RET/PTC-induced gene expression in thyroid PCCL3 cells reveals early activation of genes involved in regulation of the immune response.

RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response.

An extracellular matrix response element in the promoter of the LpS1 genes of the sea urchin Lytechinus pictus.

The extracellular matrix (ECM) has been shown to play an important role in development and tissue-specific gene expression, yet the mechanism by which genes receive signals from the ECM is poorly understood. The aboral ectoderm-specific LpS1-alpha and -beta genes of Lytechinus pictus , members of the Spec gene family, provide an excellent model system to study ECM- mediated gene regulation. Disruption of the ECM by preventing collagen deposition using the lathrytic agent beta-aminopropionitrile (BAPN) inhibits LpS1 gene transcription. LpS1 transcription resumes after removal of BAPN and subsequent collagen reformation. Using a chloramphenicol acetyltransferase (CAT) reporter gene assay, we show that a 125 bp region of the LpS1-beta promoter from -108 to +17 contains an ECM response element (ECM RE). Insertion of the 125 bp region into the promoter of the metallothionein gene of L. pictus, a gene unaffected by ECM disruption, caused the fused promoter to become ECM dependent. As with the endogenous LpS1 genes, CAT activity directed by the fused LpS1-beta promoter resumed in embryos recovered from ECM disruption. A mutation in a cis -acting element called the proximal G-string, which lies in the 125 bp region, caused CAT activity levels in ECM-disrupted embryos to equal that of the wild-type LpS1-bet apromoter in ECM-intact embryos. These results suggest that the intact ECM normally transmits signals to inhibit repressor activity at the proximal G-string in aboral ectoderm cells. Consistent with these results were our findings which showed that in addition to expression in the aboral ectoderm, the proximal G-string mutation caused expression of the CAT gene in oral ectoderm cells. These studies suggested that the proximal G-string serves as a binding site for negative regulation of the LpS1 genes in oral ectoderm during development. We also examined trans -acting factors binding the proximal G-string following ECM disruption. Band shift gels revealed a predominant set of slower migrating nuclear proteins from ECM-disrupted embryos which bound the proximal G-string. This work suggested that ECM disruption initiates signaling that induces a repressor to bind the ECM RE and/or modifies ECM RE binding proteins, which in turn represses LpS1 gene activity.

Disruption of gastrulation and oral-aboral ectoderm differentiation in the Lytechinus pictus embryo by a dominant/negative PDGF receptor.

Little is known about the cell signaling involved in forming the body plan of the sea urchin embryo. Previous work suggested that PDGF-like and EGF-like receptor-mediated signaling pathways are involved in gastrulation and spiculogenesis in the Lytechinus pictus embryo. Here we show that expression of the human PDGF receptor-beta lacking the cytoplasmic domain disrupted development in a manner consistent with a dominant/negative mechanism. The truncated PDGF receptor-beta inhibited gut and spicule formation and differentiation along the oral-aboral axis. The most severely affected embryos arrested at a developmental stage resembling mesenchyme blastula. Coinjection into eggs of RNA encoding the entire human PDGF receptor-beta rescued development. The truncated PDGF receptor-beta caused the aboral ectoderm-specific genes LpS1 and LpC2 to be repressed while an oral ectoderm-specific gene, Ecto-V, was expressed in all ectoderm cells. The results support the hypothesis that a PDGF-like signaling pathway plays a key role in the intercellular communication required for gastrulation and spiculogenesis, and in cell commitment and differentiation along the oral-aboral axis.

USF in the Lytechinus sea urchin embryo may act as a transcriptional repressor in non-aboral ectoderm cells for the cell lineage-specific expression of the LpS1 genes.

