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)-I-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.

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 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; Pikó & 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) 4-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.

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 –20 °C. They were embedded in paraplast, sectioned at 8 μm 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 10μg/ml proteinase K (10 min 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.

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 et al. 1980) from Drosophila melanogaster. The 4-6 kilobase (kb) DNA fragment of the Dm-500 complex contains complete sequences of the genes for Hl, 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 × 103 Ci/ mmole; New England Nuclear, Boston, MA) to a specific activity of about 1–5 × 107d.p.m./μg 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 (2·10 μg/ml; 20 μl 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.

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.

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 of fixed and 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.

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 of fixed and 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.

Figs 9, 10.

In 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. ×400. See Table 1 for details of the RNase treatment. Ectoplasm (ec).

Figs 9, 10.

In 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. ×400. See Table 1 for details of the RNase treatment. Ectoplasm (ec).

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). –400. Fig. 12. An autoradiograph of a primary oocyte showing clusters of grains (arrows) over ectoplasmic granules in the endoplasm. –1000. 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). ×400. Fig. 14. An autoradiograph of parapodial oocytes of various sizes showing cortical ectoplasmic grains. ×150.

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). –400. Fig. 12. An autoradiograph of a primary oocyte showing clusters of grains (arrows) over ectoplasmic granules in the endoplasm. –1000. 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). ×400. Fig. 14. An autoradiograph of parapodial oocytes of various sizes showing cortical ectoplasmic grains. ×150.

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 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.

Table 1.

In situ hybridization of Chaetopterus eggs with [3H]poly(U) and I125-labelled actin and histone DNA probes

In situ hybridization of Chaetopterus eggs with [3H]poly(U) and I125-labelled actin and histone DNA probes
In situ hybridization of Chaetopterus eggs with [3H]poly(U) and I125-labelled actin and histone DNA probes
Figs 15, 16.

In 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). ×600. 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). ×400.

Figs 15, 16.

In 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). ×600. 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). ×400.

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. ×1000. 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. ×600.

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. ×1000. 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. ×600.

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. ×400. Fig. 20. An autoradiograph of a section through a trefoil stage embryo treated with the histone DNA probe showing 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. ×400. 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. ×400. Animal ectoplasm, (AE); vegetal ectoplasm, (VE); AB blastomere, (AB); CD blastomere, (CD).

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. ×400. Fig. 20. An autoradiograph of a section through a trefoil stage embryo treated with the histone DNA probe showing 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. ×400. 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. ×400. Animal ectoplasm, (AE); vegetal ectoplasm, (VE); AB blastomere, (AB); CD blastomere, (CD).

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 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 10- and 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 Onco-peltus poly(A)+ RNA is present in the anterior and posterior cortical cytoplasms (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 animal-vegetal 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 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 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 & 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 cyto-skeletal 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.

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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