ABSTRACT
Removal of the polar lobe at the trefoil stage of the first cleavage division of Ilyanassa embryos causes abnormalities much later in development. To determine if the developmental differences between normal and delobed embryos were reflected in alterations in protein synthesis and at what stages of development these become evident, protein solutions were separated by disc electrophoresis on basic acrylamide gels. For the analysis of the newly synthesized proteins, two protein samples, one labelled with 14C and the other with 3H, were combined in the same electrophoretic column. Each was prepared from normal embryos or lobeless embryos at different stages of development.
The distribution of the two groups of differentially labelled proteins was compared by a determination, for each fraction, of the ratio of the normalized 3H/14C counts for that particular fraction (R =3H/14C). The plot of R versus fraction number was studied for various combinations of samples.
During normal development the profile of labelled proteins remains unchanged until the onset of visible differentiation. At this stage, around day 4 of development, there are changes in biosynthesis revealed by a greater emphasis on the synthesis of slow moving proteins. The profile of labelled proteins of lobeless embryos remains unchanged up to the 5th day of development. This result is correlated with the absence, in the lobeless embryos, of many of the visible differentiations.
Preliminary studies revealed that the spectrum of labelled proteins of the polar lobe is identical to the one present in lobeless embryos and in normal embryos in early stages of development. This suggests the possibility that the morphogenetic factors associated with the polar lobe are not among the newly synthesized proteins. A hypothesis is presented to account for the effects on morphogenesis and protein synthesis which are produced by removal of the polar lobe.
INTRODUCTION
The role played by the egg cytoplasm or its component organelles in establishing the initial patterns of differentiation is classically exemplified in the spiralian egg of molluscs and annelids. Studies on the normal development of spiralian blastomeres in ovo and in vitro (Conklin, 1896; Wilson, 1892, 1904a,b; Hatt, 1932; Rattenbury & Berg, 1954; Clement, 1952, 1956) indicated that during the early cleavage divisions of these eggs there is a segregation of qualitatively different cytoplasmic areas, resulting in the formation of daughter cells with different morphogenetic potential.
One of the early manifestations of the regional differences in the egg is the appearance, either during maturation or after fertilization, of distinct cytoplasmic regions called pole plasms. In Ilyanassa these pole plasms are transferred to transitory lobulations, polar lobes, which are formed in the vegetal region of the egg during maturation and the first three cleavage divisions. During the first cleavage division, the polar lobe and the two blastomeres form a trefoil figure (trefoil stage). The polar lobe is later resorbed into blastomere CD (see Clement, 1952, for detailed description of early cleavage stages). It is formed again in the 2nd cleavage division and is then included in the D blastomere. In later divisions, the pole plasm is transferred to descendants of the D cell.
The removal of the polar lobe results in the formation of a viable larva, which lacks bilateral symmetry, mesodermal bands, eyes, foot, heart, intestine and a normal shell (Wilson, 1904; Clement, 1952; Cather, 1967). These results indicate that both the D blastomere and the polar lobe contain factors required for the differentiation of most of the post-gastrular structures. In Ilyanassa, Clement (1962) found that the determining effect of the polar lobe is exerted in the period extending from the formation of the 3rd to the 4th quartet of micromeres (5th–6th cleavage divisions).
Although many studies have been carried out at the cytochemical and histological level demonstrating certain structural features of this localization (Clement & Lehman, 1956; Weber, 1961; Pucci-Minafra, Minafra & Collier, 1969; Humphreys, 1964; Crowell, 1964), they do not provide any causal evidence for the unique morphogenetic potentialities of this region of the egg.
The possibility of detecting biochemical differences in the egg has been explored by several investigators (Berg & Kato, 1959; Collier, 1960, 1961). In Ilyanassa, Davidson et al. (1965) found that synthesis of what probably is informational RNA is higher in normal than in lobeless embryos after only 10 h of development, even though the initial 10 h period includes the formation of the D cell derivatives. If the activation of RNA synthesis represents an increase in the amount of functional genome, it would indicate that factors present in the polar lobe are responsible for the activation of genes which determine the differentiation of lobe dependent characteristics. The lobeless embryo, then, would fail to establish these required specific patterns of gene activity.
If the role of the polar lobe is, in effect, to elicit specific patterns of gene activation, there should be differences in the types of proteins synthesized during development by the normal and lobeless embryo. The present report is an analysis of the normal spectrum of newly synthesized proteins throughout early development and an examination of the effects of polar lobe deletion on this pattern of changes.
