Range and stability of cell fate determination in isolated sea urchin blastomeres

We have examined the developmental potential of blastomeres isolated from either the animal (mesomeres) or vegetal (macromeres-micromeres) half of 16-cell embryos of the sea urchin Lytechinus pictus. We have also examined the effects of two known vegetalizing agents on the development of isolated mesomeres; LiCl treatment and combination with micromeres, the small blastomeres found at the vegetal pole of the 16-cell embryo. The markers for differentiation used were both morphological (invaginations, spicules and pigment cells) and molecular (gut-specific alkaline phosphatase activity, and monoclonal antibodies against antigens specific for gut and oral ectoderm). Embryoids derived from isolated mesomeres expressed markers characteristic of vegetal differentiation only at very low levels. They did express an antigen characteristic of animal development, the oral ectoderm antigen, but with an altered pattern. Isolated macromere-micromere pairs expressed all markers characteristic of vegetal development, but did not express the marker characteristic of animal development. Increasing concentrations of LiCl caused isolated mesomeres to give rise to embryoids with an increasing tendency to express vegetal markers of differentiation, and it was found that expression of different vegetal markers begin to appear at different concentrations of LiCl. LiCl also caused the marker for oral ectoderm to be expressed in a more normal pattern. Combining micromeres with mesomeres also induced mesomere derivatives to differentiate in a vegetal manner. Micromeres were not completely effective in inducing a more normal pattern of expression of the marker for oral ectoderm. The treatment of isolated mesomeres with both LiCl and micromeres produces a synergistic effect resulting in embryoids expressing markers not induced by either treatment alone.     

Limited complexity of the RNA in micromeres of sixteen-cell sea urchin embryos

The sequence complexity of sea urchin embryo micromere RNA is about 75% of that of total 16-cell embryo cytoplasmic RNA, as reported earlier by Rodgers and Gross [Rodgers, W. H., and Gross, P. R. (1978) Cell, 14, 279–288]. In contrast to the rest of the embryo, there are few, if any, complex maternal RNA species in the micromere cytoplasm which are not represented in the polysomes. The micromeres do not contain detectable quantities of high-complexity nuclear RNA, though such RNA exists in other cells of the fourth-cleavage embryo.     

Injection of myo-inositol reverses the effects of lithium on sea urchin blastomeres

Lithium is known to cause sea urchin blastomeres destined to give rise to epithelium rather than to differentiate into gut or skeleton. While it has been proposed that lithium alters development by interfering with the inositol-tris phosphate-protein kinase C (IP₃-PKC) signaling pathway, the mechanism of action of lithium in sea urchins has remained elusive. Here we describe experiments that examine the hypothesis that lithium exerts its effect on sea urchin embryos via the IP₃-PKC pathway. We make use of methods developed to isolate epithelial precursor cells from the animal hemisphere of cleavage 16-cell stage embryos. Pairs of cells were isolated and one of each pair was injected with either myo-inositol or its inactive isomer, epi-inositol. Rhodamine dextran was co-injected as a lineage tracer to follow the fate of injected cells. We demonstrate that injected myo-inositol, but not epi-inositol, can reverse the effects of lithium on sea urchin blastomeres. This is direct evidence that lithium affects the IP₃-PKC pathway in sea urchins, and that this pathway plays an important role in cell fate determination.     

Potential of veg2 blastomeres to induce endoderm differentiation in sea urchin embryos

Two different modes of gastrulation in sea urchin embryos have been reported. The first mode, reported in Hemicentrotus pulcherrimus and some other species, consists of two phases: a primary and a secondary invagination. The second mode involves gastrulation with a continuous convolution of cells near the blastopore; this mode has been reported to occur in the embryos of the sand dollar, Scaphechinus mirabilis. The rudimentary gut is comprised of fewer cells in the embryos of the former species than in the latter. We assumed that the differences in gastrulation modes could be related to the different potentials of the veg2 layer to induce endoderm differentiation in the upper layer. In the present study, we produced chimeric embryos consisting of an animal cap recombined with veg2 layer blastomere(s) to compare the inductive effect of the veg2 layer and/or the blastomere(s) in H. pulcherrimus and S. mirabilis embryos. Our results showed that the inductive effect of the veg2 layer is stronger in S. mirabilis embryos than in H. pulcherrimus embryos. Moreover, it was suggested that the difference in the strength of inductive effects of veg2 layers is related to the difference in gastrulation modes.     

