Support for this scenario could come from surveying the
diversity of the yolk-rich regions of embryos of both amphibians and reptiles. One question is whether any amphibian
exhibits vascular invasion of its yolk-rich endodermal region.
The second question is whether cellularized yolk, such as the
one reported here, is found in embryos of more basal amniotes.
The yolk-rich regions of embryos of terrestrial vertebrates merit
more intensive investigation.
Protocols approved by ETSU IACUC.
Acknowledgements. We thank D.G. Blackburn (Trinity College, Hartford,
CT, USA) for eggs, N.B. Ford for adult snakes and Jerry Thomsen
(SUNY Stony Brook, NY, USA) for laboratory facilities.
Funding statement. This project was initiated under National Science
Foundation grant no. IOS-0841720 to R.P.E.
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Biol. Lett. 10: 20130870
4. Discussion
the yolk and generates blood vessels. The yolk cleft is distinct
from the vascular invasion we describe here, but the yolk cleft
indicates that mesodermal invasion of yolk is a property of
squamate development.
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of cells of large size and unusual appearance. Many endodermal
cells were attached to blood vessels, but some were not (figure 2).
The corn snake yolk sac at stage 31 belies the image generated by
the word ‘sac’. Rather than a sac containing yolk, this structure is
a vascularized tissue with endodermal cells.
By contrast, the chicken yolk sac splanchnopleure is a true
sac. Liquid yolk was surrounded by a vascularized yolk
splanchnopleure. Blood vessels did not invade the interior.
Endodermal cells formed an intermediary between blood
vessels, the outer epithelium and the non-cellular yolk.
When chicken endodermal cells were isolated from the yolk
splanchnopleure, the cells floated on top of the aqueous
Ringer’s solution, indicating their lipid-richness. Like the
corn snake endodermal cells, the chicken cells were filled
with vesicles and were nucleated (figure 1g,h).
The cellularization of the yolk sac at stage 31 led us to
examine other stages. One embryo was examined at stage
23/24, 8 days after oviposition. When the eggshell was cut,
liquid yolk flowed out, but there was also a thick mass of endodermal cells beneath the embryo. DAPI staining confirmed the
presence of nuclei. As development proceeded, there was less
liquid yolk and more vesicle-rich endodermal cells. At stage
28–29, most of the yolk was contained in cells, with a small
amount of non-cellular yolk in the centre of the cellular area.
Until about stage 30, the yolk splanchnopleure looked similar
to that in chicken, namely a thin outer membrane with blood
vessels. By stage 30, the non-cellular yolk was gone. This was followed by the extensive invasion of the endodermal cell mass by
blood vessels (figure 2). Stage 30 represented both the completion
of endodermal cellularization and the beginning of increased
vascularization. In stages 34–36, all of the endodermal cells
were attached to blood vessels.
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