Download PDF

J. Embryol. exp. Morph. 91, 153-168 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
153
Localization of specific mRNA sequences in Xenopus
laevis embryos by in situ hybridization
EVA DWORKIN-RASTL, DARCY B. KELLEY AND MARK B.
DWORKIN
Department of Biological Sciences, Sherman Fairchild Center, Columbia University,
New York, NY 10027, USA
SUMMARY
In situ hybridization of cloned cDNA probes to frozen sections of Xenopus laevis stage-42
tadpoles has been used to determine the tissue localization of several mRNAs. Nine out of
sixteen probes tested hybridized to most or all tadpole tissues; seven probes exhibited tissuespecific hybridization. The non-tissue-specific sequences hybridized to RNA species that are
also present in maternal RNA while the tissue-specific sequences hybridized to embryonic RNA
species induced after gastrulation and undetectable in maternal RNA. Tissue-specific hybridization was observed with muscle (five clones), epidermis (one clone), and the nervous system
(one clone). All muscle-specific sequences hybridized to somites and lateral plate muscles, but
they differed in their hybridization to heart muscle.
INTRODUCTION
In situ hybridization to tissue sections has become a widely used method
to determine the tissue localization of mRNAs (Harding et al. 1977; Venezky,
Angerer & Angerer, 1981; Scheller et al 1982; Akam, 1983; Edwards & Wood,
1983; Gee et al. 1983; Hafen, Levine, Garber & Gehring, 1983; Levine, Hafen,
Garber & Gehring, 1983; Zimmerman, Petri & Meselson, 1983; Cox, DeLeon,
Angerer & Angerer, 1984; Jamrich, Mahon, Gavis & Gall, 1984). The technique is
especially powerful in the study of embryos where RNA cannot easily be prepared
from single tissues due to the difficulty of dissection and the small amounts of
material obtained. In contrast, by in situ hybridization the localization of several
different sequences can be determined with a single embryo. We used in situ
hybridization to X. laevis embryonic sections to characterize several mRNA
species with respect to their tissue distribution.
Mature eggs of X. laevis contain a large amount of mRNA (maternal mRNA).
This RNA is synthesized during oogenesis and it is used, at least in part, to support
protein synthesis in the early embryo. RNA synthesis does not occur in the embryo
until the midblastula stage (Newport & Kirschner, 1982). At the onset of neurulation, increases in the titers of many RNA species are observed, as well as the
appearance of qualitatively new RNA species (Dworkin & Dawid, 1980b;
Dworkin & Hershey, 1981; Dworkin, Shrutkowski, Baumgarten & DworkinKey words: in situ localization, Xenopus development, cDNA clones, mRNA, hybridization.
154
E. DWORKIN-RASTL, D. B. KELLEY AND M. B. DWORKIN
Rastl, 1984). These RNA species persist for the next two days of development,
during which time no further burst of this magnitude of qualitatively new RNA
synthesis is observed (Dworkin & Dawid, 19806). These measurements were done
by colony hybridization, dot blot, and Northern blot hybridization and reflect the
behaviour of moderately abundant to abundant sequences. RNA species present
at moderate abundance in total embryonic RNA may be present at moderate copy
numbers in all cells of the embryo or at high copy numbers in a particular tissue or
cellular subtype. These patterns can be distinguished by in situ hybridization
autoradiography.
Those mRNA species whose titers increase dramatically at the beginning of
neurulation are candidates for mRNAs coding for structural proteins specific to
the developing organ systems of the embryo. We selected cloned sequences with
such a developmental expression pattern, derived from X. laevis embryonic libraries (Dworkin & Dawid, 1980a), as probes for the in situ hybridization experiments. The selection included eight sequences that can already be found (in lower
titers) in maternal mRNA, as well as seven sequences found only in embryonic
mRNA after gastrulation. All sequences of this latter group exhibited tissuespecific localization.
MATERIALS AND METHODS
Preparation of embryo sections
Xenopus laevis stage-42 wild-type embryos or stage-35/36 albino embryos were fixed for
30min at room temperature in 4% formaldehyde in PBS (130mM-NaCl, 7mM-Na2HPO4,
3mM-NaH2PO4), with occasional agitation. Then 1 volume of 15 % sucrose (w/v, in H2O) was
added and the fixation continued for a further 15min. Several embryos were aligned in TissueTek II embedding medium (Miles Laboratories) within a hemicylindrical mould and frozen on
dry ice. The frozen embryos were stored at -70°C for up to several months and used for cutting
as needed. Cryostat sections (10 to 12pan) were cut at -18°C to -22°C, collected on subbed
slides (Gall & Pardue, 1971), air dried, and subsequently fixed onto the slides by immersion in
4 % formaldehyde in PBS for 20min, followed by one rinse in 3xPBS and two rinses in lxPBS,
5 min each. The sections were then dehydrated in an ethanol series of increasing concentration
(30 %, 60 %, 80 %, 95 %, 100 %; 5 min each) and air dried. They were stored at -70°C in slide
boxes containing drying material, until needed for hybridization (up to 2 months).
