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779
Development 105, 779-786 (1989)
Printed in Great Britain © T h e Company of Biologists Limited 1989
Differential gene expression in the anterior neural plate during gastrulation
of Xenopus laevis
MILAN JAMRICH and SHERYL SATO
Laboratory of Molecular Genetics, N1CHD, National Institutes of Health, Bethesda, Maryland 20892, USA
Summary
We have isolated three cDNA clones that are preferentially expressed in the cement gland of early Xenopus
laevis embryos. These clones were used to study processes involved in the induction of this secretory organ.
Results obtained show that the induction of this gland
coincides with the process of neural induction. Genes
specific for the cement gland are expressed very early in
the anterior neural plate of stage-12 embryos. This
suggests that the anteroposterior polarity of the neural
plate is already established during gastrulation. At later
stages of development, two of the three genes have
secondary sites of expression. The expression of these
genes can be induced in isolated animal caps by incubation in lOmM-NHtCl, a treatment that is known to
induce cement glands.
Introduction
dorsal mesodermal cells form the chordamesoderm
(notochord and somites). This is called the primary or
mesodermal induction, though it is possible that other
inductive processes precede this step. In the process of
gastrulation, the involuting chordamesoderm induces
the overlying ectoderm to form the neural plate. This is
called secondary or neural induction. As O. Mangold
noticed in 1933, neural induction is not a uniform
process, because different regions of the archenteron
roof induce different structures in the overlying ectoderm. These structures are formed in a predetermined
order (head structures, forebrain, midbrain, hindbrain,
spinal cord), although it is not known at what stage of
development they are specified. The first morphological
signs of differentiation of the neural plate into subregions are observable during neurulation. At this time,
as the embryo begins to elongate, the neural folds
increase in elevation and subdivide the originally uniform plate into a sense plate, neural plate and gill
plates. In order to establish how early in development
the different regions of neural plate are specified, we
attempted to isolate molecular markers that would
allow a study of this question. In this paper, we describe
three cDNA clones that are suitable for the study of
cement gland development from the earliest stage of
formation of this organ. The cement gland (also called
adhesive organ, mucous gland, or sucker) is derived
from the outside ectodermal cells of the lower sensory
plate. It is an induced structure (Spemann & Schotte,
1932; Picard, 1975a,b), consisting of elongated columnar cells which secrete adhesive substances such as
The development of the metazoan organism is a continuous process involving the generation of new cell
types and their subsequent differentiation until the final
body plan is realized. In amphibians, the formation of
new cell types depends on the utilization of localized
maternal factors and on inductive interactions between
neighbouring cells. Although most of the developmentally important maternal components are likely to be
uniformly distributed in the egg, there is good evidence
that at least some of them are localized (Bonoure, 1934;
Spemann, 1938). More recently, Melton (1987) demonstrated the presence of localized maternal RNA in the
vegetal pole of developing Xenopus oocytes. This RNA
is transcribed from a gene which shows similarity to the
TGF-beta gene family (Weeks & Melton, 1987). Members of this gene family are likely to play an important
role in mesoderm formation (Kimmelman & Kirschner,
1987; Rosa et al. 1988). The data of Sargent et al. (1986)
and Jamrich etal. (1987) suggest the presence of a
localized maternal component in the animal hemisphere of Xenopus embryos which regulates the synthesis of keratin genes in the embryonic ectoderm.
Embryonic induction has been one of the most
popular topics for study in the amphibian embryo since
its discovery by Spemann & Mangold in 1924. Embryonic induction can be divided into at least two major
phenomena. The first is the induction of mesodermal
cells by vegetal cells, primarily in the dorsal but also
including the lateral and ventral marginal zone. The
Key words: cement gland, gastrulation, in situ
hybridization, neural induction, NH4CI induction, Xenopus
laevis.