Studies on the effect of polar lobe on the rate of protein synthesis in Ilyanassa embryos, as well as a comparison of the detailed pattern of bulk proteins of normal embryos, lobeless embryos and isolated polar lobes will be reported in another publication.
MATERIALS AND METHODS
The experiments were performed with the prosobranch gastropod Ilyanassa obsoleta Stimpson (Nassarius obsoletas Say, 1822). The animals were purchased from the Marine Biological Laboratory (Woods Hole, Mass.), kept in a 15 gal (57 1.) recirculating sea-water aquarium in an air-conditioned room at 23°C and fed fresh clams on alternate days. The culture methods followed were those suggested by Costello et al. (1957).
The eggs, in numbers ranging from 60 to 200, are enclosed in capsules which are deposited on the walls of the aquarium. Egg capsules were collected every morning, the age of the eggs was determined and only those capsules containing fertilized but uncleaved eggs were used. After cleaning the capsules by rolling them over filter paper, they were deposited in Syracuse watch-glasses containing Millipore filtered sea water (MFSW) and cut with fine curved scissors. The eggs were then flushed away from the capsules with a water current from a fine pasteur pipette.
Culture of control embryos
The eggs were collected, washed twice in MFSW and allowed to develop, with gentle agitation, at 17 ± 2°C. The sea water was changed daily.
Preparation of lobeless embryos
The uncleaved eggs were transferred to Van’t Hoff Ca-free sea water (NaCl 25·23 g/1.; KC1 0·71 g/1.; CaCl2.2H2O 0·64 g/1.; MgCl2.6H2O 6·86 g/1.; MgSO4.7H2O 4·07 g/1.). The polar lobes were removed at the trefoil stage by pipetting the eggs through a fine bore pipette, under mouth pressure, once or twice. The diameter of the tip of the pipette was only slightly larger than that of a single blastomere -that is, smaller than the whole egg.
Lobeless eggs were collected with a pipette at the end of the day, washed once in MFSW and cultured for the desired period of time. The polar lobes can be distinguished from isolated blastomeres by their content of yolk and the presence of a nuclear region in the blastomeres. Furthermore, at the time when the lobeless eggs were collected, the blastomeres had already cleaved once or twice.
Incorporation of radioactive precursors
When the eggs were in the proper stage of development, they were washed twice with MFSW and incubated in a solution containing 8 μCi/ml of either a [14C]amino acid mixture (New England Nuclear, Boston, Mass. 02118) (NEN, 0·1 mCi/ml) or a [3H]amino acid mixture (NEN, 1·0 mCi/ml). The culture mixture was maintained at pH 8 by the addition of the necessary amount of a saturated solution of NaHCO3 in sea water. Phenol red was used as a pH indicator.
The eggs were incubated with isotopes for 2 h at 18°C with gentle agitation. At the end of this period, the eggs were collected with a pipette, washed twice in ice cold MFSW, pelleted by a short centrifugation, all the sea water removed and storedin a freezer. This procedure was used for both normal and lobeless embryos.
Polar lobes remained in Ca-free sea water for approximately 10 h. They were then collected and immediately afterwards placed in MFSW solution containing 4 μCi/ml of a [3H]amino acid mixture (NEN, 1·0 mCi/ml) plus phenol red and NaHCO3 as described above. After 4 h the polar lobes were removed from the isotope medium and washed twice with ice cold MFSW. They were then collected with a pipette, pelleted by centrifugation and stored in a freezer.
Preparation of the protein sample
The eggs, in numbers ranging from 500 to 1000, were allowed to thaw and were homogenized in cold buffer (0·02M Tris pH 9; 0·1 M-KCI; 0·006M-MgCl and 0·25 M sucrose). The homogenization was carried out with a loose-fitting, motor-driven, fine glass rod at 5°C. The homogenates were centrifuged at 5000 rev/min for 20 min and then at 39000 rev/min for 1 h. The clear supernatant was removed and stored frozen.
Protein concentration was determined by the Lowry technique (Lowry, Rosebrough, Farr & Randall, 1951) in 0·05 ml samples. Standards were prepared from crystallized bovine plasma albumin (Calbiochem) diluted in the homogenization buffer in different concentrations.
The incorporation of radioactive amino acids into trichloroacetic acid (TCA) insoluble protein was determined with the paper-disc method of Mans & Novelli (1961).