Preparation of the sectioned tissue for hybridization by HC1 and pronase treatment was done
as described by Hafen et al. (1983), except that for fixation formaldehyde was used instead of
paraformaldehyde.
Preparation of hybridization probes
Circular plasmid DNA was labelled with 3H-TTP by nick translation (Rigby, Dieckmann,
Rhodes & Berg, 1977). A typical 10//I reaction contained 5 ^Ci 3H-TTP (Amersham, ca. 100 Ci
mmol"1; dried in a Speedvac), about 50ng plasmid DNA, IOJUM each of dATP, dCTP, dGTP
(Boehringer/Mannheim), 50mM-Tris-HCl, pH7-8, 5mM-MgCl2, 10mM-/?-mercaptoethanol,
5ng-DNAase I (Miles Laboratories) and 2 units E. coli DNA polymerase I (New England
Biolabs). In addition, 1 to 2[id 32P-dCTP was added to the reaction to facilitate monitoring of
the DNA with Geiger-counter and X-rayfilm-autoradiography.After incubation for 2 h at 14 °C
the reaction was stopped by the addition of 10mM-EDTA and 5jug salmon sperm carrier DNA.
The enzymes were denatured by incubation at 70°C for 10 min. The DNA was separated from
unincorporated label on Sephadex G50 in 0-lxSET (lxSET= 150mM-NaCl, 5mM-EDTA,
50mM-Tris-HCl, pH8) and brought to lxSET. Yeast carrier RNA (250^, i.e. the amount of
Ill situ hybridization to X. laevis embryos
155
carrier RNA needed for 500 fA hybridization solution) was added and the nucleic acids
precipitated with 2-5 volumes of 95 % ethanol. The precipitate was pelleted, washed once with
70% ethanol, dried under vacuum and dissolved in 30^1 H2O. To determine the mean single
strand length of the nick-translated DNA, a denaturing 8% polyacrylamide gel (DNAsequencing gel formula, Maxam & Gilbert, 1980) was run with 0-5 jul sample and Hae III cut
pBR322 as marker, and analysed by autoradiography of the 32P-labelled fragments. The desired
single-stranded probe length (50-250 bases) was obtained in most experiments. It is necessary to
determine for every batch of DNAase I the amounts required in nick-translation reactions to
achieve optimal probe size. The specific activity (3H) of the probe was about 2xl0 7 c.p.m. jug"1.
Hybridization solutions contained (in a volume of 500 jul) 50 ng nick-translated DNA, 0-5 mg
ml" 1 yeast carrier RNA, 50% formamide (recrystallized at 0°C and deionized), 10 mMTris-HCl, pH7-5, 0-6M-NaCl, lmM-EDTA, O-Smgml"1 poly(U) (Sigma), lxDenhardt's
solution (Denhardt, 1966), and 10% dextran sulphate (Sigma, Mx 500000).
Hybridization of3H-labelled DNA to tissue sections
This step was carried out as described by Hafen et al. (1983). Briefly, the hybridization
solution was heated to 100 °C for 1-2 min to denature the DNA, 50 fA of solution were applied to
each slide and covered with 22x40 mm coverslips. The edges of the coverslips were sealed with
rubber cement to prevent evaporation. The slides were incubated in a humid chamber at
34-36°C for 36-40 h. After hybridization, the slides were washed in 50% formamide (Fisher
F-82), 10mM-Tris-HCl, pH7-5, 0-6M-NaCl, 1 mM-EDTA at 35°C for 20h, with four changes of
solution. The slides were dehydrated in an ethanol series containing 0-3M-ammonium acetate,
pH70, air dried, and dipped in Kodak NTB-2 emulsion, diluted 1:1 with 0-6M-ammonium
acetate, pH 7-0. Exposure was for 2-8 weeks at 4°C in a dry chamber; developing time in Kodak
D-19 developer was 2 min. The sections were stained with methylene blue and scanned under
darkfield and brightfield illumination using a Zeiss Universal microscope to identify radiolabelled tissues.
Genomic DNA blots
Genomic DNA, prepared either from adult Xenopus laevis liver or from total X. laevis embryos, was digested to completion with Eco RI or Hind III, separated on 0-8 % agarose gels
(15 ng per gel lane) and transferred to nitrocellulose (Southern, 1975). Filter hybridizations with
nick-translated plasmid DNA of a specific activity of 2-5xl0 8 c.p.m. jug"1 (Rigby, Dieckmann,
Rhodes & Berg, 1977) were carried out at 37°C as described in Dworkin et al. (1984).