Expression of the aboral ectoderm-specific LpS1 gene in Lytechinus was used to study lineage-specific transcriptional regulation during sea urchin development. Band shift assays using anti-USF antibody showed that a USF-like protein bound the USF core sequence 5'-CACGTG-3' in the promoter of the LpS1 gene. DNA constructs consisting of a wild-type LpS1 promoter and the same LpS1 promoter with a mutated USF binding site fused to the bacterial chloramphenicol acetyltransferase reporter gene were tested. The mutation in the USF binding site caused an increase in chloramphenicol acetyltransferse activity. We selected a clone that encodes USF, LvUSF, from a gastrula-stage cDNA library representing Lytechinus variegatus. Transactivation experiments, in which LvUSF RNA or a DNA construct consisting of the LvUSF cDNA clone fused to the Lytechinus pictus metallothionein promoter coinjected with the wild-type or mutated LpS1 promoter-chloramphenicol acetyltransferase gene construct, showed that chloramphenicol acetyltransferase activity from the wild-type construct was repressed, while the construct mutated at the USF binding site was active. The same wild-type and mutated LpS1 promoter DNA fragments ligated to the green fluorescent protein reporter gene were used to examine spatial expression. The reporter gene constructs containing the mutated USF binding site were expressed inappropriately in all cell types including the gut and oral ectoderm in gastrula and larva stage embryos, while the wild-type constructs were expressed primarily in the aboral ectoderm. USF was expressed in all cells of the early embryo and in all tissues except the aboral ectoderm in later embryos. The data are consistent with a model depicting Lytechinus USF, as a temporal and spatial regulator by repressing LpS1 gene transcription in non-aboral ectoderm cells.

Two distinct forms of USF in the Lytechinus sea urchin embryo do not play a role in LpS1 gene inactivation upon disruption of the extracellular matrix.

Recent studies in our laboratory indicated that the upstream stimulatory factor (USF) in the sea urchin embryo of Lytechinus acts as a transcriptional repressor for the aboral ectoderm-specific expression of the LpS1 genes. Disruption of the extracellular matrix (ECM) arrests development prior to gastrulation and inactivates the LpS1 genes. We wanted to determine whether the inactivation of the LpS1 genes by ECM disruption may be due to an increase in USF expression. In the course of the investigation, a second L, variegatus USF cDNA clone (LvUSF2) was isolated and sequenced. The deduced amino acid sequence of LvUSF2 is nearly identical to LvUSF1 except at the amino end, where they are sharply divergent. Like LvUSF1, LvUSF2 has a USF-specific, a basic/hefixloop-helix, and a leucine zipper domain. Genomic DNA blots indicated that the two cDNA clones are derived from one gene, which suggested that the Lytechinus USF1 and USF2 mRNAs, of approximately 6.0 and 4.0 kb, respectively, are the result of differential RNA splicing. ECM disruption in Lytechinus embryos caused a relative drop in USF RNA accumulation levels to approximately 60% of control embryos, while LpS1 RNA accumulation levels dropped to less than 5%. USF protein levels and DNA binding activities in ECM-disrupted embryos also dropped to approximately 60% to that of control embryos. A mutation at the USF binding site in an LpS1 promoter-chloramphenicol acetyl transferase (CAT) fusion DNA construct did not cause a relative increase in CAT activity in ECM disrupted embryos. These results suggest that the induced drop in LpS1 gene expression by ECM disruption is not due to an increase in the repressive activity of USF.

A tissue-specific repressor in the sea urchin embryo of Lytechinus pictus binds the distal G-string element in the LpS1-beta promoter.

LpS1 RNA transcripts and proteins are expressed exclusively in the aboral ectoderm of the embryo in the sea urchin Lytechinus pictus. We have characterized the LpS1-beta promoter to identify the cis-acting elements that may be involved in the aboral ectoderm-specific expression of the LpS1-beta gene. The distal G-string site, composed of six contiguous guanine deoxynucleotides located at -721 to -726, was analyzed. A mutation at the distal G-string caused over a two-fold increase in reporter chloramphenicol acetyltransferase gene activity and inappropriate expression of reporter green fluorescent protein in nonaboral ectoderm cells in L. pictus embryos. These results suggest that the proteins that bind the distal G-string act as a spatial repressor in the nonaboral ectoderm cells of the developing embryo.

