J. Embryol. exp. Morph. 75, 225-239 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
225
Localization of messenger RNA in the cortex of
Chaetopterus eggs and.early embryos
By WILLIAM R. JEFFERY 1 AND LINDA J. WILSON
From the Department of Zoology, University of Texas, Austin, and the Marine
Biological Laboratory, Woods Hole, Massachusetts
SUMMARY
The distribution of mRNA in Chaetopterus pergamentaceus eggs was examined by in situ
hybridization with poly(U) and specific cloned DNA probes. Eggs contain three distinct
regions; the cortical ectoplasm, endoplasm, and a plasm released from the germinal vesicle
(GV) during maturation. The ectoplasm of the mature egg showed a 15-fold enrichment in
poly(A) and in histone and actin mRNAs relative to the endoplasm and the GV plasm after
in situ hybridization. More than 90% of the total mass of egg poly (A) + RNA and histone and
actin messages was estimated to be present in the ectoplasm. The mRNA molecules codistributed with ectoplasmic inclusion granules during ooplasmic segregation. During the
extensive cytoplasmic rearrangements which occur at the time of the first cleavage the
ectoplasm was divided into animal and vegetal portions. The animal portion was segregated
evenly between the AB and CD blastomeres, whereas the vegetal portion entered the polar
lobe and was preferentially segregated to the CD blastomere. Histone and actin mRNA
entered both the AB and CD blastomeres of the 2-cell embryo. The results demonstrate that
mRNA is quantitatively localized in the cortex of the Chaetopterus egg and early embryo.
INTRODUCTION
The localization of maternal mRNA molecules and their segregation to different embryonic cells has been proposed to mediate cell determination during
early development (see Davidson, 1976 and Jeffery, 1983 for reviews). The
existence of localized maternal mRNA molecules, however, is still a controversial issue in most embryonic systems. Solution hybridization studies have uncovered differences in the complexity of poly(A)+ RNA molecules located in
different regions of sea urchin (Rodgers & Gross, 1978; Ernst et al. 1980) and
Xenopus laevis (Carpenter & Klein, 1982) embryos. Qualitative differences,
however, are yet to be detected between the in vitro translation products of
prevalent poly(A)+ RNA molecules isolated from different parts of embryos
(Ilyanassa; Brandhorst & Newrock, 1981; Collier & McCarthy, 1981), suggesting that the localized RNA sequences detected by hybridization are either very
rare or do not serve as messages. Conflicting results were also obtained when the
1
Author's address: Department of Zoology, University of Texas, Austin, Texas 78712,
U.S.A.
226
W. R. JEFFERY AND L. J. WILSON
spatial distribution of total poly(A)+ RNA was examined by in situ hybridization with poly(U). Poly(A)+ RNA sequences were found to be evenly
distributed in sea urchin (Angerer & Angerer, 1981) and mouse (Sternlicht &
Schultz, 1981; Piko & Clegg, 1982) embryos. In contrast, poly(A)+ RNA appeared to be localized when other embryos, particularly mosaic embryos, were
analysed by in situ hybridization with poly(U). For instance, about half the mass
of poly (A) + RNA was present in the plasm derived from the germinal vesicle
(GV) of Styela eggs and remained localized in this region after maturation,
ooplasmic segregation and cleavage (Jeffery & Capco, 1978). The localized
poly(A)+ RNA of Styela embryos was primarily segregated to the animal
hemisphere blastomeres during early embryogenesis. Localizations of poly (A)+
RNA have also been reported in the cortex of Oncopeltus fasciatus (Capco &
Jeffery, 1979) and Xenopus laevis (Capco & Jeffery, 1982) oocytes after in situ
hybridization.
In the present study we have continued the comparative analysis of the spatial
distribution of mRNA during early embryonic development. The egg of
Chaetopterus has been selected for further study because, like that of Styela, it
contains a GV plasm and distinct cytoplasmic regions which are subject to
ooplasmic segregation and differential partitioning between the embryonic cells
during early development (Lillie, 1906). In situ hybridization with poly(U) and
cloned DNA probes for specific messages has revealed that most of the mRNA
of eggs and early embryos is localized in the cortex.