The scintillation cocktail used was Spectrafluor (Nuclear Chicago) diluted in 1 gal (3·8 1.) of toluene to give the standard concentration of 4 g PPO and 50 mg POPOP.
Electrophoresis
Electrophoresis in basic gels was carried out using 7% acrylamide gels and buffers according to standard procedures (Davies, 1964). The electrophoretic columns had a 6 mm internal diameter and a length of 16 cm. The spacer gel and separating gel were 3 cm long and 10 cm long respectively.
For the analysis of the distribution of the newly made, pulse-labelled proteins, the electrophoretic columns contained both the 3H-and 14C-labelled protein samples which were mixed in various combinations. The ratio of the 3H/14C counts was always at least 2:1, and when the availability of the material made it possible, this ratio was changed to 10:1. This variation in the ratio between the two markers did not affect the final results since they were repeatable independently of the ratio used.
Electrophoresis took place at 5°C with an initial voltage of 100 V (1 mA/tube), and when the tracking dye formed a band in the spacer gel the current was increased to 3 mA/tube (600 V). The run took approximately 4h. The gels were removed from the electrophoretic columns and left overnight in 10% solution of TCA at 5°C. The gels were then washed for 5 min with tap water and fractionated with the Maizel apparatus (Maizel, 1966). The eluent used was 0·1M-NH4OH. The fractions, collected in scintillation vials, were air-dried in a 20°C incubator. To each vial was added 0·1 ml of 0·1 N-NaOH and, after 0·5 h, the gels were solubilized with 0·5 ml of NCS (Nuclear Chicago Solubilizer, Amersham/Searle). Two hours later 10 ml of scintillation fluid were added to each vial.
Counting was performed with a Packard Scintillation Spectrometer, appropriately set for double label techniques (Packard Manual, 1967). Preliminary experiments revealed a small, and reproducible, amount of quenching. The counting efficiencies were 15% and 40% for the low-and high-energy isotope respectively with a 4% spillover of 14C counts in the 3H channel. The final 3H counts were corrected for this effect. The samples were counted for 10 min each. The background level was 200/10 min and only those samples with 100 above this level were considered significant.
In order to check that the radioactivity present in the gels corresponded only to that incorporated into proteins, electrophoretic columns were prepared in the standard way and on top of the separating gel was added a solution of 0·8 μCi of amino acid mixture diluted in sample gel. After a 5 h electrophoresis the gels were removed from the glass tubes and immediately fractionated with the Maizel apparatus. The fractions were then solubilized as previously described. From an input of 1·12× 106 counts, 0·9×106 were recovered, indicating that at least 80% of the amino acids were retained in the gels at the end of the electrophoretic period. This evidence prompted us to include a step in which the gels were left overnight in a 10% solution of TCA prior to the fractionation. In this way the proteins were precipitated in situ and the amino acids could diffuse from the gel. When gels contained only amino acids, the number of counts was at background level after the 10% wash.
Analysis of bulk proteins
The gels were stained overnight with a solution of 1% AmidoSchwartz 10B in 7% acetic acid. Destaining was carried out by repeated washing with 7% acetic acid. The gels were scanned and recorded with a Densicord (Recording Electrophoretic Densitometer, Model 542, Photovolt Co.).
RESULTS
Analysis of the spectra of labelled proteins synthesized by normal embryos at different days of development
Samples of radioactive proteins were obtained from normal embryos from the 1st to the 7th day of development. The electrophoretic columns were loaded with two protein solutions. One of them was prepared from 4-day embryos labelled with [14C]amino acids (standard). The other sample, labelled with [3H]amino acids, was obtained from normal embryos at other developmental stages. All gels were processed as described in Materials and Methods.
Electrophoretic (acrylamide gel) profiles of radioactivity of proteins extracted from 4-day-old and 7-day-old Ilyanassa embryos after incorporation of [14C-L-] amino acids (……) and [3H-L-]amino acids (_______ ) respectively. Total radioactivity in protein per applied sample: 7-day-old embryo, 1·2 × 105 cpm; 4-day-old embryo, 5·8 × 104 cpm.
Electrophoretic (acrylamide gel) profiles of radioactivity of proteins extracted from 4-day-old and 7-day-old Ilyanassa embryos after incorporation of [14C-L-] amino acids (……) and [3H-L-]amino acids (_______ ) respectively. Total radioactivity in protein per applied sample: 7-day-old embryo, 1·2 × 105 cpm; 4-day-old embryo, 5·8 × 104 cpm.