780
M. Jamrich and S. Sato
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Fig. 1. (A) Localization of XCG 2, XCG 7, and XCG 13 RNA in dissected stage-24 Xenopus embryos. Northern blot
analysis of equal amounts of RNA (1 jig per lane) using nick-translated XCG 2, XCG 7, and XCG 13 probes. In every case,
the corresponding RNA is present in the head region only. (B) Localization of XCG 2, XCG 7, and XCG 13 in dissected
regions of stage-24 Xenopus heads. Northern blot analysis of equal amounts of RNA (1 jig per lane) using nick-translated
probes. RNA corresponding to the XCG 2, XCG 7, and XCG 13 is present in the cement glands.
mucoproteins (Eakin, 1963). It is homologous to the
balancers of urodeles and it is the first organ in Xenopus
embryos to become functionally differentiated. Knowing the exact timing of cement gland induction would
help to establish when in development the regional
identity of the original neural plate is specified. Since
the cement gland is not morphologically noticeable
before stage 15, we monitored the expression of
cement-gland-specific RNAs as an indicator of tissue
differentiation.
The study of cement gland induction offers additional
advantages. Although inductive phenomena are frequently studied, only a few experimental models of
induction exist that deal with a homogeneous cell
population and the cement gland is one of them. In
contrast to mesodermal and neural induction, which
result in the formation of a variety of tissues, cement
gland induction represents a homogeneous transition
from one cell type into another. Furthermore, this
transition can be achieved by treatment of the isolated
animal caps of gastrulae in 10 mM-NtLjCl, which causes
the outside ectodermal cells to differentiate into cement
gland cells (Picard, 1975a,b). The ability to study this
inductive process in explants makes this system especially suitable for biochemical analysis.
Materials and methods
Preparation of RNA
RNA was prepared as previously described by Sargent et al.
(1986). Poly(A)+ RNA was purified according to Aviv &
Leder (1972). In the plus-minus screening, single-stranded
32
P-cDNA probes were prepared by using Moloney murine
leukemia virus (Mo-MLV) reverse transcriptase (Sargent,
1988).
Filter blotting and hybridization
RNA electrophoresis and Northern blot hybridization was
performed as previously described (Bailey & Davidson, 1976;
Church & Gilbert, 1984). Filters were washed as described in
LaFlamme et al. (1988).
Gene expression in the neural plate during gastrulation
781
B
CG
Fig. 2. (A) In situ hybridization of XCG 2 antisense transcripts to a stage-34 embryo viewed with dark-field optics.
Hybridization is to the cement gland. The plane of section is indicated in B. (C) A phase-contrast view of the same section;
B, brain; CG, cement gland; E, eye. (D) In situ hybridization of XCG 7 probe to a sagittal section of a stage-25 embryo.
Though most of the hybridization is to the cement gland, secondary hybridization to the olfactory pit can be observed
(arrow). (E) A phase-contrast view of the section in Figure D; CG, cement gland; O, olfactory placode; P, pharynx. (F) In
situ hybridization of XCG 13 probe to a cross section of a stage-34 embryo. The plane of section is indicated in B.
Hybridization is to the cement gland. Arrows indicate the concentration of pigment in the embryonic eyes. These areas
diffract light in darkfield, but do not show any real hybridization. (G) Phase-contrast picture of Figure F; CG, cement gland;
E, eye; N, notochord; P, pharynx.
782
M. Jamrich and S. Sato
Fig. 3. (A) In situ hybridization of a clone XCG 7 probe to a section of a stage-12 embryo. This dark-field view shows the
hybridization to the anterior portion of the neural plate (arrow). (B) Phase-contrast view of the section of stage 12. Boxed
area shows the region enlarged in A; A, archenteron; BL, blastocoel; Y, yolk plug. (C) A higher magnification of the neural
plate to show the position of the future brain; B, brain. The apparent signal in the archenteron floor is due to trapped air
bubbles and not to silver grains.
