Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis

We have determined spatial patterns of expression of individual actin genes in embryos of the sea urchin Strongylocentrotus purpuratus. Radioactively labeled probes specific for each of five cytoplasmic-type (Cy) and the single muscle-type (M) mRNAs were hybridized in situ to sections of fixed embryos. M actin mRNA appears only late in development and is confined to a few cells associated with the coelomic rudiments. The five Cy mRNAs fall into three sets, whose times and sites of expression during development are highly distinctive. Different cell lineages express messages of one or more of these sets, but never all three. Although all Cy actin mRNAs exhibit monophasic accumulation in the RNA of whole embryos during the course of development, such accumulation in many cases results from the summation of both increases and decreases in abundance within individual sets of cells. Within the genomic linkage group CyI-CyIIa-CyIIb, expression of CyI and CyIIb appears to be co-ordinate, and quite distinct from that of CyIIa. CyI and CyIIb are expressed in all lineages at some point in embryogenesis, but confined mainly to oral ectoderm and portions of the gut of the pluteus larva. CyIIa mRNAs are restricted to mesenchyme lineages throughout late gastrula stage, and subsequently accumulate in parts of the gut. The CyIIIa and CyIIIb genes, which form a separate linkage group, are expressed only in aboral ectoderm and its precursors. Furthermore, CyIII messages are the only detectable actin mRNAs in this cell lineage after late blastula stage.     

Sea urchin actin gene subtypes. Gene number, linkage and evolution

The actin gene family of the sea urchin Strongylocentrotus purpuratus was analyzed by the genome blot method, using subcloned probes specific to the 3' terminal non-translated actin gene sequence, intervening sequence and coding region probes. We define an actin gene subtype as that gene or set of genes displaying homology with a given 3' terminal sequence probe, when hybridized at 55 degrees C, 0.75 M-Na+. By determining the often polymorphic restriction fragment band pattern displayed in genome blots by each probe, all, or almost all of the actin genes in this species could be classified. Our evidence shows that the S. purpuratus genome probably contains seven to eight actin genes, and these can be assigned to four subtypes. Studies of the expression of the genes (Shott et al., 1983) show that the actin genes of three of these subtypes code for cytoskeletal actins (Cy), while the fourth gives rise to a muscle-specific actin (M). We denote the array of S. purpuratus actin genes indicated by our data as follows. There is a single CyI actin gene, two or possibly three CyII genes (CyIIa, CyIIb, and possibly CyIIc), three CyIII actin genes (CyIIIa, CyIIIb, CyIIIc), and a single M actin gene. Comparative studies were carried out on the actin gene families of five other sea urchin species. At least the CyIIa and CyIIb genes are also linked in the Strongylocentrotus franciscanus genome, and this species also has a CyI gene, an M actin gene and at least two CyIII actin genes. It is not clear whether it also possesses a CyIIc actin gene, or a CyIIIc actin gene. The genome of a more closely related congener, Strongylocentrotus dr?bachiensis, includes 3' terminal sequences suggesting the presence of a CyIIc gene. In S. franciscanus and S. dr?bachiensis the first intron of the CyI gene has remained homologous with intron sequences of both the CyIIa and CyIIb genes, indicating a common origin of these three linked cytoskeletal actin genes. Of the four S. purpuratus 3' terminal subtype probe sequences only the CyI 3' terminal sequence has been conserved sufficiently during evolution to permit detection outside of the genus Strongylocentrotus. An unexpected observation was that a sequence found only in the 3' untranslated region of the CyII actin gene in the DNA of S. dr?bachiensis and S. purpuratus is represented as a large family of interspersed repeat sequences in the genome of S. franciscanus.     

DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchinStrongylocentrotus purpuratus

Summary The general organization and primary amino acid sequences of theS. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26–27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage.The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.     

Sea urchin actin gene linkages determined by genetic segregation

Genetic linkage between the actin genes of Strongylocentrotus purpuratus was investigated by observing the segregation of restriction fragment length polymorphisms (RFLPs). Specific RFLPs of actin gene pairs CyI/CyIIa and CyIIIa/CyIIIb always cosegregated, confirming the linkage groups CyI-CyIIa-CyIIb previously previously determined by molecular cloning. In contrast, RFLPs of actin genes CyI/CyIIa, CyIIIa/CyIIIb, and M all segregated at random with respect to one another. This demonstrates that the known actin gene clusters CyI-CyIIa-CyIIb, CyIIIa-CyIIIb, and the M actin gene are not closely linked.     