RESULTS
A number of cDNA cloned probes from X. laevis embryonic libraries were
selected for an in situ localization study of specific tadpole mRNAs. The 16 clones
chosen for this study are listed in Table 1. They originated from a gastrula or a
stage-42 tadpole library (Dworkin & Dawid, 1980a). As indicated in Table 1, all
but one of these sequences (C20, named according to Dworkin & Dawid, 1980ft)
show an increased liter in embryos after the first half day of development
(Dworkin & Hershey, 1981; Dworkin et al. 1984) and are examples of moderately
abundant mRNA sequences in postneurulation embryos (ranging from about
20pg to more than 100 pg per stage-37 tadpole; Dworkin & Hershey, 1981).
Sequences with this developmental expression pattern were chosen since they are
likely to code for tissue-specific structural components of the developing embryo.
Sequence C20, although it does not increase in mRNA titer during development
(Dworkin & Hershey, 1981), was included in this study because its coding capacity
156
E . D W O R K I N - R A S T L , D . B . K E L L E Y AND M. B .
DWORKIN
Fig. 1. Hybridization of a non-tissue-specific clone to sections of stage-42 embryos.
(A) Photomicrograph of a transverse section through the gut region of an embryo,
hybridized with probe G17. Sections were stained with methylene blue. Scale bar
equals 100 \ivs\. (B) Same section as in A, darkfield illumination. Note the high density
of labelling over all tissue types: e.g. spinal cord, somite and epidermis. Cells of the
notochord do not survive the histological procedures. (C) Control section, at a level
similar to that shown in A and B, hybridized with labelled plasmid pBR322 DNA not
containing a cDNA insert. Note the reflectance of melanocytes in the epidermis and
dorsal to the spinal cord and in the coelomic wall. Cells of the gut are not present in this
particular section. No non-specific hybridization of plasmid sequences to embryonic
tissue is seen. (D,E) Bright- and darkfield photomicrographs of a section through the
embryonic head region, hybridized with probe G17. Scale bar equals 100 jum.
Abbreviations: di, diencephalon; e, epidermis; g, gut; n, notochord; oc, optic cup; ph,
pharynx; s, somite; sc, spinal cord.
In situ hybridization to X. laevis embryos
157
has been determined to be ubiquitin (Dworkin-Rastl, Shrutkowski & Dworkin,
1984).
(A) Clones representing rnRNAs without tissue-specific localization
Nine of the sixteen clones included in this study did not show tissue-specific
localization. An example of a sequence which hybridized to all tissues in stage-42
tadpoles is shown in Fig. 1. Panels A and B are the brightfield and the corresponding darkfield photomicrographs of a transverse section through the gut
region of a stage-42 tadpole, hybridized with probe G17. In the darkfield photomicrograph hybridization to somites, spinal cord and epidermis can be seen. An
autoradiograph of a section through the head region of an embryo hybridized with
G17 is shown in Fig. 1D,E. Again, hybridization to all tissues is observed.
One difficulty in viewing such autoradiographs is the presence of pigment
granules in the embryo which resemble the grains in the emulsion. These pigment
granules, like emulsion grains, reflect light under darkfield conditions. Control
hybridizations with pBR322 of adjacent sections were routinely performed in
every experiment to localize these pigment granules (Fig. 1C, a section neighbouring the one shown in Fig. 1A,B). There is an area of strong pigmentation
surrounding the dorsal half of the spinal cord, and there are pigment patches
throughout the epidermis and the coelomic wall. A high density of isolated
granules can usually be found in the gut (not seen in Fig. 1C, because the gut
was lost in this particular section). Hybridization with pBR322 also served to
determine the level of non-specific probe retention in these experiments which
usually was negligible.
(B) Clone A4, hybridizing specifically to epidermis
The in situ hybridization of clone A4 to embryonic sections is shown in Fig. 2.
Panels A,B,C represent sections through the tail and gut regions of stage-42
tadpoles, hybridized with A4; labelling of the epidermis can be seen. In Fig. 2A,B
brightfield and darkfield photomicrographs of the same section are shown. Since
pigmentation varies between different animals and between different regions
in one animal, corresponding control sections, hybridized with pBR322, are
displayed (Fig. 2E,F,G). Sections from other regions of the embryo showed
similar hybridization to epidermis. To exclude completely any effects of pigment
on the visualization of the A4 hybridization pattern, the experiment was repeated
with albino embryos of stage 35/36. The results are shown in Fig. 2D. In albino
embryos hybridization is also epidermis-specific. Control sections, hybridized with
pBR322 (Fig. 2H) are completely free of pigmentation. The diffuse reflection of
light by the embryonic tissue, which is always seen, is easily distinguished from
autoradiographic grains under the microscope.