Similarity of proteins synthesized by isolated blastomeres of early sea urchin embryos

The 16-cell sea urchin embryo has blastomeres of three distinct size classes: micromeres, mesomeres, and macromeres. Each class is already restricted in its developmental fate, micromeres being committed to formation of primary mesenchyme cells. The three classes of blastomeres were isolated in high purity and incubated in [³⁵S]methionine until the next cleavage. Nearly all the radioactive protein was solubilized and subjected to two-dimensional electrophoresis according to O'Farrell. Of approximately 1000 spots resolved, there are no qualitative differences among the three blastomeres. When embryos were labeled between the first and fourth cleavages and blastomeres then isolated, no qualitative differences in protein synthesis were observed. Moreover, there are very few changes when unfertilized eggs are compared to 16-cell embryos. Thus cellular determination during embryonic development is not accompanied by qualitative changes in the distribution within the embryo of abundantly synthesized proteins, virtually all of which are coded for by sequences present in the egg.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

Histones and histone synthesis in sea urchin development

Histones are synthesized and become a part of the chromatin as early as the first cleavage in sea urchins. Reproducible changes in relative amounts of individual histone fractions synthesized are observed during development. A new and electrophoretically distinct very lysine rich fraction appears at hatching in Arbacia and in the early gastrula of Lytechinus. When RNA synthesis is blocked by actinomycin D, maternal mRNA alone can direct a quantitatively and qualitatively changing pattern of histone synthesis as cleavage proceeds. Inhibition of DNA synthesis by hydroxyurea reduces synthesis of histones; the arginine-rich histones are more severely affected than the lysine-rich ones.     

RNA synthesis in unfertilized sea urchin eggs

We have examined the synthesis of messenger-like RNA in unfertilized sea urchin eggs. Most of the RNA synthesized is restricted to the nucleus and sediments from 16 to 30S. A small fraction can be isolated from the postmitochondrial supernatant and displays a sedimentation profile typical of embryonic mRNA with peaks at 9 and 18S. This cytoplasmic RNA is largely present as free RNPs and we estimate that less than 20% of the RNA is in polysomes. The RNA made in the egg is unstable and reaches a steady state with a half-time of about 30 min. We have examined the accumulation of RNA in the egg and have calculated a rate of synthesis of 1.4 × 10^?14 g of RNA/min/egg which is similar, on a per-nucleus basis, to that found in the just-fertilized egg and very early embryo. It is approximately 10 times greater than the rate of RNA synthesis in the blastula nucleus. We estimate that the RNA synthesized by the unfertilized egg amounts to a maximum of 3 × 10^?13 g of potential mRNA at the time of fertilization, or 10–15% of its immediate needs. This RNA cannot account for the increase in protein synthesis that occurs after fertilization, which must be the result of the translation of another population of more stable egg or oogenic mRNA that is kinetically distinct from the RNA we have measured. The steady-state level of labeled RNA present in the egg does not change upon fertilization until after the first cleavage, at about 2.5 hr after fertilization. Thus the RNA synthesis that occurs in the just-fertilized zygote appears to be merely a continuation (at least quantitatively) of the RNA synthesis taking place in the egg.     

