Susan G. Ernst

Whole mount in situ hybridization shows Endo 16 to be a marker for the vegetal plate territory in sea urchin embryos

We have used whole mount in situ hybridization to analyze the pattern of expression of the gene Endo 16 in S. purpuratus embryos. The mRNA is first detectable at 18 h post-fertilization in the cytoplasm of blastomeres derived from the Veg2 6th cleavage tier. The number of Endo 16 positive cells increases gradually through the beginning of gastrulation, and these cell numbers are in agreement with estimates of the number of cells that should be in the vegetal plate at these stages. We conclude that Endo 16 expression is indeed an early vegetal plate marker and that this gene is expressed by all Veg2 tier derivatives while they are part of the vegetal plate. The progressive regionalization of Endo 16 expression that occurs in normal embryos is also seen in lithium chloride induced exogastrulae, leading to the conclusion that genetic regulation of endoderm differentiation is programmed into the vegetal plate cells once they have been specified. Finally, we report a reproducible phenomenon seen in cultures of LiCl exogastrulae, in which the tips of the everted archenterons fuse, followed by the induction of supernumerary pigment cells.

Appearance and persistence of maternal RNA sequences in sea urchin development

This paper deals with the relationship between the single copy transcripts represented in mature oocytes of the sea urchin and the RNA sequences present in immature oocytes and embryos. We term the oocyte transcripts from single copy DNA the maternal single copy sequence set. A single copy [^3H]DNA fraction ([^3H]oDNA) enriched for sequences complementary to the maternal single copy sequence set was prepared and reacted with the different RNA preparations. The complexity of the mature oocyte RNA is estimated to be 37 × 10^6 nucleotides. At kinetic termination, [3H]oDNA reacted with the polysomal mRNA of 16-cell embryos to 73% of the reaction with mature oocyte RNA, indicating that 27 × 10^6 nucleotides of the maternal sequence set are present. With blastula mRNA the reaction equals about 56%, a complexity of 21 × 10^6 nucleotides; with gastrula mRNA, 53%, a complexity of 19 × 10^6 nucleotides. The relative amount of hybridization of [^3H]oDNA was 100% with cytoplasmic RNA of the 16-cell stage and became progressively less with the cytoplasmic RNAs of later stages. The total RNA of immature oocytes was found to include about 26 × 10^6 nucleotides of the maternal sequence set. Results of these experiments are discussed, and an interpretation of the pattern of utilization of structural genes during oocyte and embryo development is suggested.

Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation☆

We have isolated and characterized a new endoderm-specific gene, designated Endo16, from a sea urchin gastrula stage cDNA library. Northern blot analysis and in situ hybridization experiments indicate that this gene is first expressed in the vegetal plate, a group of endodermal and mesenchymal precursor cells that are poised to invaginate in the first movement of gastrulation. Expression becomes progressively restricted to a subset of endodermal cells as development proceeds. To study the Endo16 gene product, a polyclonal antiserum was raised against bacterially expressed Endo16 protein. Indirect immunofluorescence experiments in midgastrula stage embryos reveal that the Endo16 protein is localized to the surface of endoderm and secondary mesenchyme cells. In Western blot experiments, the antiserum detects a small set of high molecular weight proteins ranging from 180 to greater than 300 kDa. Analysis of the nucleotide-derived amino acid sequence from a partial Endo16 cDNA clone reveals a highly repetitive, extremely acidic protein segment that includes the Arg-Gly-Asp (RGD) tripeptide known to be important in cell binding domains of a number of extracellular proteins. Taken together, these data suggest that the Endo16 protein may be an adhesion molecule involved in gastrulation of the sea urchin embryo.

Mitochondrial tRNAthr mutation in fatal infantile respiratory enzyme deficiency

The mitochondrial DNA (mtDNA) of two unrelated infants with lethal respiratory chain defects was studied using denaturing gradient gel analysis. This analysis revealed melting behavior differences suggesting a point mutation(s) in a restriction fragment containing the apocytochrome b and tRNA(thr) genes. Sequencing revealed that patient 1 had an A to G mutation at nt 15924 which is the last base pair of the anticodon stem adjacent to the anticodon loop of tRNA(thr). Patient 2 had an A to G mutation at nt 15923 which is the last base of the anticodon loop. The results suggest that mtDNA mutations affecting the anticodon loop structure of tRNA(thr) cause mitochondrial disease that is fatal in infancy.

Altered cell fate in LiCl-treated sea urchin embryos

Endo16 is a lineage-specific protein expressed during embryogenesis in the sea urchin, Strongylocentrotus purpuratus. We have examined the effects of various concentrations of lithium chloride, a well-known vegetalizing agent, on Endo16 expression in whole sea urchin embryos. Our results show that treatment with LiCl causes increased steady-state levels of Endo16 transcripts. An increase in the number of endodermal cells in treated embryos demonstrates a change in cell lineage. In addition, we observed a delay in development of lithium-treated embryos that is accompanied by a delay in the expression of a late class histone H2B gene.