In situ hybridization
The hybridization procedure used is a modification of existing
protocols (Angerer & Angerer, 1981; Akam, 1982; Jamrich
etal. 1984; Ingham etal. 1985; Kintner & Melton, 1987).
Albino embryos were dejellied in 2% cysteine pH7-8 and
fixed in 4 % paraformaldehyde in phosphate-buffered saline
(PBS) for 30min. They were dehydrated in an ethanol series
(70%, 95%, 100%), cleared in xylene and embedded in
paraffin (Paraplast-Plus with DMSO). 5/an sections were cut
and transferred to a drop of water on a polylysine-coated
slide. After sections had dried down and attached to the slide,
they were incubated in xylene to remove paraffin and rehydrated through an ethanol series. Slides were incubated in
2xSSC for 30min at 65 °C, treated with Proteinase K
(2//gmr 1 in 10mM-Tris-HCl pH7-5, 5mM-EDTA) for
15 min at 37°C. Digestion was terminated by placed slides in
2mgml~1 glycine in PBS for lmin. Slides were washed in PBS
and acetylated (Hayashi et al. 1978). They were washed in
2 X SSC, dipped in water and hybridized overnight with 35Slabelled transcripts (Green et al. 1983). Hybridization was
carried out in 50 % formamide, 5 x SSC, 0-1 M-sodium-potassium phosphate pH7-0, 1 x Denhardt's solution, 5 % dextran
sulphate, 100mM-dithiothreitol (DTT), lOOfigmr1 E. coli
tRNA at 50°C under glass coverslips. Typically 7/il hybridization solution was used per slide containing 300000 cts min"1.
Coverslips were sealed with rubber cement. After hybridization, rubber cement was manually removed and the coverslips were floated off in 2 x SSC and 10mM-DTT. Slides were
incubated in this solution for 2 h and then in 50 % formamide,
1 x SSC, lOmM-DTT for lh at 50°C. Sections were treated
with RNase A (2/igmP 1 in 2 x SSC for 30min at 37°C),
washed in 2 x SSC for 2h, dipped in water and covered with
autoradiographic emulsion (Kodak NTB-2 diluted 2:1 with
water). Slides were typically exposed for 2-10 days, developed in D-19 developer for 60 s at 18°C,fixedin Rapid Fix for
2 min and rinsed in water. They were dried and viewed in a
microscope using dark-field optics or stained with Giemsa
using standard procedures.
NH4Cl induction
Animal caps of stage-10 embryos were dissected and incubated for 6h in lOmM-NKtCl. They were transferred into
67 % L15 medium (GIBCO) and collected 12 h later for RNA
preparation and Northern blot analysis. Staging of embryos
was done according to Nieuwkoop & Faber (1967).
Results
Cement-gland-specific cDNA clones were obtained as
Gene expression in the neural plate during gastruladon
neural plate
783
notochord
i
B
endoderm
Fig. 4. In situ hybridization of XCG 13 probe to a section of a stage-17 embryo. The plane of section is indicated in B;
A, anterior; P, posterior.
part of an experiment designed to isolate cDNA clones
specifically expressed in the head region of Xenopus
embryos. Heads were separated from the trunks of
stage-24 embryos, and RNA was prepared from both
fractions. 32P-labelled cDNA was prepared from both
RNA preparations and duplicate filters of a Xenopus
neurula cDNA library (Richter et al. 1988) were
screened using these probes. Plaques hybridizing with
head cDNA but not trunk cDNA were isolated and
purified. Head region specificity was confirmed by
hybridizing the isolated clones to Northern blots of
head and trunk RNA. Fig. 1A shows three clones
preferentially hybridizing to head RNA. Clone XCG 13
hybridizes to a very large RNA. The hybridization
signal appears smeared in all our RNA preparations.