Altered expression of spatially regulated embryonic genes in the progeny of separated sea urchin blastomeres

We have examined the importance of the extracellular environment on the ability of separated cells of sea urchin embryos (Strongylocentrotus purpuratus) to carry out patterns of mRNA accumulation and decay characteristic of intact embryos. Embryos were dissociated into individual blastomeres at 16-cell stage and maintained in calcium-free sea water so that daughter cells continuously separated. Levels of eleven different mRNAs in these cells were compared to those in control embryos when the latter reached mesenchyme blastula stage, by which time cells in major regions of the intact embryo have assumed distinctive patterns of message accumulation. Abrogation of interactions among cells resulted in marked differences in accumulation and/or turnover of the individual mRNAs, which are expressed with diverse temporal and spatial patterns of prevalence in intact embryos. In general, separated cells are competent to execute initial events of mRNA accumulation and decay that occur uniformly in most or all blastomeres of the intact embryo and are likely to be regulated by maternal molecules. The ability of separated cells to accumulate mRNAs that appear slightly later in development depends upon the presumptive tissue in which a given mRNA is found in the normal embryo. Messages that normally accumulate in cells at the vegetal pole also accumulate in dissociated cells either at nearly normal levels or at increased levels. In one such case, that of actin CyIIa, which is normally restricted to mesenchyme cells, in situ hybridization demonstrates that the fraction of dissociated cells expressing this message is 4- to 5-fold higher than in the normal embryo. In contrast, separated cells accumulate significant levels of a message expressed uniformly in the early ectoderm but are unable to execute accumulation and decay of different messages that distinguish oral and aboral ectodermal regions. These data are consistent with the idea that interactions among cells in the intact embryo are important for both positive and negative control of expression of different genes that are early indicators of the specification of cell fate.     

Histone synthesis by cleavage arrested sea urchin eggs.

A change in Hl histone synthesis occurs in blastulae, from Hlm to a faster moving Hlg in acrylamide gel electrophoresis. The experiments below indicate that this shift occurs in the absence of normal cleavage. Hybrid embryos containing paternal Hl histone markers along with homospermic embryos were studied. Both were labeled with L-[3H]lysine. Some cultures were kept at 11 degrees C to inhibit cleavage. It was found that Hlm and Hlg are synthesized sequentially in time by embryos grown at 20 degreet C as well as by those grown at 11 degrees C. The hybrid data establish that Hlm is translated at least in part from mRNA newly transcribed from paternal DNA. This observation also holds for cleavage inhibited hybrid embryos. Hlg is made by both hybrid and homospermic embryos during the later phases of development at both 11 and 20 degrees C. These results confirm and extend those of Seale et al. (1973), Ruderman et al. (1974) and Easton et al. (1974).     

Immunocytochemical evidence for the presence of two domains in the plasma membrane of sea urchin blastomeres

Summary The blastomeres of sea urchin embryos have two surface regions with different properties. Numerous microvilli are present in the apical surface region, while the baso-lateral surface region, either on adjoining adjacent cells or facing the blastocoel, is smooth. When blastomeres are isolated from embryos and stained with fluorescein-isothiocyanate-labelled anti-(egg surface) antibody (anti-ES) prepared against membranes isolated from fertilized eggs, the apical microvillous region fluoresces while the smooth region does not [Yazaki I (1984) Acta Embryol Morphol Exp 5∶3–22]. In order to study quantitatively the ‘bindability’ of the membrane in the two regions to anti-ES, immunoelectron microscopy was used. Blastomeres isolated from embryos ofHemicentrotus pulcherrimus at the eight-cell stage were treated with rabbit anti-ES serum or pre-immune serum and then with ferritin-conjugated goat anti-(rabbit IgG) for 10 min at 0°C, mainly before fixation. About 10 times (maximally 45 times) more ferritin particles were counted per contour length in the microvillous surface region than in the smooth surface region. These results suggest that the membrane of the blastomeres of sea urchin embryos is a mosaic of two different membrane territories: one represented by the microvillous surface originating from the unfertilized egg, which binds anti-ES, the other by the smooth surface newly organized after the first cleavage, which does not react with anti-ES. The mechanism of segregation of the membrane into these two regions is discussed.