The plot of the normalized ratio (R) versus fraction number was studied for different combinations of samples.
If the labelled proteins present in the two samples which were run together are quantitatively and qualitatively identical, the R values for each fraction should be equal to one or show very small oscillations around this number.
Large increases in the synthesis of some 14C proteins would be indicated by R values lower than 1 for those particular fractions. On the other hand, R values greater than 1 would point to a relative increase in the synthesis of the proteins labelled with 3H (recall that R = 3H/14C).
To determine the normal variations of R values to be expected with this technique, one sample of 4-day-old embryos was divided in two parts, one labelled with 14C and the other with [3H]amino acid mixtures respectively. Fig. 2 shows the small oscillations of R around the line of identical distribution (R = 1). The data of the experiment shown in Fig. 2 were used to determine what constitutes a significant deviation of R. Considering a confidence level of 99·7%, R = 1·004 ± 0·3.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from one batch of 4-day-old normal embryos divided into two groups, one labelled with [14C-L-]amino acids and the other with [3H-L-]amino acids respectively. The gel was fractionated into 60 fractions. R values were obtained as described in the text.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from one batch of 4-day-old normal embryos divided into two groups, one labelled with [14C-L-]amino acids and the other with [3H-L-]amino acids respectively. The gel was fractionated into 60 fractions. R values were obtained as described in the text.
Fig. 3 demonstrates a comparison of 1-day embryos labelled with 3H and 4-day embryos (standards) labelled with 14C. The small oscillations of R around the line of identical distributions indicate that these two samples, as well, are nearly identical. Moreover, the same results were obtained when the labels were reversed; that is, when the 1-day and the 4-day embryos were labelled with 14C and 3H respectively. This indicates that the results are independent of the type of isotope used to label the various samples.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 1-day-old and 4-day-old normal Ilyanassa embryos, labelled with [3H-L-]amino acids and [14C-L-]amino acids respectively. R values were obtained as described in the text.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 1-day-old and 4-day-old normal Ilyanassa embryos, labelled with [3H-L-]amino acids and [14C-L-]amino acids respectively. R values were obtained as described in the text.
Fig. 4 compares 3-day-old and 4-day-old embryos. The small oscillations of R values from 1 indicates great similarity between the two samples being compared. Standards (4-day embryos) labelled with 14C were then compared with 5-, 6-and 7-day embryos labelled with 3H. The results were similar in all cases;
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 3-day-old normal embryos (labelled with [3H-L-]amino acids) and 4-day-old normal embryos (labelled with [14C-L-]amino acids). Both samples were run in the same gel. lvalues were calculated as described in the text.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 3-day-old normal embryos (labelled with [3H-L-]amino acids) and 4-day-old normal embryos (labelled with [14C-L-]amino acids). Both samples were run in the same gel. lvalues were calculated as described in the text.
Fig. 5 shows a typical example of the relative increase in the proportion of the slow-moving proteins, labelled with 3H synthesized by the older embryos (7-day-old in this instance). Comparison of Fig. 5 with Fig. 1, both obtained using the same data, reveals the advantage of the use of the technique employed here in the analysis of the results.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 7-day-old normal embryos labelled with [3H-L-]amino acids and 4-day-old embryos labelled with [14C-L-]amino acids. Both samples were run in the same gel, as in all other cases. R values were calculated as described in the text.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 7-day-old normal embryos labelled with [3H-L-]amino acids and 4-day-old embryos labelled with [14C-L-]amino acids. Both samples were run in the same gel, as in all other cases. R values were calculated as described in the text.
From these observations it can be concluded that after the 4th day of development there is a dramatic change in the biosynthetic pattern of normal embryos, reflected as a relative increase in the synthesis of proteins at the cathodic pole, i.e. slow-moving proteins. This change is coincidental with the onset of visible differentiation. Gastrulation is completed in the 2nd day of development. The 3-day embryos show stomodeal invagination and the shell gland begins to form (Cather, 1967); while secretion of shell material starts 1 day later. The primordia of foot, heart and other organs also make their appearance during the fourth day. Two days later the embryos develop into a fully differentiated veliger larvae.