Expression of alpha- and beta-tubulin genes during development of sea urchin embryos

Mature unfertilized eggs of the sea urchin Lytechinus pictus contain multiple alpha-tubulin mRNAs, which range in size from 1.75 to 4.8 kb, and two beta-tubulin mRNAs, 1.8 and 2.25 kb. These mRNAs were found at similar levels throughout the early cleavage stages. RNA gel blot hybridizations showed that prominent quantitative and qualitative changes in tubulin mRNAs occurred between the early blastula and hatched blastula stages. The overall amounts of alpha- and beta-tubulin mRNAs increased two- to fivefold between blastula and pluteus. These increases were due mainly to a rise in a 1.75-kb alpha RNA and a new 2.0-kb beta RNA. Other, minor changes also occurred during subsequent development. All size classes of alpha- and beta-tubulin RNAs in early and late embryos contained poly(A)+ translatable sequences. As reported earlier, some of each of the alpha RNAs, but neither of the beta RNAs, are translated in the egg and a small portion of each of the stored alpha and beta RNAs is recruited onto polysomes within 30 min of fertilization. In the work described here, subsequent development up to the morula stage was accompanied by a gradual recruitment of tubulin mRNAs into polysomes. By the early blastula stage, most of the maternal tubulin sequences were associated with polysomes. In contrast to the gradual recruitment of maternal sequences throughout cleavage, the tubulin mRNAs which appeared at the blastula stage showed no delay in entering polysomes. The exact fraction of each mRNA that was translationally active at later stages varied somewhat among the individual mRNAs. From the differential hybridization patterns of egg, embryo, and testis RNAs to various tubulin cDNA and genomic DNA probes, it is concluded that at least one gene producing maternal alpha mRNA is different from a second one which is expressed only in testis. Each of the three embryonic beta RNAs is encoded by a different beta gene; at least two of these different beta genes are also expressed in testis.     

The sequence of a sea urchin muscle actin gene suggests a gene conversion with a cytoskeletal actin gene

Summary We report the nucleotide sequence of the single muscle actin gene of the sea urchinStrongylocentrotus purpuratus. Comparison of the protein-coding sequence of this muscle actin gene (pSpG28) with that of two linked sea urchin cytoskeletal actin genes (pSpG17 and CyIIa) reveals a region of exceptional sequence conservation from codon 61 through codon 120. Furthermore, when silent nucleotide changes are compared, the conservation of this region is still evident (7.9% silent site differences in the conserved region vs 43.3% silent site differences in the rest of the gene when pSpG28 and CyIIa are compared), indicating that the conservation is not due to particularly stringent selection on the portion of the protein encoded by this region of the genes. These observations suggest that a gene conversion has occurred between the muscle actin gene and a cytoskeletal actin gene recently in the evolution of the sea urchin genome. Gene conversion between nonallelic actin genes may thus play a role in maintaining the homogeneity of this highly conserved gene family.     

Territorial expression of three different trans-genes in early sea urchin embryos detected by a whole-mount fluorescence procedure.

We have developed a new procedure for detection of the protein product of chloramphenicol acetyltransferase (CAT) reporter genes in whole mounted sea urchin embryos. The position of a commercially available anti-CAT antibody is visualized by video or confocal microscopy, and thus the spatial domains of exogenous reporter gene expression can be determined with regard to the intact three-dimensional structures of the embryo. We show that in pluteus stage embryos CAT protein expression patterns for SM50 . CAT or CyIIIa . CAT reporter genes are similar to those previously obtained by in situ hybridizations with radioactive probes. Taking advantage of the superior resolution of cellular CAT expression patterns using the antibody visualization method, we found for the first time that, in addition to the expression in aboral ectoderm, some cells in the ciliated band of the pluteus express CyIIIa . CAT. The expression of a new fusion construct, CyIIa . CAT, was also examined. As expected from the localization of endogenous CyIIa mRNA, CAT protein was expressed under control of the CyIIa promoter in gut and skeletogenic mesenchyme cells.     

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.     

Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs

A fusion gene construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of CyIIIa actin gene regulatory sequences was injected into unfertilized eggs of the urchin Strongylocentrotus purpuratus, and early pluteus stage embryos that developed from these eggs were fixed and sectioned for analysis by in situ hybridization. A [3H]RNA antisense probe for CAT mRNA was hybridized to 5-micron embryo sections. Autoradiographic signal denoting the presence of CAT mRNA was detected only over aboral ectoderm cells, in which the CyIIIa gene is normally expressed, and not over any recognizable regions of gut or oral ectoderm included in the same sections.