Arthur H. Whiteley

Isolation, culture, and differentiation of echinoid primary mesenchyme cells

SummaryMethods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.

A change in permeability and uptake of [14C]uridine in response to fertilization in Strongylocentrotus purpuratus eggs

Abstract 1. 1. The extent to which the unfertilized Strongylocentrotus purpuratus egg is permeable to uridine has been investigated by experiments measuring the partitioning of [ 14 C]uridine between the inside and outside of the cell while the eggs remained suspended in the radioactive uridine and after the eggs were washed with ice-cold sea water. It was found that very little uridine entered the unfertilized egg. After fertilization, the label became concentrated in the egg and depleted from the environment. The label was not lost from the eggs by washing with sea water. 2. 2. Uridine uptake after fertilization has been in part characterized by its time course, dependence on exogenous uridine concentration and response to 2,4-dinitrophenol. A lag phase, acceleration phase and constant accumulation phase was found in the rate of uridine uptake which reached a plateau within the first hour of development. Once the maximal rate of accumulation was attained, fertilized eggs were shown to be very efficient in concentrating very small amounts of exogenous [ 14 C]uridine. As the concentration of uridine was raised beyond 0.5–1.0 μM in the sea water, there was no further increase in the rate of uptake. 2,4-Dinitrophenol-inhibited uridine uptake. At −3° only a very small amount of uridine penetrated into the eggs, and they did not reach equilibrium with the outside concentration, indicating penetration involves more than diffusion. 3. 3. Most of the radioactivity taken up by the fertilized egg remained soluble in ice-cold 0.2 M perchloric acid. No appreciable free [ 14 C]uridine was detected inside the fertilized eggs. The soluble radioactivity was identified on the basis of anion-exchange column chromatography to be in the form of phosphorylated nucleosides, primarily triphosphates. 4. 4. The observations are believed to indicate that fertilized eggs accumulate uridine by phosphorylating the nucleoside at the surface of the cell, a process which may be deficient in unfertilized eggs because of lack of phosphate donors or of the necessary enzymes.

Differential expression of the msp130 gene among skeletal lineage cells in the sea urchin embryo: a three dimensional in situ hybridization analysis

In order to examine the ontogeny of tissue-specific expression of the msp130 gene during early embryogenesis of the sea urchin, we have developed a whole-mount, non-radioactive in situ hybridization protocol suitable for these embryos. This protocol is adapted from the existing technology of immunohistochemical localization of digoxygenin-labelled hybridization probes in tissue sections. Transcript distribution patterns in the whole embryo are seen in three dimensions, and at much higher resolution and sensitivity than can be achieved using radioactive probes and sectioned material. We have traced the ontogeny of expression of the skeleton-specific gene, msp130, during the development of Strongylocentrotus purpuratus. Transcripts are first detected at the blastula stage, in micromere-lineage cells just prior to ingression. Appearance of msp130 transcripts remains strictly limited to this lineage through the pluteus stage. Estimated from the relative intensity of staining of the PMCs of an embryo, the relative abundance of msp130 transcripts is uniform among the 32 cells of this lineage in secondary mesenchyme blastulae and in gastrulae, indicating that expression is homogeneous among these cells up to the early prism stage. However, the relative intensity of stain, and therefore abundance of transcripts, changes dramatically and in a consistent pattern among the PMCs of an embryo during prism and pluteus stages, suggesting that these cells switch from an autonomous mode of regulation of the msp130 gene, to an inductive mode. In the pluteus larva, the highest levels of expression occur in those cells associated with the rapidly growing tips of the spicular skeleton.

The program of protein synthesis during the development of the micromere-primary mesenchyme cell line in the sea urchin embryo

Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [3H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. this peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of grastrulation.

Pheromonal stimulation of spawning release of gametes by gonadotropin releasing hormone in the chiton, Mopalia sp.