MATERIALS AND METHODS
Chaetopterus pergamentaceus was obtained from the Marine Resources
Department of the Marine Biological Laboratory, Woods Hole, MA. Adults,
gametes and embryos were maintained and handled by established procedures
(Costello & Hendley, 1971). Parapodia, oocytes and embryos at the desired stage
of development were fixed at -20°C for 30min in Petrunkewitsch's fluid. This
fixative, which we have found to quantitatively preserve poly(A)+ RNA in the
specimens, was freshly prepared before use by mixing one part of an aqueous
solution containing 12 % nitric acid (v/v)-8 % cupric nitrate (w/v) with three
parts of an aqueous solution containing 76 % ethanol (v/v), 5-7 % ethyl ether
(v/v) and 3-8 % crystalline phenol (w/v). The fixed specimens were dehydrated
in ethanol and cleared in toluene at - 2 0 °C. They were embedded in paraplast,
sectioned at 8 fim and attached to glass slides for histology or in situ hybridization.
In situ hybridization with [3H]poly(U) [4-65 Ci/mmole; New England
Nuclear, Boston, MA.] was carried out as described previously (Capco & Jeffery, 1978), except that a step involving treatment of the slides with 10/ig/ml
proteinase K (lOmin at 20 °C) was inserted between the DNase I pretreatment
and the annealing. This step was necessary to obtain the highest efficiency of
poly(A) + RNA detection in sections of Chaetopterus eggs.
Cortical mRNA
227
The DNA probes were prepared by Hind III restriction of plasmids containing
the Dm-500 histone gene complex (Lifton et al. 1977) or the Dm-A2 actin gene
(Fyrberg etal. 1980) from Drosophila melanogaster. The 4-6 kilobase (kb) DNA
fragment of the Dm-500 complex contains complete sequences of the genes for
HI, H2a, H2b, H3, and H4 and their spacers (Lifton et al. 1977) and was used
as a probe for histone mRNA. The 1-8 kb DNA fragment of the Dm-A2 actin
gene contains almost the entire coding sequence of an actin mRNA (Fyrberg et
al. 1980) and was used as a probe for actin mRNA. The Hind III digests were
separated by agarose gel electrophoresis and bands containing the appropriate
DNA fragments were excised from the gel. The DNA was extracted from the gel
slices and nick translated with [125I]deoxycytidine triphosphate (2-5 x lt^Ci/
mmole; New England Nuclear, Boston, MA) to a specific activity of about
1-5 x 10 7 d.p.m./^g DNA as described by Maniatis et al. (1975). The DNA
probes were dissolved in hybridization buffer, denatured by heating at 90 °C for
5 min and applied to the sections at saturating concentrations (2-10ng/ml; 20 [A
per slide). In situ hybridization with cloned histone and actin DNA probes was
carried out according to the method of Jeffery (1982).
The autoradiographs and sections for cytological observation were stained
with Harris haematoxylin-eosin for 2-5 min. This stains the ectoplasmic granules
red, the endoplasm and yolk granules light blue, and the GV plasm dark purple.
RESULTS AND DISCUSSION
Behaviour of cytoplasmic regions during early development
The cytoplasmic regions of Chaetopterus eggs and their movements during
maturation and early embryogenesis were originally described by Lillie (1906).
Lillie's published account did hot consider the behaviour of the cytoplasmic
regions during the entire period between maturation and the first cleavage. Thus
it was necessary to extend Lillie's cytological description of early Chaetopterus
development before examining the distribution of mRNA during early development. A summary of Lillie's and our own cytological observations is presented
in Figs 1-8.
Chaetopterus eggs are released from the parapodia as primary oocytes containing three regions, the GV plasm, endoplasm and ectoplasm (Fig. 1). The
endoplasm consists of large blue-staining, yolk particles and clusters of smaller,
red-staining granules. The ectoplasm, situated in the animal two-thirds of the
oocyte cortex, is densely packed with these same red-staining granules (ectoplasmic granules). Within 10 min after the oocytes are exposed to sea water the GV
ruptures initiating maturation and ooplasmic segregation (Fig. 2). The GV plasm
moves into the animal pole region where an ectoplasmic defect (Lillie, 1906)
appears at the place where the polar bodies are formed. Simultaneously, the
clusters of internal ectoplasmic granules move from the endoplasm into the
cortex and join with the rest of the ectoplasm to form a cortical layer which
228
W. R. JEFFERY AND L. J. WILSON
ec
Figs 1-8
Cortical mRNA
229
surrounds the entire surface of the egg, except for the region of the ectoplasmic
defect (Figs 2, 3).
