Download Head Ectodermal Patterning and Axial Development in Frogs1

AMER. ZOOL., 33:417^23 (1993)
Head Ectodermal Patterning and Axial Development in Frogs1
THOMAS A. DRYSDALE AND RICHARD P. ELINSON
Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario M5S 1A1, Canada
SYNOPSIS. TWO transient glands, the hatching and cement glands, define
critical boundaries on the head of the frog embryo. They can be used to
monitor formation of the head, which in turn is a sensitive indicator of
development of the dorsal axis, characteristic of chordates. Experimental
treatment of embryos generates a variety of head abnormalities. Alteration
of inductive patterns can produce large heads (macrocephaly), and comparable alterations may yield new phenotypes naturally. Several paths
lead to decreased head development, and one of these may mimic in
reverse the path which led to the evolution of the vertebrate head.
neural plate is widest and forms the neural
structures of the head. The narrower, posterior neural plate gives rise to the spinal
cord and tail.
After the neural plate has been internalized, three new cell types appear on the surface: hatching gland, cement gland, and ciliated cells. The hatching gland secretes a
protease which weakens the fertilization
envelop, helping the embryo to hatch (Carroll and Hedrick, 1974; Yoshizaki and
Katagiri, 1975; Yoshizaki, 1991). The
cement gland (adhesive organ, sucker)
secretes a glue enabling the emergent tadpole to stick to substrates. Ciliated cells generate water currents over the embryo's body.
Although hatching gland cells were known
to be in the head and to have a distinct
morphology (Yoshizaki, 1973), their exact
distribution was not obvious until markers
became available (Sato and Sargent, 1990;
Hemmati-Brivanlou et al, 1990). We found
SURFACE CELLS OF THE EMBRYONIC
that an antibody against tyrosine hydroxyFROG HEAD
When the Xenopus embryo finishes gas- lase identified these cells and have used this
trulation, the surface of the embryo appears to analyze their development (Drysdale and
as a sheet of uniform cells. This apparent Elinson, 1991). Hatching gland cells appear
uniformity hides the precise patterning as a Y-shaped collection of cells on the top
which has taken place in the surface cells, of the head after the embryo begins to elonparticularly in the head region. With neu- gate. The "stalk" of the Y runs along the
rulation, the most dramatic change in the dorsal midline of the embryo, but does not
embryonic surface is the internalization of extend into the trunk (Fig. 2).
Cement gland and ciliated cells appear on
the neural plate. The anterior portion of the
the surface at about the same time as hatching gland cells. Once the development of
1
From the Symposium on Development and Evo- hatching gland cells was known, it became
lution of the Vertebrate Head presented at the Annual
Meeting of the American Society of Zoologists, 27-30 obvious that the pattern of these three cell
types was interrelated. Using hatching and
December 1991, at Atlanta, Georgia.
INTRODUCTION
The vertebrate head develops at the anterior end of the dorsal axis of the embryo.
Its development is so coupled with that of
the dorsal axis, that a single dorsoanterior
index can be used to describe axial variations in the embryo (Fig. 1). Increases or
decreases in the degree of dorsal development lead to corresponding increases or
decreases in the degree of head development
(Kao and Elinson, 1988). As a result, the
head is a sensitive indicator of axial development.
Head development in the frog can be
monitored by observing two transient ectodermal organs, the hatching and cement
glands, which delineate regions of the head
(Fig. 2). We will use these glands to follow
patterning of the head and its reliance on
axial development.
417
418
T. A. DRYSDALE AND R. P. ELINSON
10
FIG. 1. Dorsoanterior Index. Experimentally produced variations in axial development of Xenopus embryos
can be arranged as a continuous series. The ground state (DAI 0) has no dorsal or anterior structures. As more
dorsal development occurs (DAI 1 to DAI 4), more anterior (head) structures appear, until normal embryos
(DAI 5) result. Overexpression of dorsal development leads to progressive loss of posterior dorsal structures
and gain of anterior structures (DAI 6 to DAI 9). This series culminates in an embryo with radial head structures
and a central beating heart (DAI 10). Only a few of these phenotypes have been noted in other vertebrates
(Elinson and Kao, 1993), but patterns of gastrulation suggest that comparable series should exist. (From Kao
and Elinson, 1988, by permission of Academic Press.)
cement glands as boundaries, we can divide tion to accomplish the transformation (Durthe embryo into specific zones, character- ston et ah, 1989). It has been suggested that
ized by whether they contain ciliated cells retinoic acid is present in a gradient with
(Fig. 2). As will be described, the boundaries the highest level at the posterior end, and
formed by the hatching and cement gland that this gradient imparts axial information
represent fundamental borders in the to the embryo (Cho and De Robertis, 1990;
Sive etai, 1990; Green, 1990; Balling, 1991;
embryo.