(C) Clone D8, hybridizing specifically to nervous tissue
The hybridization of clone D8 to sections of stage-42 tadpoles is illustrated in
Fig. 3. In a section through the head, at the level of the eyes, hybridization to
158
E. DWORKIN-RASTL, D. B. KELLEY AND M. B. DWORKIN
the brain (mesencephalon and diencephalon) and to neural retina can be seen
(Fig. 3A,B)- At the level of the otic vesicles, hybridization to the metencephalon
and to cranial nerve ganglia 8 and 9 is apparent (Fig. 3C,D). A higher magnification of a section showing hybridization of D8 to cranial nerve ganglion 7 is
shown in Fig. 3E,F. Thus, D8 hybridizes to derivatives of the neural tube (brain,
retina) as well as to neural crest derivatives (ganglia). D8 does not hybridize to
non-neural neural crest derivatives (e.g. head cartilage). Hybridization of D8 is
also seen with cells of the olfactory pit, i.e. cells of placodal origin (data not
shown). In the absence of additional markers (e.g. antibodies to glia-specific
proteins) we cannot state whether or not D8 hybridizes to glia.
(D) Clones hybridizing specifically to muscle tissue
Five of the sixteen clones we examined hybridized selectively to muscle tissue
(A2, B5, B9, D l , H2). The hybridization pattern of D l to sections through the tail
and gut region of stage-42 tadpoles is shown in Fig. 4, panels A,B, and C,D. These
autoradiographs are representative for all five muscle-specific clones we have
characterized and show intense hybridization to the somites. Sequence B5
hybridized with a slightly lower intensity than the other four sequences. All five
clones also hybridized to smooth muscle; an example is shown in Fig. 4E. This
photomicrograph shows the skin over the gut and the underlying lateral plate
muscles of a section hybridized with sequence A2. In Fig. 4F a control hybridization with pBR322 is shown.
The five muscle-specific clones exhibited significant differences in their hybridization to heart muscle. In Fig. 5, in situ hybridizations of clones A2, D l and H2 to
sections through the heart region of stage-42 tadpoles are displayed (Fig. 5A,D;
Fig. 5B,E and Fig. 5C,F, respectively). Hybridization to somites is observed in all
cases. Branchiomeric muscle, present in the sections in Fig. 5A,B laterally to the
heart, also shows hybridization as expected. Nevertheless, hybridization to heart
muscle is below the level of detection with clone A2 (Fig. 5A,D). Clone D l
(Fig. 5B,E) does hybridize to heart muscle, but less intensely than it does to
skeletal muscle. Sequence B5 (not shown) did not hybridize detectably with heart
muscle, but because it hybridized less intensely to somites than the other four
sequences its lack of signal with heart muscle could place it into a class with either
Fig. 2. Photomicrographs of embryonic sections hybridized with probe A4 (A-D) or
pBR322 DNA (E-H). (A,B) Section through the cloacal region of a stage-42 embryo,
hybridized with A4. Scale bar equals 100 (im. Note hybridization, apparent with
darkfield illumination, to the epidermis. (E,F) Control section, hybridized with
pBR322 DNA. The presence of melanocytes in the epidermis givesriseto patches of
reflectance, unlike the even reflectance due to silver grains overlying the epidermis
after hybridization with probe A4 (A,B). (C) Transverse section through the gut
region of a stage-42 embryo, hybridized with A4. Compare with control section,
hybridized with pBR322 DNA, in Fig. 2G. (D,H) Adjacent sections through the gut
region of a stage-35/36 albino embryo. Albino embryos were used because they have
less pigment. The epidermal localization of probe A4 is clearly visible (compare D,
hybridized with A4, with H, hybridized with pBR322 DNA). Abbreviations: c, cloaca;
e, epidermis; n, notochord; sc, spinal cord.
In situ hybridization to X. laevis embryos
2AV
—
159
160
E . D W O R K I N - R A S T L , D . B . K E L L E Y AND M. B .
DWORKIN
Fig. 3. Hybridization of probe D8 to cells of the nervous system of stage-42 embryos.
(A,B) Section through the head region at the level of the eyes. Hybridization to cells of
the central nervous system and neural retina is apparent. Scale bar equals 100 jum.
(C,D) Section through the head region at the level of the otic vesicles. Hybridization to
cells of the metencephalon and to cranial nerve ganglia 8 (acoustic-vestibular) and 9
(vagal-lateralis) is apparent. Scale bar equals 100 jan. (E,F) Hybridization to cells of
the ganglion of cranial nerve 7 (facial). Scale bar equals 20jian. Abbreviations: di,
diencephalon; mes, mesencephalon; met, metencephalon; n, notochord; ov, otic
vesicle; ph, pharynx; 7, 8, 9, ganglia of cranial nerves 7, 8, 9.
Fig. 4. Hybridization of muscle-specific probes Dl and A2 to sections of stage-42 embryos. (A,B) Probe Dl hybridizes intensely to tail
muscle in the caudal end of the embryo. Note the lack of hybridization to neural tissue (spinal cord) and epidermis. Scale bar equals 100 jum.
(C,D) More anteriorly, Dl hybridizes to the somites which are largely myogenic at this point. Scale bar equals 100 jum. (E) Hybridization of
probe A2 to smooth muscle beneath the epithelium overlying the gut. Pigment granules in the epidermis are visible. Scale bar equals 20 jum.