An ECM-bound, PDGF-like growth factor and a TGF-alpha-like growth factor are required for gastrulation and spiculogenesis in the Lytechinus embryo.

Growth factors and the extracellular matrix have been shown to fulfill vital developmental roles in many embryonic systems. Our hypothesis is that a developmental role played by the extracellular matrix in sea urchins may be the binding of a PDGF-like growth factor to promote signaling activity. We report here that anti-human PDGF-B antibodies and anti-human TGF-alpha antibodies immunoprecipitated specific proteins isolated from Lytechinus embryos. Addition of these antibodies to Lytechinus embryos inhibited gastrulation and spiculogenesis. The embryos are sensitive to the antibodies from the four-cell through the hatching blastula stages, which suggests that the TGF-alpha-like and PDGF-like ligands are required for the early differentiation of the gut and spicules. We present evidence that the PDGF-like growth factor depends on the extracellular matrix for signaling activity. Synthetic peptides representing the heparan sulfate proteoglycan binding sequence on human PDGF-B were added to Lytechinus embryo cultures to compete for binding sites with the endogenous PDGF-like growth factor. The experimental peptide inhibited gastrulation and caused radially arranged multiple spicules to form. Development was unaffected by a control peptide. These studies support our hypothesis and suggest that TGF-alpha-like and PDGF-like growth factors induce signaling events required for sea urchin gastrulation and spiculogenesis and suggest that an extracellular matrix-associated PDGF-like growth factor is involved in differentiation along the oral-aboral axis.

Role for platelet-derived growth factor-like and epidermal growth factor-like signaling pathways in gastrulation and spiculogenesis in the Lytechinus sea urchin embryo.

The mechanisms underlying sea urchin gastrulation and spiculogenesis have been sought for decades. We have identified two growth factor signaling pathways that are involved in these developmental events. Antibodies against mammalian platelet-derived growth factor (PDGF) receptor-beta inhibited gastrulation and spiculogenesis, and antibodies against human epidermal growth factor (EGF) receptor disrupted gastrulation and spicule placement in Lytechinus pictus and L. variegatus embryos. Our studies suggested that the antibodies affect development by inhibiting rather than activating the signaling pathways. Polyclonal and monoclonal antibodies against the mammalian receptors recognized specifically Lytechinus proteins of the expected size of 170-180 x 10(3) M(r). Growth factor binding assays indicated that there are approximately 1.25 x 10(4) platelet-derived growth factor-like receptors per cell at the mesenchyme blastula stage of L. pictus, and human platelet-derived growth factor bound with an apparent affinity of KD = 4.4 nM to dissociated cells at the mesenchyme blastula stage. Immunolabelling experiments showed that at the gastrula stage, the Lytechinus platelet-derived growth factor-like receptors are located on the primary mesenchyme cells, the gut, and most prominently on the secondary mesenchyme cells and the stomodeum. The epidermal growth factor-like receptors stained less intensely on the gut and primary and secondary mesenchyme cells. Both receptors are expressed on the ciliary band and the gut of the pluteus larva but only the PDGF-like receptor is expressed on the primary mesenchyme cells. Pulse studies showed that the embryos are sensitive to the platelet-derived growth factor receptor-beta and epidermal growth factor receptor antibodies from the blastula to sometime between the mesenchyme blastula and midgastrula stages. We show that antibodies enter the blastocoel as late as the gastrula stage. Our results suggest that platelet-derived growth factor-like and epidermal growth factor-like signaling pathways are involved in the early differentiation and morphogenesis of the sea urchin gut and spicules.

Identification of an antennapedia-like homeobox gene in the ascidians Styela clava and S. plicata.

Homeobox genes from the urochordates Styela clava (AHox2) and S. plicata (AHox3) were cloned and analyzed. The two genes are homologous and Antennapedia-like. The homeobox regions have 87% identity at the nucleotide level and are identical at the amino-acid level. No introns are present in the homeobox region of AHox3, and AHox3 is represented at a low copy number per haploid genome. AHox2 and AHox3 represent the second type of homeobox gene found in this evolutionarily and developmentally important group of organisms.