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.

Sea urchin oocytes possess elaborate cortical arrays of microfilaments, microtubules, and intermediate filaments

Extensive arrays of microfilaments, microtubules and cytokeratin-type intermediate filaments were detected in the cortex of Strongylocentrotus droebachiensis oocytes using fluorescently labeled antibodies on both cortex and whole mount preparations. All three filament systems undergo dramatic structural reorganization during meiotic maturation of the egg. Microfilaments form a dense meshwork within the cortex of the oocyte. After meiosis, the filaments rearrange and shorten, resulting in a more loosely organized network. Both cortical microtubules and microtubules associated with a microtubule-organizing center are observed within the oocyte. After meiosis, the number and length of the cortical microtubules gradually diminish. A microtubule organizing center is found situated between the germinal vesicle and the plasma membrane in many oocytes. A network of filaments extends from the microtubule organizing center and radiates peripherally toward the germinal vesicle, presumably marking the animal pole. Cytokeratin-like intermediate filaments form a reticular network within the oocyte cortex, then solubilize during meiosis. In whole mounts of oocytes there is a single focal center of cytokeratin staining from which filaments radiate. Indirect immunofluorescence experiments, using anti-tubulin and anti-cytokeratin antibodies simultaneously, reveal the intermediate filament focal center to be localized within the microtubule organizing center. These results demonstrate the presence of a complex cortical cytoskeleton in premeiotic eggs of the sea urchin, Strongylocentrotus droebachiensis.

Applied DC Magnetic Fields Cause Alterations in the Time of Cell Divisions and Developmental Abnormalities in Early Sea Urchin Embryos

Most work on magnetic field effects focuses on AC fields. The present study demonstrates that exposure to medium-strength (10 mT-0.1 T) static magnetic fields can alter the early embryonic development of two species of sea urchin embryos. Batches of fertilized eggs from two species of urchin were exposed to fields produced by permanent magnets. Samples of the continuous cultures were scored for the timing of the first two cell divisions, time of hatching, and incidence of exogastrulation. It was found that static fields delay the onset of mitosis in both species by an amount dependent on the exposure timing relative to fertilization. The exposure time that caused the maximum effect differed between the two species. Thirty millitesla fields, but not 15 mT fields, caused an eightfold increase in the incidence of exogastrulation in Lytechinus pictus, whereas neither of these fields produced exogastrulation in Strongylocentrotus purpuratus.

Mitochondrial Disorder Associated with Newborn Cardiopulmonary Arrest

ABSTRACT: A female infant who died 2.5 d after birth with hypoglycemia, lactic acidosis, and sudden multisystem failure was studied. Biochemical studies showed complex III and IV deficiency in liver, kidney, and muscle, with muscle most severely affected. Southern blot analysis of the patients mitochondrial DNA did not reveal any deletions. Denaturing gradient gel analysis, which detects single base changes by differences in melting behavior, showed an extra band that was not seen in mitochondrial DNA from the mother, the mothers identical twin sister, or an unrelated normal subject. This extra band indicated heteroplasmy for a restriction fragment containing the apocytochrome b and transfer RNAthr genes. Sequencing revealed an A to G mutation at nucleotide 15923, the last base of the anticodon loop of the transfer RNAthr gene. The mutation lengthens the anticodon stem by added pairing and reduces the anticodon loop size from 7 to 5 nucleotides, potentially compromising transfer RNAthr function in translation and/or in processing the polycistronic RNA transcript. The patients mother previously had a male infant who also died at 1.5 d postnatal, and both the mother and her twin have had multiple miscarriages. Amniocentesis for a genetic screen was performed on the mothers twin sister during a recent pregnancy; some of the cultured cells were made available for this study. The mutation was not found in the amniocytes or in umbilical cord blood obtained at birth; the baby was normal at birth and remains healthy. It is concluded that the mutation at nucleotide 15923 was most likely the cause of the fatal disease in the index case. The timing of the illness was consistent with postnatal depletion of glycogen reserves. The findings suggest that a mitochondrial disorder should be considered for infants who experience sudden cardiopulmonary arrest within the first few days of life. (Pediatr Res 33: 433–440, 1993)

Three sea urchin actin genes show different patterns of expression: muscle specific, embryo specific, and constitutive.

The expression of three different actin genes in the sea urchin, Strongylocentrotus purpuratus, was monitored in embryos and adult tissues by using untranslated mRNA sequences as specific hybridization probes. Three distinct patterns of expression were found: muscle specific, embryo specific, and constitutive (i.e., present in all tissues examined). The actin genes encoding the muscle-specific and constitutively expressed genes were each found to be present once in the haploid genome. The embryo-specific probe could derive from either a single gene or a small subset of actin genes. These data demonstrate that at least three members of the sea urchin actin gene family are expressed in distinct ways and thus are probably associated with different regulatory programs of gene expression necessary for development of this metazoan.