We presume that this is due to the partial degradation of
this RNA; however, it is possible that this clone
hybridizes to multiple transcripts. Further analysis of
the expression of these clones by microdissection of the
head region into brain, eyes and cement gland revealed
that they are preferentially expressed in the cement
gland (Fig. IB). More detailed analysis by in situ
hybridization to sections of embryos confirmed that all
three clones are predominantly expressed in the cement
gland (Fig. 2). We hybridized these clones to embryos
of progressively earlier stages of development to determine when and in what region these clones are first
activated. The earliest detectable hybridization was
limited to the anterior neural plate of stage-12 embryos
(Fig. 3). This is an important result as it shows that the
different regions of the neural plate are already specified during gastrulation, well before the different regions can be recognized as morphological entities. A
few hours later, at stage 17, the hybridization is restricted to the anterior ventral region of the embryo in what
can now be recognized as the lower part of the sense
plate (Fig. 4).
Whereas clone XCG 13 appears to be expressed
exclusively in the cement gland throughout development, secondary sites of expression were observed for
clones XCG 2 and XCG 7 in older embryos (past stage
25). At least some of these expression sites were
common to both of the genes. The most prominent
secondary sites of expression were the pharynx (with
the highest expression in the branchial arches)
(Fig. 5A), a part of the olfactory placode that appears
to be the olfactory pit (Fig. 2D), an endodermal region
between the pharynx and heart mesoderm which is
probably involved in the formation of the trachea or
esophagus (Fig. 5C), and the ear vesicle (not shown).
One of the requirements for understanding the molecular detail of the processes involved in inductive
phenomena is the availability of defined model systems
for induction. Induction of the cement gland was
achieved by treating microdissected animal caps of
Xenopus gastrulae in a solution of 10 mM-NI-LtCl for few
hours. Fig. 6 shows a strong activation of the gene
XCG 7 in the treated caps using Northern blot analysis
of isolated RNA. The other two genes (XCG2 and
XCG13) are activated by this treatment as well (not
shown). In contrast, transcription of the epidermal
cytokeratin gene XK 81 (Jonas et al. 1985; Miyatani et
al. 1986; Jamrich et al. 1987) declined after this induction period (Fig. 6).
Discussion
We have studied the expression of three genes preferentially transcribed in the cement gland during the embry-
784
M. Jamrich and S. Sato
B
Fig. 5. (A) 7n sita hybridization of XCG 7 probe to a section of a stage-38 embryo. Hybridization is to the pharyngeal
arches. Arrow shows the accumulation of pigment in the eye. There is no hybridization to this region. (B) Phase-contrast
view of the same section; E, eye; NT, neural tube; P, pharynx. (C) In situ hybridization of XCG 7 to a section of a stage-28
embryo. Arrow indicates the pigment in the neural crest cells. Most of the hybridization is to the pharynx with a high
concentration of grains dorsal to the heart mesoderm. (D) Phase-contrast view of the same picture; H, heart; N, notochord;
NT, neural tube.
onic development of Xenopus laevis. We found that
these genes are initially expressed at stage 12 (gastrula)
in the anterior neural plate. This result suggests that the
induction of this gland coincides with, or is part of,
neural induction. Furthermore, our results demonstrate
that gene expression specific for the anterior neural
plate is already in progress during gastrulation. We do
not see expression of these genes in the ectoderm prior
to the contact of ectoderm with chordamesoderm,
suggesting that the chordamesoderm is inducing the
transcription of cement-gland-specific genes in the ectoderm. This agrees well with transplantation exper-
iments by Spemann & Mangold (1934) and Mangold
(1933), suggesting that the contact of mesoderm and
ectoderm is necessary to induce the ectoderm to form
neural structures. More recently, it was shown that the
entire ectoderm is programmed to express epidermisspecific products unless it is prevented from doing so by
contact with chordamesoderm (Jones & Woodland,
1986; Jamrich et al. 1987). It was also demonstrated that
N-CAM, a neural-specific marker, is not synthesized in
presumptive neural ectoderm before induction by chordamesoderm (Kintner & Melton, 1987). However, the
presumptive neural ectoderm might not be totally
Gene expression in the neural plate during gastrulation
S o
o
Q
o
i- o° ?9 °o
XCG7 XK81
Fig. 6. Induction of XCG 7 gene by treatment of animal
caps with NH4Cl. Northern blot analysis of RNA from
NH4Cl-treated caps shows a strong induction of
transcription of this gene whereas cytokeratin XK 81 RNA
accumulation is suppressed.