Ruiz i Altaba and Jessel, 1991).
HATCHING GLAND AND THE
A consequence of such a gradient would
HEAD/TRUNK BOUNDARY
be that addition of exogenous retinoic acid
Hatching gland cells are found only in the should cause posterior structures to be
head, and are unique in that they run from formed in more anterior positions (Sive et
the anterior tip of the head to the head/trunk ah, 1990). We, however, did not see any
boundary. During gastrulation, the invo- shift forward of the hatching gland pattern
luting head organizer is thought to induce when retinoic acid was applied to embryos.
the head along the entire dorsal axis, a pro- Rather, retinoic acid caused a suppression
cess termed activation. The head is then of the hatching gland cells leaving the Y
suppressed in the trunk by a transforming shape of the gland unaltered (Fig. 3) (Drysfactor, resulting in the posterior neural dale and Elinson, 1991). Retention of the
structures of the trunk (Nieuwkoop, 1952). normal Y pattern suggests that retinoic acid
Hatching gland may demarcate the bound- does not alter the size or folding movements
ary formed by this putative transforming of the anterior neural plate.
factor.
HATCHING GLAND AND THE
Retinoic acid is considered a good canNEURAL/EPIDERMAL BOUNDARY
didate for the transforming factor because
it can suppress anterior structures, and it is
The hatching gland also marks the boundpresent in sufficient levels during gastrula- ary between the anterior neural plate and
419
HEAD AND AXIS DEVELOPMENT IN FROGS
St.14
St.20
St32
FIG. 2. Face Morphogenesis in Xenopus. The morphogenesis of the face can be followed by observing the
formation of two transient glands, the hatching gland (HG) and the cement gland (CG). Although not visible
when the neural plate is open (St. 14), both HG and CG have been determined by this stage. Following neural
fold closure (St. 20), these glands surround a non-ciliated zone (NZ), known as the face plate, where the mouth
and olfactory pits form. The rest of the embryo's surface is covered with ciliated epidermis (CZ). (From Drysdale
andElinson, 1991.)
the surrounding ectoderm (Fig. 2). Because
our marker only detects hatching gland cells
after they differentiate, their presence at the
neural plate edge was determined experimentally. We removed surface cells from
the embryo, prior to the time when these
hatching gland cells were detectable, and
cultured them away from the embryo.
Hatching gland cells arose in explants from
the anterior, but not transverse, neural folds
(Fig. 2).
After neural fold closure, hatching gland
cells form a single line along the dorsal midline, due to the meeting of hatching gland
FIG. 3. Xenopus Hatching Gland. A. The hatching gland is visualized with an antibody to tyrosine hydroxylase
and appears as a Y. B. When axial development is inhibited (DAI 2/3, Fig. 1), the angle between the arms of
the Y narrows. This indicates that reduced dorsal development results in reduction of the anterior neural plate.
C. When embryos are treated with retinoic acid, the Y pattern remains intact with a reduction in number of
hatching gland cells. This result demonstrates that retinoic acid does not alter the shape of the anterior neural
plate, but inhibits differentiation of dorsoanterior tissues, like the hatching gland. (From Drysdale and Elinson,
1991.)
420
T. A. DRYSDALE AND R. P. ELINSON
FIG. 4. Head Reduction Target. The contour lines
indicate losses of head structures associated with each
level of axis deficiency on the dorsoanterior index (Fig.
1). At DAI 4, part of the transverse neural fold, which
contributes to the face plate, is removed. At DAI 3,
cement gland (CG) is reduced, and the embryo is
cyclopic due to the fusion of the retinal primordia (R).
At DAI 2, the transverse neural fold is removed, so
that the arms of the Y shaped hatching gland (HG) are
brought together (Fig. 3B). At DAI 1, all head structures
are lost.
cells from both sides of the embryo. Because
the stalk of the Y contains no gaps or spaces
and is only a few cells wide, the hatching
gland cells must be formed from cells that
are directly adjacent to the lateral edge of
the anterior neural plate. When neural fold
fusion is prevented, hatching gland cells still
appear on either side of the neural plate,
indicating that neural fold fusion is not
required for hatching gland development
(Drysdale and Elinson, 1991).