When the gels were stained for bulk protein analysis, the same profile was obtained throughout development. Fig. 6 shows a typical densitometer recording of the protein spectra of a normal embryo. This result is not surprising since most of the bulk proteins are probably inherited from oogenesis and the level of new synthetic activity is probably too low to be detected with the staining techniques used. Under these circumstances it is not possible to compare the staining profile with the distribution of labelled proteins.
Proteins synthesized by the lobeless embryos
The spectra of soluble proteins synthesized by lobeless embryos at different stages of development were studied. Protein samples were obtained from lobeless embryos labelled with 3H and normal embryos labelled with 14C. All gels were processed as described in Materials and Methods.
Fig. 7 compares the distribution of labelled proteins of 1-day lobeless and normal embryos. It is evident that the distribution and relative proportion of the newly made proteins in both samples is remarkably similar. The same result was obtained when 1-day lobeless and 3-day lobeless were compared to 4-day normal.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profile of radioactive proteins extracted from 1-day-old lobeless and normal embryos labelled with [3H-L-]amino acids and [14C-L-]amino acids respectively. R values were calculated as described in the text.
Important differences were noted when 5-day normal and lobeless were compared (Fig. 8). In this case the fluctuations of the R values about the line of identical distribution (R = 1) indicate a relative increase in the proportion of the [14C]proteins (from normal embryos), in the cathodic side of the gel (i.e. R < 1) with a concomitant diminution of these proteins towards the anodic side of the gel.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 5-day-old lobeless embryos labelled with [3H-L-]amino acids and 5-day-old normal embryos labelled with [14C-L-]amino acids. R values were calculated as described in the text.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profiles of radioactive proteins extracted from 5-day-old lobeless embryos labelled with [3H-L-]amino acids and 5-day-old normal embryos labelled with [14C-L-]amino acids. R values were calculated as described in the text.
The temporal changes in the protein profile which characterize normal development appear to be absent in the lobeless series, which appears to retain continuously the younger conditions.
Fig. 9 illustrates, in a highly simplified fashion, the results obtained in the comparison of the newly made, pulse-labelled proteins. It assumes the existence of the same two proteins in each of the samples analysed. Graph C illustrates the hypothetical profile obtained when these two samples are quantitatively identical, i.e. when the comparison is made between two samples of normal embryos younger than the 4th day of development, or between these young normal embryos and lobeless ones.
Hypothetical model. In the C graph both samples are quantitatively identical. In the A graph, there is a proportional increase in the 3H-labelled proteins in the cathodic side of the gel. A1 shows a plot of the number of counts (normalized against the total number of counts of that isotope present in the gel) against fraction number, while the A2 graph indicates the corresponding R values v. fraction number. In the B scheme there is a proportional increase of the [14C]proteins in the cathodic side of the gel. See text for more details.
Hypothetical model. In the C graph both samples are quantitatively identical. In the A graph, there is a proportional increase in the 3H-labelled proteins in the cathodic side of the gel. A1 shows a plot of the number of counts (normalized against the total number of counts of that isotope present in the gel) against fraction number, while the A2 graph indicates the corresponding R values v. fraction number. In the B scheme there is a proportional increase of the [14C]proteins in the cathodic side of the gel. See text for more details.
In the graphs on the right it is assumed that changes had already occurred. Our results for the control series are exemplified by the A diagram while those of the lobeless series follow the B scheme. In part A the proteins of normal embryos older than the 4th day of development are labelled with [3H]amino acids and the plot of R(R =3H/14C) versus fraction number indicates that R > 1 in the region of the slow-moving proteins while the converse situation is shown in the anodic pole of the gel (R < 1). In the B diagram the proteins of the lobeless embryos are labelled with 3H while those of normal embryos older than the 4th day of development are marked with 14C. When the R values are obtained (Fig. 9, B2) it is observed that in the region of the slow-moving proteins R < 1, indicating the presence of more [14C]-than [3H]proteins. In the region of the fast-moving proteins, on the contrary, R > 1, indicating the presence of more 3H than 14C proteins. These results suggest that lobeless embryos synthesize proportionally more fast-and less slow-moving proteins than normal (14C) embryos older than the 4th day of development.
Proteins synthesized by the polar lobe
The significance of the polar lobe in development has been revealed in a complete series of experiments by Clement (1952, 1956). Clement & Tyler (1967) showed that the polar lobe was active in protein synthesis. No evidence was presented to indicate whether the proteins synthesized by the polar lobe are different from those synthesized by the rest of the egg. To analyse this problem the distribution of labelled proteins synthesized by the polar lobe was studied. The 3H-labelled polar lobe proteins were compared with the ones synthesized by normal 4-day-old embryos labelled with 14C (Fig. 10).