(F) Control hybridization of an adjacent section with pBR322. Abbreviations: e, epidermis; g, gut; n, notochord;/?, pigment granules; s,
somite; sc, spinal cord; sm, smooth muscle; tm, tail muscle.
O\
I
X
3.
'.
)
V
_
••
•
•,.•>••
,.
r.'..
.• 1
'•-•
E •r
•
,-'•'
—. B
'•••V
^-. V
:
.
Fig. 5. Hybridization of muscle-specific probes A2, Dl and H2 to skeletal and myocardial muscle. All probes hybridize to skeletal muscle
but differ in hybridization to myocardium. Panels D,E,F are higher magnifications of panels A,B>Q respectively. Corresponding regions of
the sections are marked with V. Scale bar equals (A), 100 [an; (D), 2O.jUm. (A,D) Hybridization of probe A2 to a section ihrough the
heart at the level of the otic vesicles. Hybridization to somitic and branchiomeric muscles can be seen. However, as is indicated in the
higher magnification photomicrograph (D) hybridization to heart muscle does not exceed background. (B,E) Hybridization of probe Dl to
somites, laryngeal muscle and heart. Dl hybridizes at above background levels to heart muscle but less intensely than to somitic
musculature. (C,F) Hybridization of probe H2 (a-cardiac actin) to somites and heart. H2 hybridizes intensely to heart muscle.
Abbreviations: bm, branchiomeric muscle; /, laryngeal muscle; me, myocardial muscle; n, notochord; s, somite.
5A
€\9
S
z
o
o
w
as
D
o
o
to
In situ hybridization to X. laevis embryos
A2
"
B5 B9
D1
H2
A4
163
D8
•
5.04.3-
-
r
an
EH
EH
-a
2.01.61.4-
EH
EH
EH
EH
EH
Fig. 6. Hybridization of tissue-specific cloned sequences to genomic DNA. Genomic
DNA was digested with Eco RI (E) or Hind III (H), separated on 0-8 % agarose gels,
transferred to nitrocellulose and hybridized with nick-translated plasmid DNA as
indicated above each set of lanes. Marker sizes are in kilobase pairs and are derived
from Eco RI+Hind III digested phage A DNA.
A2 or with D l . Sequences B9 (not shown) and H2 (Fig. 5C,F) display the same
intensities of hybridization to skeletal and to heart muscle. Sequence H2 has been
identified as ar-cardiac actin (Tom Sargent, personal communication). Thus, we
can divide the five muscle-specific clones into three categories with respect to their
hybridization to heart muscle: not reactive (A2, possibly B5), weakly reactive
(Dl, possibly B5), reactive (B9, H2).
To exclude that any two of the five muscle-specific clones represented the
same mRNA, we determined their hybridization to genomic DNA by 'Southern
blotting' (Southern, 1975). The result (Fig. 6, panels A2 through H2) shows
distinctive hybridization patterns for all five clones, indicating that each clone
represents a different mRNA. Fig. 6 also displays the Southern hybridization
patterns of A4 (epidermis-specific clone) and D8 (nerve-specific clone). All tissuespecific sequences are represented in the genome as low copy genes.
Table 1 summarizes the results of this investigation as well as the developmental
expression patterns of each clone as determined previously (Dworkin et al 1984,
and unpublished results). All probes studied showed predominantly cytoplasmic
labelling. It is interesting to note that all clones exhibiting tissue-specific expression fall into a class of sequences whose expression is undetectable or
qualitatively different in maternal mRNA (see Discussion).
DISCUSSION
In this paper we describe the in situ hybridization pattern of cDNA clones from
X. laevis embryonic libraries to tissue sections of 3-day-old tadpoles. Nine out of
164
E. DWORKIN-RASTL, D. B. KELLEY AND M. B. DWORKIN
sixteen cDNA clones showed hybridization to all tissue types of the embryo and
seven clones displayed localized hybridization to either epidermis, the nervous
system, or muscle tissue. We have thus demonstrated the feasibility of in situ
hybridization to cryostat sections of X. laevis embryos, using nick-translated
double-stranded plasmid DNA as probe. The protocol we employed is very
economical in the usage of 3 H-TTP. For one nick translation of 50 ng plasmid
DNA, yielding 500 /il of hybridization solution, only 5 juCi 3 H-TTP are needed. We
have never experienced significant background due to non-specific sticking of the
probe to the tissue, probably due to the low amount of radioactivity in the
hybridizations. Control hybridizations with pBR322 were included with each
experiment.
The intense pigmentation of X laevis embryos presents a difficulty when analysing autoradiographs of in situ hybridizations, especially with stages earlier than
stage 42. One way to avoid this problem is the use of albino embryos. This has
been demonstrated with stage-35/36 embryos in Fig. 2. A second problem is the
difficulty of sectioning embryos up to about stage 35, due to the high yolk content
Table 1. Tissue specificity and developmental expression of 16 sequences*
... .