Cytoplasmic rearrangements also occur in fertilized eggs during cleavage and
polar lobe formation. The cleavage associated movements are first detected at
metaphase when the egg elongates into a pear-shaped mass with the narrowest
portion of the cell in the animal hemisphere (Fig. 4). Many of the ectoplasmic
granules are temporarily dislodged from the cortex at this time and accumulate
at the boundary between the GV plasm and the endoplasm (Fig. 4). The pearshaped cell is generated by a cortical constriction which begins to form in the
animal hemisphere and proceeds vegetally, eventually resulting in polar lobe
protrusion (Figs 5,6). Prior to polar lobe formation the ectoplasmic granules
return to the cortex and are split into animal and vegetal fields by the advancing
cortical constriction (Fig. 5). At cleavage the animal ectoplasmic field is divided
about equally between the AB and CD blastomeres (Figs 6-8). In contrast, most
of the vegetal ectoplasmic field, along with the other contents of the polar lobe,
enter the CD blastomere (Figs 6, 7). After polar lobe regression at telophase its
ectoplasmic granules accumulate in the vegetal region of the constricting
cleavage furrow and become localized in the cortex of the CD blastomere (Figs
7,8). Although we have not examined the fate of the vegetal ectoplasmic field
beyond the 2-cell stage, according to Lillie (1906) it is also present in the second
polar lobe and is thus likely to enter the D quadrant of the embryo.
Localization of mRNA in the cortical ectoplasm
The distribution of grains in mature eggs after in situ hybridization with
poly(U) is shown in Fig. 9 and quantified in Table 1. The grains were concentrated about 15-fold in the cortical ectoplasm relative to the GV plasm or the
endoplasm. The interaction of poly(U) with the eggs was substantially reduced
Figs 1-8. The behaviour of cytoplasmic regions during maturation and early
embryogenesis of Chaetopterus pergamentaceus eggs. These drawings represent sections through the animal-vegetal axis offixedand stained oocytes, eggs and embryos.
They are drawn in the style of Lillie (1906) and are a composite of Lillie's and our
own histological studies. Fig. 1. A primary oocyte as it appears shortly after its
release into sea water. Fig. 2. An egg at the first maturation division. Fig. 3. An egg
which has completed the first maturation division. Fig. 4. A zygote at the pear stage
showing the transient accumulation of cortical ectoplasmic granules at the boundary
between the endoplasm and the residual germinal vesicle plasm. Fig. 5. A zygote at
metaphase showing the equatorial constriction which splits the ectoplasm into animal
and vegetal portions. Fig. 6. A trefoil embryo showing the accumulation of vegetal
ectoplasm in the polar lobe. Fig. 7. A telophase embryo showing the accumulation
of vegetal ectoplasmic granules along one side of the vegetal cleavage furrow following the retraction of the polar lobe. Fig. 8. A two-cell embryo showing the
distribution of animal and vegetal ectoplasms in the AB (right) and CD (left) blastomeres. Germinal vesicle or residual plasm of the germinal vesicle (GV); endoplasm
(en), ectoplasm (ec); polar lobe (PL). The filled spheres represent the ectoplasmic
granules. The open spheres represent the endoplasmic yolk platelets.
230
W. R. JEFFERY AND L. J. WILSON
era
Figs 9, 10. /n situ hybridization of Chaetopterus eggs with poly(U). Fig. 9. An
autoradiograph of a mature egg showing grains in the cortical ectoplasm. X400. Fig.