PDGF-BB and TGF-alpha rescue gastrulation, spiculogenesis, and LpS1 expression in collagen-disrupted embryos of the sea urchin genus Lytechinus.

Development and LpS1 transcription in Lytechinus embryos are arrested at the mesenchyme blastula stage when collagen deposition is inhibited by the lathrytic agent beta-aminopropionitrile (BAPN) or by proline analogs. We found that human recombinant platelet derived growth factor-BB (PDGF-BB) and transforming growth factor-alpha (TGF-alpha) synergistically rescue collagen disrupted/developmentally arrested L. pictus and L. variegatus embryos so that development and RNA accumulation of LpS1 proceed. In addition, nonspecific antagonists of PDGF block gastrulation and LpS1 RNA accumulation. The embryos recover and LpS1 RNA accumulation resumes when the antagonists are removed. These data suggest that a growth factor mediated pathway, associated with the ECM, is required for sea urchin gastrulation, spiculogenesis, and LpS1 gene activation.

A characterization of the H3 and H4 histone genes from the ascidian Styela plicata.

Information about histone sequences and histone gene organization from invertebrate chordates is completely lacking. A genomic clone containing linked H3 and H4 histone genes from the urochordate Styela plicata was analyzed. The nucleotide sequence indicates that the two genes are transcribed in opposite directions and have structural similarities to cell cycle-dependent histones. Unique amino acid replacements occur in H3 at positions 88 (serine) and 98 (arginine). In H4, the methionine at position 84 is replaced by a leucine, a replacement found only in fungi. Codon usage patterns are nonrandom and resemble invertebrate patterns. The H3 and H4 genes are polymorphic and are represented five times per haploid genome.

Evolution of the chordate muscle actin gene.

The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage.

Structure and promoter activity of the LpS1 genes of Lytechinus pictus. Duplicated exons account for LpS1 proteins with eight calcium binding domains.

The LpS1 genes of the sea urchin Lytechinus pictus are activated early in development in aboral ectoderm cells. They therefore have ontogenic properties similar to their counterparts in Stronglyocentrotus purpuratus, the Spec genes. Both gene families encode proteins belonging to the calmodulin superfamily as evidenced by the presence of distinct EF-hand (helix-loop-helix) domains. The presence of eight EF-hand domains in LpS1 proteins suggests that the LpS1 genes arose from a duplication of an ancestral Spec-like gene. The LpS1 genes were further analyzed to increase our understanding of the mechanisms underlying their evolution and activation in aboral ectoderm cells. Genomic DNA blot analysis showed two LpS1 genes, LpS1 alpha and LpS1 beta, which did not appear to be closely linked. LpS1 genomic clones were isolated by screening an L. pictus genomic library with an LpS1 cDNA clone, and partial gene structures for both LpS1 alpha and LpS1 beta were constructed. These revealed internal duplication of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Duplication of exon 1 in both genes suggested four different LpS1 proteins could be derived from the LpS1 genes. Primer extension to map the transcriptional initiation sites of the LpS1 genes and sequencing analysis showed there was little in common among the 5'-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF. A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5'-flanking DNA and 17 bp of 5'-untranslated leader sequence of the LpS1 beta gene were sufficient for correct temporal and spatial expression of reporter chloramphenicol acetyltransferase and lacZ genes in sea urchin embryos. Deletions at the 5' end to either 511 or 368 bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase activity and disrupted the exclusive activation of the lacZ gene in aboral ectodermal cells. Based on a lineage analysis among the LpS1 and Spec gene families and other related genes, we propose a model in which LpS1 genes evolved from a series of duplications of an ancestral Spec-like gene.

Ectoderm nuclei from sea urchin embryos contain a Spec-DNA binding protein similar to the vertebrate transcription factor USF.