Presumptive hatching gland cells are first
found on the surface of the embryo at the
end of gastrulation (Drysdale and Elinson,
1991). Their appearance on the surface could
be due either to induction of the surface cells
or to migration of deep cells to a surface
position. To distinguish between these two
possibilities, we transplanted fluorescently
labelled surface cells onto an unlabelled
embryo, early in gastrulation. After the
embryos had healed and developed into a
tadpole, hatching gland cells were labelled,
indicating that hatching gland cells arose
from surface cells by induction (Drysdale
and Elinson, 1992).
Using the same methods employed for
analyzing the hatching gland, we showed
that ciliated cells migrate from the deep
ectoderm into the surface (Drysdale and
Elinson, 1992). Ciliated cells begin to adhere
to the surface layer at the end of gastrulation
but do not appear on the surface until the
neural folds are closing (Drysdale and Elinson, 1992; Chu and Klymkowsky, 1989).
The lack of ciliated cells along the dorsal
midline (Chu and Klymkowsky, 1989) and
in the face plate (Fig. 2) (Drysdale and Elinson, 1991), can be explained by the deep
cells of the dorsal midline and face region
being diverted from a ciliated cell fate.
Cement gland was found to be the product
of induced surface cells with a small contribution of deep cells (Drysdale and Elinson, 1992).
When there is less axial development (Fig.
1), the arms of the Y shaped hatching gland
come together, leading to a single line of
cells (Fig. 3). This narrowing results from
loss of the transverse neural fold, the most
anterior neural structure, which separates
the front of the Y (Fig. 4). This loss illustrates the interrelationship between the dorsoventral and anteroposterior axis.
CEMENT GLAND AND THE
HEAD/VENTRAL BOUNDARY
The cement gland lies between the face
and the ventral surface (Fig. 2) and represents the furthest anterior spread of neural
induction signals (Sive et ai, 1989). Despite
its location, cement gland is not the first
structure lost when axial development is
inhibited. Eyes and much of the face disappear, before the last vestige of cement
gland is gone (Fig. 4). This pattern of loss
suggests that structures along the transverse
neural fold, such as the anterior pituitary
and telencephalon (Eagleson and Harris,
1990), are actually the most dorsoanterior
ones.
Embryos with very large cement glands
and other head structures can be produced
experimentally. This syndrome, known as
macrocephaly arises in certain species
hybrids, such as when eggs of the mink frog,
Rana septentrionalis, are fertilized by sperm
of the bull frog, Rana catesbeiana (Fig. 5).
The reciprocal cross produces microcephaly, embryos with small heads (Elinson,
1977).
HEAD AND AXIS DEVELOPMENT IN FROGS
421
Fio. 5. The Macrocephalic Syndrome. A. Mink frog embryos (left) have small heads relative to macrocephalic
mink frog-bull frog hybrids (right). Note in particular the huge cement gland (CG) in the hybrid. B. The
macrocephalic hybrid (bottom) has a large head and a huge cement gland, secreting copious amounts of glue.
A mink frog embryo (top) is included for comparison. Scale lines: lmm. (From Elinson, 1991, by permission
of Academic Press.)
The head arises by inductive interactions,
so macrocephaly could be due either to the
inducing activities of the mesoderm or the
responding activities of the ectoderm.
Transplantations demonstrated that the
hybrid mesoderm is responsible (Elinson,
1991), and ectoderm from R. septentrionalis
can form large cement glands, when induced
by hybrid mesoderm. Cement gland inducing activity is more spread through the
hybrid mesoderm than in normal embryos,
since a large cement gland forms in the
hybrids, even when the dorsal axial mesoderm is eliminated (Elinson, 1991). This
analysis indicates that macrocephaly results
from alterations in inductive patterns.
forming the neural plate. Another possibility concerns frog embryos developing in
rapidly flowing streams rather than calm
water. The former may have more prominent cement glands, enabling them to attach
to rocks and not be swept away. A simple
way to form large cement glands would be
to change inductive patterns as in the mink
frog-bull frog hybrids.