10. An autoradiograph of a mature egg treated with RNase T2 prior to in situ
hybridization. Cortical grains are not apparent. x400. See Table 1 for details of the
RNase treatment. Ectoplasm (ec).
when the sections were treated with RNase (Fig. 10; Table 1) or alkali (data not
shown) prior to in situ hybridization suggesting that the probe binds to RNA in
the section. It is unlikely that the concentration of mRNA in the cortex is due
to the differential extraction of RNA during fixation since we have shown that
poly(A)+ RNA is quantitatively retained in eggs fixed with Petrunkewitsch's
fluid. When sections of eggs were subjected to in situ hybridization with the
histone or actin DNA probes, the grain concentration also ranged between 10and 15-fold higher in the ectoplasm than in the other cytoplasmic regions (Table
1; Figs 19 and 21). To estimate the proportion of the egg poly(A) present in the
cortical ectoplasm the grain counts were multiplied by the volume comprised by
each of the cytoplasmic regions (as derived from their areas in egg sections;
Jeffery & Capco, 1978) and these values were expressed as percentages of the
total grains. Although the ectoplasm comprises only 35 % of the egg volume, it
was estimated to contain about 95 % of the mass of egg poly(A). These results
indicate that a population of mRNA molecules, comprising most of the egg
poly(A)+ RNA, histone mRNA and actin mRNA, is localized in the cortical
ectoplasm.
The case of Chaetopterus now represents the fourth example of quantitative
mRNA localization during early development. In vitellogenic oocytes of Oncopeltus poly(A)+ RNA is present in the anterior and posterior cortical cytoplasms
Cortical mRNA
231
Table 1. In situ hybridization of Chaetopterus eggs with [3H]poly(U) and
I125-labelled actin and histone DNA probes
Grain Number ± S.D.
Probe and Pretreatment
Poly(U)
Poly(U), RNase T2
Histone DNA
Histone DNA, RNase A
Actin 1-8 kb. DNA
Actin 1-8 kb. DNA, RNase A
pBR322
Ectoplasm
Endoplasm
Residual
GV plasm
62-3 ±2-1
3-5 ±1-3
72-6 ±9-2
3-7 ±0-8
42-4 ±3-3
3-9 ±1-6
2-4 ±1-0
3-7 ±0-9
3-1 ±1-4
8-112-5
1-1 ±0-6
4-611-3
0-4
2-2 ±0-7
0-5
1-2 ±0-3
1-7 ±0-9
0-5
2-5 ±1-3
0-2
2-5 ±1-6
Grain numbers are expressed as the mean of counts in 15-25 different 100 ^m2 areas ±
standard deviation (S.D.). The indicated slides were pretreated with 50jUg/ml pancreatic
RNase A for 24 h at 37 °C or 50^g/ml RNase T2 for 24 h at 37 °C. The RNases were dissolved
in lOmM-Tris-HCl (pH7-6), 50mM-KCl, lmM-MgCl2.
(Capco & Jeffery, 1979). In stage-6 oocytes of Xenopus laevis poly(A)+ RNA
is localized in the cortex of the animal hemisphere (Capco & Jeffery, 1982). In
the latter two cases localization of poly(A)+ RNA is a transient phenomenon
and cannot be detected in the mature egg. In Styela about half of the poly(A)+
RNA mass is concentrated in the GV plasm of oocytes and after maturation it
becomes localized in the ectoplasm (Jeffery & Capco, 1978). The lack of a
significant accumulation of poly(A) + RNA in the GV plasm of Chaetopterus or
Xenopus (Capco & Jeffery, 1982) oocytes suggests that this feature is not a
general developmental phenomenon. It is also notable that in three of the four
cases examined mRNA localizations are present in the egg cortex, a finding of
interest in light of the morphogenetic significance ascribed to this area of the egg.
Origin of cortical mRNA
Two major possibilities exist for the origin of the cortical poly(A)+
molecules. They could be present in the cortex of the primary oocyte before
maturation or they could migrate into the egg cortex from sites such as the GV
plasm or the endoplasm during ooplasmic segregation. To resolve this issue
primary oocytes were fixed and subjected to in situ hybridization with poly(U)
during the period between their release from the parapodia and the completion
of maturation. As shown in Fig. 11 grains were already concentrated in the
cortex of the primary oocyte. The co-distribution of these grains with the
cortical ectoplasmic granules is demonstrated by sections through the animalvegetal axis showing heavy labelling in the animal two-thirds of the cortex (Fig.
13). Very few grains were observed in the GV plasm of the primary oocyte or
the mature egg (Figs 11-14). In contrast to the mature egg, however, the
232
W. R. JEFFERY AND L. J. WILSON
primary oocyte also showed significant labelling over specific areas of the
endoplasm (compare Figs 11 and 12 to Fig. 13). The endoplasmic grains were
always positioned immediately above clusters of ectoplasmic granules (Fig. 1).