The Spec gene family of Stronglyocentrotus purpuratus is expressed exclusively in aboral ectoderm cells during embryogenesis. To investigate the regulation of Spec gene activity, the region around the Spec1 transcriptional initiation site was analyzed for sites of protein-DNA interaction. One high-affinity site bound a factor termed SpF1 within the Spec1 5' untranslated leader region at position +39 to +60. The core sequence recognized by SpF1, CACGTG, is the same as that of the upstream stimulatory factor (USF), a widely occurring vertebrate transcription factor containing a myc-HLH motif. A comparison of USF- and SpF1-binding activities suggested that SpF1 was a sea urchin version of USF. SpF1 activity was detectable only in ectoderm cells of the embryo, implying that it has a role as a cell type-specific transcription factor. SpF1-binding sites were also found upstream of the Spec2a and Spec2c genes in the same conserved sequence block as Spec1. Extracts from Lytechinus pictus embryos showed an SpF1-like activity, suggesting that SpF1 is conserved in sea urchins. Surprisingly, changes in the Spec1, Spec2a, or Spec2c genes that removed or modified the SpF1-binding site had no effect on expression when reporter gene fusions containing these mutations were injected into sea urchin eggs and analyzed for expression during embryogenesis. We propose that, while SpF1 may not be essential for expression of the exogenously introduced reporter genes, it may be required for proper regulation of the endogenous Spec genes.

Temporal and spatial transcriptional regulation of the aboral ectoderm-specific Spec genes during sea urchin embryogenesis.

mRNAs for Spec 1 and Spec 2 of Strongylocentrotus purpuratus and LpS1 of Lytechinus pictus accumulate only in the aboral ectoderm of developing embryos. In vitro nuclear transcription assays were done to study the transcriptional regulation of these cell type-specific genes. Spec 1, Spec 2c, and Spec 2d genes all appeared to be transcriptionally activated at the late cleavage-early blastula stage of S. purpuratus. Differences in the relative transcription rates during development appeared to play a major role in determining the relative levels of the various Spec mRNAs. The L. pictus LpS1 gene was transcriptionally activated at a similar developmental time as the corresponding S. purpuratus genes. Nuclei from gastrula or pluteus ectodermal and endodermal/mesodermal cell fractions were used to demonstrate that Spec 1 and LpS1 genes were transcriptionally active in ectoderm nuclei but not in endoderm/mesoderm nuclei, suggesting that in vivo the Spec 1 and LpS1 genes are spatially controlled at the transcriptional level. Estimations of the absolute rate constants for Spec 1 transcription were made at the late cleavage, mesenchyme blastula, and midgastrula stages. Calculations using these rate constants and the known levels of Spec 1 mRNA suggested that Spec 1 mRNA stability gradually increased throughout development.

Transcription of the Spec 1-like gene of Lytechinus is selectively inhibited in response to disruption of the extracellular matrix.

The influence of the extracellular matrix (ECM) on differential gene expression during sea urchin development was explored using cell-type-specific cDNA probes. The ECM of three species of sea urchin, Strongylocentrotus purpuratus, Lytechinus variegatus and Lytechinus pictus, was disrupted with the lathrytic agent beta-aminopropionitrile (BAPN), which inhibits collagen deposition in the ECM and arrests gastrulation (Wessel & McClay, Devl Biol. 121: 149, 1987). The levels of several mRNAs (Spec 1, Spec 2, CyIIa actin, CyIIIa actin and collagen in S. purpuratus, and metallothionine, ubiquitin and LpS3 in L. pictus and L. variegatus) were compared in BAPN-treated and control embryos. These mRNAs accumulated normally during BAPN treatment, even though the embryos did not gastrulate. To determine if the expression of any gene product is sensitive to ECM disruption, a differential cDNA screen compared poly (A+) RNA from BAPN-arrested and control embryos in Lytechinus. A cDNA clone was isolated from this screen that represented a 2.1 kb mRNA that did not accumulate during BAPN treatment. Removal of BAPN resulted in the accumulation of this transcript coincident with the onset of gastrulation. This cDNA clone encodes a L. variegatus homologue of LpS1, recently demonstrated to be an ancestral homologue of the aboral ectoderm-specific Spec 1-Spec 2 gene family in S. purpuratus. Nuclear run-on assays in L. pictus suggested that transcriptional activity of LpS1 was selectively inhibited by BAPN treatment. Thus, although the accumulation of many gene products occurred independently of the embryonic collagenous matrix, the accumulation of LpS1 and LvS1 appeared to be mediated by the ECM.