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profile of radioactive proteins extracted from polar lobes labelled with [3H-L-]amino acids and 4-day-old normal embryos labelled with [14C-L-]amino acids. Both samples were run in the same gel. R values were calculated as described in text.
Plot of R values v. fraction number obtained from electrophoretic (acrylamide gel) profile of radioactive proteins extracted from polar lobes labelled with [3H-L-]amino acids and 4-day-old normal embryos labelled with [14C-L-]amino acids. Both samples were run in the same gel. R values were calculated as described in text.
Due to the difficulty in collecting large number of polar lobes, only one run was completed with this material. Since the fluctuations around 1 of the values of R obtained are relatively small, it is possible that these data represent only the intrinsic variability of the analytical techniques used. They indicate that, within the range of the electrophoretic mobilities studied, the proteins synthesized by the polar lobe are not different from those synthesized by the rest of the egg. These data do not rule out the possibility that unique polar lobe proteins are synthesized upon maternal templates during oogenesis (G. Teitelman, in preparation) and have escaped the isotopic label.
DISCUSSION
During development of Ilyanassa embryos, the spectrum of protein synthesis seems to remain unchanged until the onset of visible differentiation, when new biosynthetic patterns appear. Changes in the types of proteins synthesized are not detected during the first few days of development, when the determination of various cell types takes place. Davidson et al. (1965) found no difference in the rate of ribonucleic acid synthesis between normal and lobeless embryos until the early post-gastrular stages.
The set of proteins which appears after gastrulation has not yet been characterized in terms of function and/or enzymic properties. Morrill & Norris (1965), using histochemical techniques for the analysis of the enzyme bands separated by starch-gel electrophoresis, reported an alteration in the pattern of activity of various hydrolytic enzymes during development of Ilyanassa embryos. Although they measured enzyme activity rather than protein synthesis per se, these data agree with the present study as to the period in development characterized by dramatic changes in the protein profile.
An analysis of the spectrum of labelled proteins synthesized by normal embryos indicates that slow-moving proteins (high-molecular-weight proteins?) are synthesized at a greater rate during later stages of development, coincident with the differentiation of tissue and organ primordia. Since the large number of proteins present in these soluble extracts makes a more detailed examination of each particular band very difficult, it is not possible to conclude that this protein increase is the result of the synthesis of novel species of proteins. There are, however, in other systems, many observations that differentiation involves the formation of new protein species (Perlmann, 1953; Westin, Perl-mann & Perlmann, 1967; Cooper, 1950; Clayton, 1953; Denis, 1961).
The evidence that factors localized in the polar lobe are responsible for the selective activation of the blastomere genome stems from the wealth of information obtained from deletion experiments (Crampton, 1896; Wilson, 1904a,b; Clement, 1952). Results from the present study are consistent with this evidence. They clearly indicate that although lobeless embryos do express some differentiation they are morphologically deficient and they fail to establish the new pattern of protein synthesis characteristic of normal development, even after 5 days. It is possible that changes related to differentiation of lobeless embryos become apparent at still later stages not studied here.
Davidson et al. (1965) have shown that the effects of polar lobe deletion on transcription become evident about the 2nd day of development, as a slower-than-normal rate of synthesis in lobeless embryos. Since the first differences in protein synthesis between normal and lobeless embryos appear after the 4th day of development, it is possible that the genetic information being transcribed by normal embryos during early development contains, in addition to the program produced by the lobeless embryos, the information required for the formation of certain larval structures. Studies on the effect of actinomycin D on the development of Ilyanassa embryos also suggest the existence of a gap between transcription and translation (Collier, 1965).
Biochemical studies of spiralian development must ultimately be concerned with the actions of morphogenetic factors localized in specific regions of the egg. The development of methods to study the process of localization may reveal answers to the problem of specification of the initial patterns of cell differentiation in Spiralia and other systems.
ACKNOWLEDGEMENTS
The author takes pleasure in acknowledging the constant encouragement and advice of her preceptor, Dr C. E. Wilde Jr, throughout the course of this work. She is also grateful to Dr R. Piddington for his criticism during the writing of this paper. This paper is based on a dissertation submitted to the Biology Department of the University of Pennsylvania in partial fulfilment of the requirements for the degree of Doctor of Philosophy.