Two factors appear to contribute to the localization of mRNA in the egg cortex.
The bulk of the oocyte mRNA must already be present in the cortical ectoplasm
prior to maturation while the remainder is likely to be incorporated into the
cortex with the internal ectoplasmic granules during ooplasmic segregation. The
cortical mRNA localization probably originates early during oogenesis since it
can be detected in parapodial oocytes of all sizes (Fig. 14).
Segregation of cortical mRNA during cleavage
Sections of fertilized eggs were fixed at intervals during the first cleavage and
subjected to in situ hybridization with poly(U) to determine the fate of the
cortical poly (A) + RNA during the unequal partitioning of ectoplasm between
the AB and CD blastomeres. The results are shown in Figs 15-18. Pear-stage
embryos showed grains concentrated in the cortical ectoplasm and in the vicinity
of ectoplasmic granules that were dislodged from the cortex and accumulated at
the edge of the residual GV plasm (Fig. 15). A few grains were still present at
this time over the ectoplasmic spherules that remain at the egg surface. Later,
when the polar lobe begins to form, heavy labelling was seen over accumulations
of ectoplasmic granules in the vegetal pole region of the egg. Grains were concentrated in two major locations at the trefoil stage, the cortical ectoplasm of the
animal hemisphere and the polar lobe (Fig. 16). After the polar lobe is retracted
into the nascent CD cell at telophase and the vegetal ectoplasmic granules move
into the region of the cleavage furrow (Figs 7 and 8), intense labelling was seen
in the furrow region directly above these granules (Fig. 17). The population of
mRNA molecules in the animal ectoplasmic field appears to be divided about
equally between the AB and CD blastomeres. In contrast, most of the mRNA
molecules in the vegetal ectoplasmic field enter the polar lobe and are preferentially distributed to the CD blastomere (Fig. 18). The results indicate that the
cortical mRNA molecules are co-distributed with ectoplasmic granules during
the first cleavage as well as ooplasmic segregation.
Two previous studies suggest that the position of mRNA molecules in the egg
Figs 11-14. In situ hybridization of Chaetopterus oocytes with poly(U). Fig. 11. An
autoradiograph of a recently shed primary oocyte showing abundant grains over the
ectoplasm (ec) and few grains over the germinal vesicle (GV). Clusters of grains also
appear in the endoplasm (en). X400. Fig. 12. An autoradiograph of a primary oocyte
showing clusters of grains (arrows) over ectoplasmic granules in the endoplasm.
xlOOO. Fig. 13. An autoradiograph of a primary oocyte sectioned through the
animal-vegetal axis showing abundant grains in the animal two-thirds of the
ectoplasm and few grains in the endoplasm and residual GV plasm. Animal pole,
(AP). x400. Fig. 14. An autoradiograph of parapodial oocytes of various sizes
showing cortical ectoplasmic grains. X150.
233
Cortical mRNA
G.¥
\
r
rt
234
W. R. JEFFERY AND L. J. WILSON
A^£*I*
«•
Figs 15, 16. /n situ hybridization of Chaetopterus embryos with poly(U) during the
period immediately prior to the first cleavage. Fig. 15. An autoradiograph of an
equatorial section through a pear stage embryo showing grains over ectoplasmic
granules in the cortex and at the interface between the endoplasm (en) and the
residual GV plasm (GVP). x600. Fig. 16. An autoradiograph of a trefoil embryo
sectioned along the animal-vegetal axis with focus over the polar lobe. Grains are
concentrated mainly in the animal ectoplasm and in the cortex of the polar lobe. AB
blastomere, (AB). CD blastomere, (CD). x400.
cytoplasm is fixed by associations with regionalized structures. First, the concentration of mRNA does not appear to change in cytoplasmic regions which
migrate extensively through the Styela egg during ooplasmic segregation (Jeffery
Cortical mRNA
235
18
Figs 17, 18. Fig. 17. In situ hybridization of cleaving Chaetopterus embryos with
poly(U). An autoradiograph of a section through the animal-vegetal axis of a cleaving embryo showing the accumulation of grains over ectoplasmic granules in the
vegetal region of the cleavage furrow. xlOOO. Fig. 18. An autoradiograph of a twocell embryo showing the distribution of grains between the animal (AE) and vegetal
(VE) ectoplasmic fields in the AB and CD blastomeres. x600.