At a more fundamental level, the experimental inhibition of head development may
mimic an evolutionary sequence. Vertebrates are hypothesized to represent protochordates to which a head has been added
(Gans and Northcutt, 1983; Langille and
Hall, 1989), so headless frog embryos superficially resemble protochordates. In embryPERTURBATIONS OF HEAD
ological terms, head evolution is viewed as
DEVELOPMENT AND EVOLUTION
the formation and expansion of prechordal
Head development can be increased or plate mesoderm with the concomitant
decreased experimentally, raising the ques- increase in anterior neural development
tion as to whether any of these develop- (Nieuwkoop and Sutasurya, 1983). There
mental patterns reflect evolutionary changes. are a variety of ways to inhibit head develFor instance, are alterations of inductive opment, so which, if any, suggest a recapitpatterns, found in experimentally produced ulatory relationship?
macrocephalic embryos, naturally present
Inhibition of axial development by UV
in any species of frog? This question has not irradiation (Malacinski et al, 1975) or by
been investigated, but there are obvious partial organizer ablation (Stewart and Gerplaces to look. One of these is the frog Lep- hart, 1990) to produce a headless DAI 1
idobatrachus laevis, whose tadpoles are car- embryo (Fig. 1), is not recapitulatory.
nivorous and have massive jaws (Ruibal and Because of the link between dorsal and anteThomas, 1988). This unusual allocation of rior development, DAI 1 embryos lack both
tissue to jaws may originate embryonically anterior head structures and notochord, the
due to alterations in inductive patterns most dorsal axial structure. An essential
422
T. A. DRYSDALE AND R. P. ELINSON
character of protochordates is presence of a
notochord, so axis deficient frog embryos
are not equivalent to protochordates.
Treatment with retinoic acid yields frog
embryos which lack heads but have a notochord (Durston et al., 1989), superficially
resembling a protochordate. Consideration
of retinoic acid treated embryos as a recapitulation model has two difficulties. First,
a head appears to form in these embryos
even though anterior structures are not visible. Treated embryos have an expanded
neural plate anteriorly (Papalopulu et al.,
1991) and a normal Y shaped hatching gland
(Fig. 3) (Drysdale and Elinson, 1991). These
patterns demonstrate that the transverse
neural fold at the anterior edge of the neural
plate is of normal size (Fig. 2). Second, most
hypotheses state that anterior neural induction requires one signal, whereas posterior
neural induction requires two signals
(Nieuwkoop, 1952; Saxen and Toivonen,
1962). It seems odd that the ancestral state,
represented by the posterior spinal cord,
requires more signals than the more recently
evolved forebrain.
Treatment with lithium after the blastula
stage also gives headless embryos with a
notochord (Backstrom, 1954; Yamaguchi
and Shinagawa, 1989). Embryos treated with
various doses of lithium may represent an
evolutionarily relevant sequence. Unlike
axial deficient embryos, anterior structures
are lost without loss of dorsal structures.
Unlike retinoic acid treated embryos, the
head is relatively smaller. Evolution and
enlargement of the prechordal plate has been
suggested as an important event in the evolution of the head (Nieuwkoop and Sutasurya, 1983), and lithium can inhibit both
responsiveness of ectoderm and development of prechordal plate mesoderm (Masui,
1960a, b). In addition, lithium affects the
IP 3 second messenger system (Berridge et
al., 1989). Protein kinase C, which is activated by the IP 3 second messenger pathway,
is implicated in induction of anterior neural
structures (Durston and Otte, 1991).
A HYPOTHESIS FOR THE
EVOLUTION OF THE VERTEBRATE HEAD
The following may represent a sequence
for the evolution of the head. Ancestral
organisms had the ability to induce posterior neural structures. Development of the
vertebrate head began with the evolution of
prechordal plate mesoderm which induces
anterior neural structures. One problem
faced by such an organism would be to
ensure that anterior neural structures are
only induced in the proper place, even
though the movements of gastrulation bring
the prechordal plate past presumptive posterior neural structures. This could be
accomplished by having another signal
which prevents anterior neural differentiation in the posterior region. The transforming factor of Nieuwkoop (1952), currently
thought to be retinoic acid, would fulfill this
role.
It is unlikely that an anterior inducing
signal and a transforming factor would
evolve simultaneously. The gastrulation
movements of Amphioxus (Conklin, 1932),
however, appear to prevent contact of anterior mesoderm with the overlying ectoderm
until it has completely involuted. This pattern of gastrulation may represent a simple
mechanical method used by ancestral chordates to prevent unwanted anterior neural
induction, prior to the evolution of a transforming factor. A transforming factor would
then have to evolve before the appearance
of more complex gastrulation patterns.
ACKNOWLEDGMENTS
We thank Rob Langille and Brian Hall
for inviting us to participate in this Symposium and NSERC, Canada for funding.
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