Tissue-specific sequences
c
Sequences without
l
_
tissue specificity
Epidermis
Nervous tissue
Developmental
Developmental
Developmental
Clone
expression
Clone expression
Clone expression
Clone
BIO
o(+)n(++)
A4
o(-)n(+)
D8
o ( - ) n(+) A2
B20
o(+)n(++)
B5
C20f
o(++)n(+)
B9
D4
o(+)|n(++)
Dl
E8
o(+)|n(++)
H2"
Fl
F9
F14
G17
Muscle
Developmental
expression
o ( - ) n(+)
o ( - ) n ( - ) t(+)
o(-)1f n(+)
o(-)in(+)
o(—)if n(+)
*o, oocytes from total ovary or full-grown oocytes or eggs; n, stage-13 to -15 neurulae; t,
3-day-old tadpoles; (+) and (+ +) indicate the presence of a sequence, ( - ) indicates the absence
of a sequence (as determined by RNA gel blots using poly(A)+RNA and total RNA); (+) and
(++) are used to indicate increases or decreases in titer of sequences present both in oocytes
and neurulae. All developmental expression data are from Dworkin etal. (1984), as well as from
unpublished gel blot experiments. The clone nomenclature refers to clone positions as described
in Dworkin & Dawid, 19806.
t C20 codes for ubiquitin (Dworkin-Rastl et al. 1984).
$ D4, E8 and F9 were detected in oocyte RNA only when using poly(A)+RNA gel blots; they
were not detected using total RNA gel blots (Dworkin et al. 1984).
§The increase in titer of F14 could be determined with total RNA gel blots, but not with
poly(A)+RNA gel blots or DNA dot blots (Dworkin & Hershey, 1981), possibly due to the lack
of poly (A) sequences in a fraction of these RNA molecules.
If B9, Dl and H2 hybridize in oocyte RNA with transcripts of different size and much lower
abundance than the respective embryonic transcripts.
"H2 codes for ar-cardiac actin (Tom Sargent, personal communication).
Ill situ hybridization to X. laevis embryos
165
of early X. laevis embryos. This difficulty can be overcome using plastic embedding
methods (Jamrich et al. 1984), but preliminary experiments in our laboratory,
using methacrylate-embedded embryos and nick-translated probes, have not yet
been successful.
The cDNA clones used in this study represent RNA sequences that increase
in titer during early development (except for C20), as shown by dot blot and
Northern blot hybridizations (Dworkin & Hershey, 1981; Dworkin etal. 1984, and
unpublished results). The specific mRNAs are present at stage 37 at concentrations ranging from about 20 pg (C20) to more than 100 pg per embryo (G17)
(Dworkin & Hershey, 1981). In a stage-37 tadpole (300000 cells per embryo,
based on 2/xg DNA per embryo and 6-3pg DNA per diploid cell (Dawid, 1965;
Thiebaud & Fischberg, 1977)), 20 pg per embryo correspond to about 60 molecules
of a 2 kb mRNA per cell. Since these probes appear to hybridize to all cells in the
embryo, we estimate that the autoradiographic method used in this study can
detect at least 60 mRNA molecules per cell.
The cloned sequences studied fall into two groups according to their presence
or absence (as determined by gel blots with total RNA and poly(A) + RNA) in
maternal mRNA (Table 1). Most interestingly, all sequences that displayed tissuespecific in situ hybridization patterns belong to the latter group; that is, they were
detectable in embryonic RNA only after gastrulation. This had originally been
shown on gel blots with total RNA (Dworkin et al. 1984, and unpublished results).
Poly(A) + RNA gel blots, which are more sensitive than blots with total RNA but
cannot be accurately quantitated during early development due to polyadenylation changes, were prepared for all clones displaying tissue-specific hybridization. These blots confirmed the original result that sequences A2, A4, B5 and D8
were not present in maternal mRNA (not shown). Sequences B9, D l and H2 did
hybridize weakly with maternal poly(A) + RNA; however, the transcript sizes were
different from the embryonic transcript sizes (Dworkin et al. 1984, and unpublished results; only occasional RNA preparations from early obcytes showed a
weak band for H2 at the position of the tadpole transcript). Sequences that did not
display tissue-specific in situ hybridization could be detected in maternal mRNA
and displayed transcript sizes similar to those of the embryonic RNA. This was
shown with total RNA blots for sequences B10, B20, F l , F14 and G17 (Dworkin
etal. 1984, and unpublished results); sequences D4, E8 and F9 are less abundant
maternal sequences and were detected in oocyte RNA only when using the more
sensitive poly(A) + RNA blots (not shown). Sequence C20 (ubiquitin) is abundant
in maternal poly(A) + RNA (Dworkin-Rastl etal. 1984).
Although the number of sequences tested by in situ hybridization is small, the
different patterns of in situ hybridization of maternal and embryo-restricted
sequences may be significant. Thus, it is tempting to think that maternal sequences
whose synthesis resumes after fertilization are likely to accumulate in all cells of
3-day-old tadpoles and to represent 'housekeeping' functions. On the other hand,
transcripts that are synthesized only after the gastrula stage are good candidates to
be tissue specific in tadpoles and to code for differentiation-specific proteins.