236
W. R. JEFFERY AND L. J. WILSON
& Capco, 1978). Second, mRNA isolated from the vegetal pole region of
Xenopus laevis eggs tends to accumulate in a vegetal pole to animal pole
gradient after microinjection into zygotes between fertilization and the first
cleavage (Capco & Jeffery, 1981). The localization of mRNA molecules in the
ectoplasm of Chaetopterus eggs could be due to an interaction with the cytoskeletal elements known to reside in the cortex of many eggs (Franke et al.
1976; Kidd & Mazia, 1980; Lehtonen & Badley, 1980; Colombo et al. 1981;
Jeffery & Meier, 1983) or organelles associated with the cortical cytoskeleton.
A possible candidate for the latter are the granular, nuage-like bodies recently
described in the ectoplasm of Chaetopterus eggs (Eckberg, 1981).
Segregation of histone and actin mRNA sequences during cleavage
The partitioning of the cortical ectoplasm into animal and vegetal fields and
their unequal division between the AB and CD blastomeres during cleavage
brings up the possibility that specific mRNA sequences may be segregated into
different parts of the embryo. As an initial test of specific mRNA segregation in
situ hybridization with histone and actin DNA probes was carried out on sections
of eggs and cleaving embryos. The signal obtained was sensitive to pretreatment
of the sections with RNase and in situ hybridization with pBR322 failed to
generate a significant autoradiographic signal suggesting that DNA does not
adventitiously bind to the sections (Table 1). Grains were found to be localized
in the cortical ectoplasm with little significant activity in the other regions of the
mature egg (Figs 19-22). In trefoil embryos the animal and the vegetal ectoplasmic fields were labelled to the same extent (Figs 20, 22). Thus the localization
and segregation of cortical histone and actin messages is identical to that of the
total poly(A) + RNA.
Several functional roles can be envisioned for mRNA localization during early
embryonic development. First, the differential segregation of mRNA could
provide metabolically-active cell lineages with an excess of maternal mRNA.
Second, differential mRNA distribution might reflect a translational-level
control mechanism in which messages are physically separated from the protein
synthetic machinery (Showman et al. 1982). Third, some localized mRNA
molecules may be cytoplasmic morphogens which dictate the developmental
choices made by embryonic cell lineages. At present we are unable to decide
between these possibilities; it is possible all three roles may be played during
early development. The Chaetopterus egg, however, provides an excellent system to test for the qualitative segregation of specific mRNA species because the
cortical mRNA mass is split into two parts during cleavage and one part is largely
delivered to the CD blastomere. Although we have shown that the histone and
actin messages are distributed to both the AB and CD cells, this result does not
exclude the possibility that other mRNA species are differentially segregated.
The distribution of many individual messages will have to be determined to
assess the possibility of selective mRNA segregation.
Cortical mRNA
CD
237
AB
7 -
AE
Figs 19-22. In situ hybridization of mature eggs and trefoil embryos with histone and
actin DNA probes. Fig. 19. An autoradiograph of a mature egg treated with the
histone DNA probe showing grains concentrated in the cortical ectoplasm. X400.
Fig. 20. An autoradiograph of a section through a trefoil stage embryo treated with
the histone DNA probe snowing grains in the animal hemisphere and the polar lobe.
Fig. 21. An autoradiograph of a section through a mature egg treated with the actin
DNA probe showing grains concentrated in the cortical ectoplasm. x400. Fig. 22.
An autoradiograph of a section through a trefoil stage embryo treated with the actin
DNA probe showing grains concentrated in the animal hemisphere and the polar
lobe. x400. Animal ectoplasm, (AE); vegetal ectoplasm, (VE); AB blastomere,
(AB); CD blastomere, (CD).
238
W. R. JEFFERY AND L. J. WILSON
Technical assistance was provided by Ms Dianne McCoig. The drawings were executed by
Ms Bonnie Brodeur. This research was supported by grant HD-13970 from the National
Institutes of Health.
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(Accepted 16 February 1983)