The chiton Mopalia sp., a mollusc, was exposed to various dilutions of gonadotropin releasing hormone (GnRH) in sea water to determine whether this peptide is capable of acting as a pheromone that could stimulate release of ripe gametes (spawning). Two of the peptides, lamprey GnRH-1 and tunicate GnRH-2, had this action at a higher concentration (1.0 mg/L) but dilutions to 50 microg/L no longer were effective. Three other GnRHs: lamprey GnRH-3, tunicate GnRH-1, and a modified chicken GnRH-2, had no such action under the same test conditions. Since the spawning response could be produced by some GnRHs and not by others, it would appear that some kind of molecular recognition is involved, possibly by specific binding to a receptor. In earlier preliminary experiments tunicate GnRH-2 rapidly stimulated gamete release in a hemichordate, Saccoglossus. Thus it is suggested that GnRHs, in at least some invertebrates, may function as pheromones, serving to stimulate simultaneous spawning of individuals in a population of animals, and in this way assure more successful fertilization in species that must release their gametes into the water in which they live.

l-malate dehydrogenase in the development of the sea urchin Strongylocentrotus purpuratus

Abstract l -Malate dehydrogenase ( l -malate:NAD oxidoreductase EC 1.1.1.37) of the sea urchin Strongylocentrotus purpuratus occurs in two forms. One form is shown to be localized in mitochondria of this species. The other form occurs exclusively in the cytoplasm and appears as a supernatant enzyme. These two enzymes of the sea urchin were separated and partially purified by ammonium sulfate fractionation and DEAE-cellulose chromatography. Of the two, the supernatant enzyme is eluted first from the chromatographic column and migrates more slowly toward the anode in disc electrophoresis. Catalytic activities of both enzymes were determined with varying concentration of substrates. Substrate inhibition occurred with the supernatant enzyme at high concentration of malate and with the mitochondrial enzyme at high concentration of oxaloacetate. These substrate-activity relationships are similar to those of corresponding enzymes of many other animals. During development, the total enzyme activity in the homogenate increases three fold at a young pluteus stage from the level of the activity in the unfertilized eggs, most of the increase occurring after hatching. Only two isozymes were found in the eggs and embryos, and the increase in the activity occurs equally in both supernatant and mitochondrial enzymes. The increase does not appear to be due to a release from inhibition by a soluble inhibitor in the unfertilized eggs. Electrophoretic mobilities of isozymes of the strongylocentrotids, S. purpuratus and S. droebachiensis and of Dendraster excentricus are sufficiently different to be of diagnostic value in differentiating species origins of the enzymes. Hybrid embryos were produced by crossing the gametes between the two species. Only the enzymes of the maternal species were found in the blocked hybrid embryos.

Conservatism of base sequences in RNA for early development of echinoderms

Abstract The homology among repetitious DNA sequences in the genomes of Strongylocentrotus purpuratus, S. droebachiensis, S. franciscanus, Dendraster excentricus, and Pisaster ochraceus has been determined by DNA-DNA annealing experiments. The extent of cross-reactivity was 70% for the DNAs of the congeners, 10% for the sand dollar and the strongylocentrotids, and about 7% for the asteroid and echinoid DNAs. Extreme conservatism is, therefore, characteristic of a small portion of these echinoderm genes despite long evolutionary separation. Some base sequence divergence has occurred in this conservative part of the genome as judged by an analysis of the thermal dissociation of DNA duplexes. Heteroduplexes had transition temperatures 0–4°C below that of the homoduplex, equivalent to 0–2.8% base pair mismatching. As previously found for sea urchin development, two general RNA populations characterize sand dollar development: a population of stable molecules in the unfertilized egg representative of genes that continue to be active as late as the prism larva, and qualitatively new transcripts that appear after the hatching blastula stage. From a comparative study of the latter populations of transcripts among five echinoderm species, it is concluded that these RNAs are species specific to an extent reflecting the systematic relationships and the DNA relatedness. The evidence was derived from a measurement of the complementarity of the RNAs to the DNAs of the five species, and by competition experiments. In contrast, the frequently represented RNAs of the unfertilized eggs of all four echinoid species are nearly identical as determined by competition experiments, and are homologous to the same DNA sites. Thus, even at superordinal levels, these eggs have substantially the same populations of the frequent RNA molecules representing genes that have been extremely conservative during evolution. A moderate population of RNA transcripts sharing these properties exists in unfertilized P. ochraceus eggs. Competition experiments using high RNA:DNA ratios indicate that the unfertilized eggs contain a smaller proportion of rare or infrequent RNA transcripts which is derived from an evolutionarily diverse, nonconserved part of the genome.