166
E. DWORKIN-RASTL, D. B. KELLEY AND M. B. DWORKIN
The tissue-specific sequences we have detected are specific for ectodermal and
mesodermal tissues. Of seven tissue-specific clones, one (A4) hybridized to the
embryonic epidermis and one (D8) to the embryonic nervous system. Thus, these
clones distinguish the two different tissues of ectodermal origin. The tissue for
which we found several (five) specific clones is muscle. This is not surprising since
muscle tissue constitutes a large percentage of the developing embryo. These
clones were all restricted to muscle tissue and did not recognize neighbouring
tissues of mesodermal origin. All five clones hybridized to somites (which, in
X. laevis, consist mostly of myotomal cells; Hamilton, 1969) and to the smooth
muscle of the lateral plate. The five clones did, however, differ in their ability to
hybridize to heart muscle. Clones H2 (ar-cardiac actin; Tom Sargent, personal
communication) and B9 hybridized to heart as strongly as to the somites. This
hybridization pattern of H2 is consistent with the finding that in X. laevis embryos
somitic mesoderm accumulates similar amounts of both <*-cardiac and ar-skeletal
muscle actin mRNA (Mohun et al. 1984). Although it is possible that in our
experiments we detected some cross-hybridization among different actin mRNA
sequences, cross-hybridization to cytoskeletal actin mRNA was not a problem,
due to the low cellular concentration of these transcripts. To obtain optimal
sensitivity in these experiments, hybridizations and washes were both carried out
at a criterion of 50 % formamide and 0-6M-NaCl at 35 °C.
Clone Dl hybridized much more weakly (but detectably) to heart muscle than
to skeletal muscle, whereas clone A2 did not hybridize above background to heart
muscle even though it hybridized very strongly to somites. Thus, somites and the
developing heart muscle can be distinguished by mRNA content at stage 42, even
though the respective actin mRNA sequences do not yet display the tissue
restriction observed in adult tissue (Mohun et al. 1984). We do not know the
proteins that A2, B5, B9 and Dl code for. However, genomic Southern blots
(Fig. 6) show that (with the possible exception of B9 and Dl) these clones are
unrelated. A2, B9, Dl and H2 are induced by the early neurula, but are still below
the level of detection in late gastrula (Dworkin et al. 1984). B5 could not be
detected in early neurula RNA by Northern blots but is present in 3-day-old
tadpoles (not shown). Thus, sequence B5 is a tadpole sequence that does not
accumulate in the late gastrula/early neurula period (Dworkin et al. 1984).
In summary, we have demonstrated that in situ hybridization can be used to
localize the expression of specific mRNA sequences in developing X. laevis
embryos. Two potential applications of the method suggest themselves. First,
cDNA probes may be used as cell-type-specific markers (analogous to cell-typespecific antibodies) to follow the choices made by cells during differentiation.
Thus, clones A4 and D8, both sequences being detectable already by the
beginning of neurulation, may be useful markers in distinguishing epidermal and
neural descendants of the ectodermal lineages. Second, the accumulation of gene
products for which cloned probes are available may be followed at the level of
single cells, in an effort to understand the basis of cell-restricted gene expression.
In situ hybridization to X. laevis embryos
167
We acknowledge the valuable technical assistance of Rachel Kraut and Anthony Shrutkowski
and we thank Drs Michael Levine and Dennis Gorlick for helpful discussions. This work was
supported by grants from the National Institutes of Health (HD 17234 and NS 19949) and a gift
from Boehringer Ingelheim GmbH.
REFERENCES
AKAM, M. E. (1983). The location of Ultrabithorax transcripts in Drosophila tissue sections.
EMBOJ. 2,2075-2084.
Cox, K. H., DELEON, D. V., ANGERER, L. M. & ANGERER, R. C. (1984). Detection of mRNAsin
sea urchin embryos by in situ hybridization using asymmetric RNA probes. Devi Biol. 101,
485-502.
DAWID, I. B. (1965). Deoxyribonucleic acid in amphibian eggs. /. mol. Biol. 12, 581-599.
DENHARDT, D. T. (1966). A membrane filter technique for the detection of complementary
DNA. Biochem. Biophys. Res. Commun. 23, 641-646.
DWORKIN, M. B. & E>AWID, I. B. (1980a). Construction of a cloned library of expressed
embryonic gene sequences from Xenopus laevis. Devi Biol. 76, 435-448.
DWORKIN, M. B. & DAWID, I. B. (1980fc). Use of a cloned library for the study of abundant
poly(A)+RNA during Xenopus laevis development. Devi Biol. 76, 449-464.
DWORKIN, M. B. & HERSHEY, J. W. B. (1981). Cellular titers and subcellular distributions of
abundant polyadenylate-containing ribonucleic acid species during early development in the
frog Xenopus laevis. Mol. Cell. Biol. 1, 983-993.