naive. It was shown that the dorsal ectoderm is more
easily induced to transcribe XlHbox6, a homeoboxcontaining neural-specific gene, and the N-CAM gene
than the ventral ectoderm (Sharpe etal. 1987). Similarly
London et al. (1988) showed that the dorsal ectoderm is
biased in its ability to express Epi 1 gene, an epidermisspecific marker. It is not clear at present what developmental significance these differences have in the embryo since it appears that this bias is neither sufficient
nor necessary for the formation of neural tissue.
The expression of the cement-gland-specific genes at
stage 12 can be visualized in the region immediately
posterior to the leading edge of the chordamesoderm,
suggesting that this induction requires only a brief
contact between the mesoderm and ectoderm. The
expression of these genes is limited to a very defined
region, implying that the induction process taking place
during gastrulation is already imprinting a region identity on the overlying ectoderm. This specificity is immediately translated into regional differences in gene
expression, although how this is accomplished is not
easily understood.
In the more posterior regions of the neural plate,
which have also had contact with the leading edge of
chordamesoderm, the expression of the cement-glandspecific genes was not observed. The following expla-
785
nations are possible: (1) the mesoderm was not transmitting the signal to the overlying ectoderm; (2) the
ectoderm was not ready to receive the signal; (3) the
signal was transmitted but immediately negated by
additional signals coming from more posterior mesoderm; (4) only a certain area of ectoderm is predetermined to become cement gland and the contact with
archenteron roof simply provides an initiating signal for
this expression. It is unlikely that the fourth alternative
is correct, since it was previously shown that any
ectoderm can form a cement gland if properly induced
(Spemann & Mangold, 1924; Spemann & Schotte,
1932). Furthermore, induction experiments by Picard
(1975a,b) showed no difference between dorsal and
ventral ectoderm in the ability to form cement gland if
properly induced; uninduced animal caps will not form
cement glands to any significant degree. Our experiments (Fig. 6) confirm these observations.
While at stage 12 the expression of the cement-glandspecific genes is in the anterior dorsal region, at stage 17
the expression is more ventral. This suggests that the
cells expressing these genes move ventrally in the
process of elongation of the embryo during neurulation.
Alternatively, the cells expressing these genes at
stage 12 and 17 are not identical. It is possible that the
hybridization signal at stage 12 visualizes a transient site
of expression (see discussion above).
After stage 25, clones XCG 2 and XCG 7 are also
expressed in additional tissues, such as pharynx, esophagus, ear vesicle and olfactory pit. These tissues are
not derived from the same germ layer and there is no
reason to assume that they are of common origin. Most
likely these tissues share a common physiological function or have similar physiological requirements. The
clones XCG 2 and XCG 7 may be useful as markers for
the induction of these tissues.
One of the most exciting aspects of the study of
cement gland induction is the ability to induce this
structure by incubating the animal caps of gastrulae in
lOmM-NELtCl. This treatment results in the expression
of all three cement-gland-specific genes. At the same
time, we observe a reduction in the expression of
epidermal cytokeratin gene XK81. This agrees well
with our previous finding that cytokeratin genes are
turned off in the neural plate during the process of
induction (Jamrich et al. 1987). In the future, we hope
to use this induction system to isolate genes which are
activated prior to the genes described here. Such genes
might provide us with information about the chain of
events involved in the process of induction.
We would like to thank Igor Dawid, Susan LaFlamme,
Klaus Richter and Tom Sargent for their advice and stimulating discussion and Kathi Mahon for a critical reading of this
manuscript.
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