Coordinate accumulation of five transcripts in the primary mesenchyme during skeletogenesis in the sea urchin embryo

The sea urchin larval skeleton is produced by the primary mesenchyme (PM), a group of 32 cells descended from the four micromeres of the 16-cell embryo. The development of this lineage proceeds normally in isolated cultures of micromeres. A complementary DNA (cDNA) library was generated from cytoplasmic polyadenylated RNA isolated from differentiated micromere cultures of Strongylocentrotus purpuratus. Five clones were selected on the basis of their enrichment in differentiated PM cell RNA as compared to the polyribosomal RNAs of other embryonic cell types and other developmental stages. Each cloned cDNA hybridized to a distinct RNA that was abundant in the polyribosomes of differentiated PM cells, but absent from larval ectoderm and from 16-cell embryos. These RNAs were encoded by single or low copy genes. In situ hybridization analysis of the most abundant of these RNAs (SpLM 18) demonstrated that it was specifically limited to the skeletogenic PM of intact embryos. During the development of the PM, all five RNAs exhibited the same schedule of accumulation, appearing de novo, or increasing abruptly just before PM ingression, and remaining at relatively high levels thereafter. This pattern of RNA accumulation closely paralleled the pattern of synthesis of PM-specific proteins in general (Harkey and Whiteley, 1983) and of the SpLM 18-encoded protein specifically (Leaf et al., 1987). These results indicate that at least five distinct genes in the sea urchin, each of which encodes a PM-enriched or PM-specific mRNA, are expressed with tight coordination during development of the larval skeleton. They also demonstrate that expression of these genes in the PM is regulated primarily at the level of RNA abundance rather than RNA utilization.

Unequal cleavage and the differentiation of echinoid primary mesenchyme

The role of unequal cleavage in echinoid micromere determination was investigated by equalizing the fourth and fifth cleavages with brief surfactant treatment. The surfactant sodium dodecyl sulfate was found to be effective in equalizing fourth cleavage when generally applied to 4-cell stage embryos of all species tested. Embryos of the sand dollar Dendraster excentricus developed normally when equalized at the fourth and fifth cleavages by surfactant treatment, as did untreated equally cleaving embryos of the sea urchin Strongylocentrotus droebachiensis. Embryos of the sea urchins Lytechinus pictus and S. purpuratus were animalized by the treatment but were capable of forming spicules after treatments which equalized the fourth cleavage. In addition, orientation of the fourth division spindles was found to have no effect on differentiation of the primary mesenchyme in D. excentricus. The results confirm that micromere determination in echinoids does not depend upon a strict cleavage pattern at the 16-cell stage.

The expression of embryonic primary mesenchyme genes of the sea urchin, Strongylocentrotus purpuratus, in the adult skeletogenic tissues of this and other species of echinoderms.

Adult tissues of the sea urchin, Strongylocentrotus purpuratus, were analyzed for the products of a set of genes whose expression, in the embryo, is restricted to the skeletogenic primary mesenchyme (PM). Three embryonic PM-specific mRNAs were found to be abundant in adult skeletal tissues (test and lantern), but not in a variety of soft tissues. Homologous mRNAs were also found in skeletal tissues of the congeneric sea urchin, S. droebachiensis, as well as a more distantly related echinoid, Dendraster excentricus, and an asteroid, Evasterias troschellii. The distributions of two of these RNAs were analyzed in regenerating spines of adult S. purpuratus using in situ hybridization. These gene products were localized primarily in the calcoblasts that accumulated at the regeneration site. In nonregenerating spines SpLM 18 RNAs, the most abundant of these gene products, were localized in a small population of noncalcoblast cells scattered through the spine shaft, and were absent from calcoblasts. These observations suggest that a program of gene expression associated with the process of calcification is conserved both developmentally through the period of metamorphosis and evolutionarily among the echinoderms.