DWORKIN, M. B., SHRUTKOWSKI, A., BAUMGARTEN, M. & DWORKTN-RASTL, E. (1984). The
accumulation of prominent tadpole mRNAs occurs at the beginning of neurulation in
Xenopus laevis embryos. Devi Biol. 106, 289-295.
DWORKIN-RASTL, E., SHRUTKOWSKI, A. & DWORKIN, M. B. (1984). Multiple ubiquitin mRNAs
during Xenopus laevis development contain tandem repeats of the 76 aminoacid coding
sequence. Cell 39, 321-325.
EDWARDS, M. K. & WOOD, W. B. (1983). Location of specific messenger RNAs in Caenorhabditis
elegans by cytological hybridization. Devi Biol. 97, 375-390.
GALL, J. G. & PARDUE, M. L. (1971). Nucleic acid hybridization in cytological preparations. In
Methods in Enzymology (ed. L. Grossman & K. Moldave), Vol. 21, pp. 470-480. New York:
Academic Press.
GEE, C. E., CHEN, C.-L. C , ROBERTS, J. L., THOMPSON, R. & WATSON, S. J. (1983).
Identification of proopiomelanocortin neurones in rat hypothalamus by in situ cDNA-mRNA
hybridization. Nature, Lond. 306, 374-376.
HAFEN, E., LEVINE, M., GARBER, R. L. & GEHRING, W. J. (1983). An improved in situ
hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its
application for localizing transcripts of the homeotic Antennapedia gene complex. EMBOJ. 2,
617-623.
HAMILTON, L. (1969). The formation of somites in Xenopus. J. Embryol. exp. Morph. 22,
253-264.
HARDING, J. D., MACDONALD, R. J., PRZYBYLA, A. E., CHIRGWIN, J. M., PICTET, R. L. & RUTTER,
W. J. (1977). Changes in the frequency of specific transcripts during development of the
pancreas. /. biol. Chem. 252, 7391-7397.
JAMRICH, M., MAHON, K. A., GAVIS, E. R. & GALL, J. G. (1984). Histone RNA in amphibian
oocytes visualized by in situ hybridization to methacrylate-embedded tissue sections. EMBO
J. 3,1939-1943.
'
,
LEVINE, M., HAFEN, E., GARBER, R. L. & GEHRING, W. J. (1983). Spatial distribution of
Antennapedia transcripts during Drosophila development. EMBO J. 2, 2037-2046.
MAXAM, A. M. & GILBERT, W. (1980). Sequencing end-labelled DNA with base-specific chemical
cleavages. In Methods in Enzymology (ed. L. Grossman & K. Moldave), Vol. 65, pp. 499-560.
New York: Academic Press.
MOHUN, T. J., BRENNAN, S., DATHAN, N., FAIRMAN, S. & GURDON, J. B. (1984). Cell type-specific
activation of actin genes in the early amphibian embryo. Nature, Lond. 311, 716-721.
168
E . D W O R K I N - R A S T L , D . B . K E L L E Y AND M. B . D W O R K I N
J. & KIRSCHNER, M. (1982). A major developmental transition in early Xenopus
embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30,
675-686.
RIGBY, P. W. J., DIECKMANN, M., RHODES, C. & BERG, P. (1977). Labelling deoxyribonucleic
acid to high specific activity in vitro by nick translation with DNA polymerase I. J. mol. Biol.
113, 237-251.
NEWPORT,
SCHELLER, R. H., JACKSON, J. F., MCALLISTER, L. B., SCHWARTZ, J. H., KANDEL, E. R. & AXEL, R.
(1982). A family of genes that codes for ELH, a neuropeptide eliciting a stereotyped pattern
of behavior in Aplysia. Cell 28, 707-719.
SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by
gel electrophoresis. /. mol. Biol. 98, 503-517.
THI£BAUD, C. H. & FISCHBERG, M. (1977). DNA content in the genus Xenopus. Chromosoma
(Berl.) 59, 253-257.
VENEZKY, D. L., ANGERER, L. M. & ANGERER, R. C. (1981). Accumulation of histone repeat
transcripts in the sea urchin egg pronucleus. Cell 24, 385-391.
ZIMMERMAN, J. L., PETRI, W. & MESELSON, M. (1983). Accumulation of a specific subset of
D. melanogaster heat shock mRNAs in normal development without heat shock. Cell 32,
1161-1170.
{Accepted 19 August 1985)
NOTE ADDED IN PROOF
Partial nucleotide sequencing of D8 and comparison of the first 94 amino acids
of the putative D8 translation product to protein databanks showed > 9 5 %
identity with the Af-terminal sequence of pig brain- and chicken brain /3-tubulin (N.
Segil, personal communication). These data, together with the pattern of its in situ
hybridization, suggest that D8 represents a neural specific form of X. laevis fitubulin.