Differential expression of a muscle actin gene in muscle cell lineages of ascidian embryos.

Specific probes were used to examine the accumulation of muscle actin mRNA during embryonic development of the ascidian Styela. Clones of a muscle actin gene were obtained from an adult mantle cDNA library. Four lines of evidence indicate that these clones correspond to a muscle actin gene. First, their coding regions share 11 of 14 diagnostic amino acid positions with mammalian smooth and skeletal muscle actins. Second, subclones that contain only the 3' noncoding region of the gene select mRNA coding for muscle actin, while subclones that include the coding region of the gene select mRNA coding for muscle and nonmuscle actins. Third, a probe that contains only the 3' noncoding region detects a single band, corresponding to a 2 kb transcript, while a probe that includes the coding region detects the 2 kb transcript and at least one other band, presumably a cytoplasmic actin transcript. Fourth, the 3' noncoding region probe detects transcripts only in muscle cells and their precursors, while the coding region probe detects transcripts in muscle and nonmuscle cells. The muscle actin transcript is present at very low levels in eggs and early embryos, begins to accumulate between the early gastrula and tailbud stages, and by the tadpole stage attains a level about 25-fold higher than in the egg. In situ hybridization showed that embryonic muscle actin transcripts are restricted to the muscle cell lineages. These transcripts were initially observed in primary muscle lineage cells (descendants of the B4.1 blastomeres) at the early gastrula stage and continued to be present in these cells throughout embryonic development. In contrast, muscle actin transcripts did not appear in secondary muscle lineage cells (descendants of b4.2 and A4.1 blastomeres) until the mid-tailbud stage, and were not detected in mesenchyme cells, the presumptive adult muscle cell precursors, at any time during embryonic development. The results suggest that muscle actin gene expression is subject to spatial and temporal regulation in the muscle cell lineages.

Development of a muscle actin specified by maternal and zygotic mRNA in ascidian embryos.

In this investigation, we characterize the embryonic and adult actins and describe the embryonic expression of a muscle actin in the ascidian Styela. Two-dimensional polyacrylamide gel electrophoresis showed that embryos, tadpole larvae, and adult organs contain three major and two minor isoforms of actin. Two of the major isoforms, which are present in the mantle, branchial sac, alimentary tract, and gonads of adults and in eggs, embryos, and heads and tails of tadpoles, are likely to be cytoplasmic actins. The third major isoform, which was enriched in the mantle and branchial sac of adults and localized primarily in the tails of tadpoles, is a muscle actin. The muscle actin isoform was not detected in eggs and early embryos. Radioactivity incorporation studies showed that the cytoplasmic actins were synthesized throughout early development, but muscle actin synthesis was first detected between the 16- and 64-cell stages, 2-3 hr after fertilization. Two lines of evidence indicate that embryonic muscle actin synthesis is directed in part by maternal mRNA. First, poly(A)+ RNA isolated from unfertilized eggs directed the synthesis of muscle actin in an mRNA-dependent reticulocyte lysate. Second, muscle actin was synthesized in anucleate egg fragments. Arguments are also presented that muscle actin synthesis is not directed exclusively by maternal mRNA. It is concluded that embryonic and adult Styela exhibit actin heterogeneity, that one of the actin isoforms is a muscle actin, and that the muscle actin is synthesized during embryogenesis under the direction of maternal and zygotic mRNA.