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The ciliary band is a distinct region of embryonic ectoderm that is specified between oral and aboral ectoderm. Flask-shaped ciliary cells and neurons differentiate in this region and they are patterned to form an integrated tissue that functions as the principal swimming and feeding organ of the larva. TGFβ signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation. We have used morpholino knockdown and ectopic expression of RNA to alter TGFβ signaling at the level of ligands, receptors, and signal transduction components and assessed the differentiation and patterning of the ciliary band cells and associated neurons. We propose that the primary effects of these signals are to position the ciliary cells, which in turn support neural differentiation. We show that Nodal signaling, which is known to be localized by Lefty, positions the oral margin of the ciliary band. Signaling from BMP through Alk3/6, affects the position of the oral and aboral margins of the ciliary band. Since both Nodal and BMP signaling produce ectoderm that does not support neurogenesis, we propose that formation of a ciliary band requires protection from these signals. Expression of BMP2/4 and Nodal suppress neural differentiation. However, the response to receptor knockdown or dominant-negative forms of signal transduction components indicate signaling is not acting directly on unspecified ectoderm cells to prevent their differentiation as neurons. Instead, it produces a restricted field of ciliary band cells that supports neurogenesis. We propose a model that incorporates spatially regulated control of Nodal and BMP signaling to determine the position and differentiation of the ciliary band, and subsequent neural patterning.

We discuss recent progress in understanding how cell fates are specified along the animal‐vegetal axis of the sea urchin embryo. This process is initiated by cell-autonomous, maternally directed, mechanisms that establish three unique gene-regulatory domains. These domains are defined by distinct sets of vegetalizing (b-catenin) and animalizing transcription factor (ATF) activities and their region of overlap in the macromeres, which specifies these cells as early mesendoderm. Subsequent signaling among cleavage-stage blastomeres further subdivides fates of macromere progeny to yield major embryonic tissues. Zygotically produced Wnt8 reinforces maternally regulated levels of nuclear b-catenin in vegetal derivatives to down regulate ATF activity and further promote mesendoderm fates. Signaling through the Notch receptor from the vegetal micromere lineages diverts adjacent mesendoderm to secondary mesenchyme fates. Continued Wnt signaling expands the vegetal domain of b-catenin’s transcriptional regulatory activity and competes with animal signaling factors, including BMP2/4, to specify the endoderm‐ ectoderm border within veg1 progeny. This model places new emphasis on the importance of the ratio of maternally regulated vegetal and animal transcription factor activities in initial specification events along the animal‐vegetal axis. © 2000 Academic Press

We have used the Xenopus embryo as a test system for analyzing the activity of SpAN, a sea urchin metalloprotease in the astacin family containing BMP1 and tolloid. Embryos expressing SpAN initiated gastrulation on a time scale indistinguishable from controls, but invagination of the vegetal pole was subsequently delayed by several hours. At tailbud stages the most severely affected embryos were completely ventralized, lacking all dorsal structures. Molecular analysis of injected embryos, using probes for both dorsal (xgsc and xnot) and ventral (xhox3 and xwnt8) mesoderm, indicates that SpAN ventralizes dorsal mesoderm during gastrula stages. These results mirror those previously obtained with BMP4, suggesting that SpAN may enhance the activity of this ventralizing factor. Consistent with this suggestion, we have shown that SpAN blocks the dorsalizing activity of noggin and chordin, two inhibitory binding proteins for BMP4, but not that of a dominant-negative receptor for BMP4. In contrast, a dominant-negative SpAN, in which the metalloprotease domain has been deleted, dorsalizes ventral mesoderm, a phenotype that can be rescued by coexpressing either SpAN or XBMP1. This suggests that SpAN is mimicking a Xenopus metalloprotease responsible for regulating the activity of Xenopus BMPs during gastrulation. Moreover, our results raise the possibility that SpAN may function to facilitate BMP signaling in early sea urchin embryos.

We have shown previously by in situ hybridization that a gene encoding a fibroblast growth factor receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis (Strongylocentrotus purpuratus) (McCoon et al., J. Biol. Chem. 271, 20119-20195, 1996). Here we demonstrate by immunostaining with affinity-purified antibody that SpFGFR protein is detectable only in muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of myoblasts. Surprisingly, we find that SpFGFR transcripts are enriched in embryo nuclei, suggesting that lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional regulatory mechanism. Western blots show that SpFGFR is also specifically expressed in adult lantern muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine, or in coelomocytes, despite the presence of SpFGFR transcripts at similar concentrations in all these tissues. We conclude that in both embryos and adults, muscle-specific SpFGF receptor synthesis is controlled primarily at a posttranscriptional level. We show by RNase protection assays that transcripts encoding the IgS variant of the ligand binding domain of the receptor, previously shown to be enriched in embryo endomesoderm fractions, are the predominant, if not exclusive, SpFGFR transcripts in lantern muscle. Together, these results suggest that only a minority of SpFGFR transcripts are processed, exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not exclusively, IgS ligand binding domain sequences.

Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus . The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.

between these genes, we have found that transcriptional feedbackloops acting within and between adjacent cells are important inestablishing the cell type pattern. Specifically, the WER MYB-typeprotein, the GL3/EGL3 bHLH-type proteins, and the TTG WD-proteinappear to interact in a transcriptional complex to positively regulatethe GL2, CPC, TRY, and ETC1 genes. The GL2 homeodomaintranscription factor is involved in regulating genes that generatethe non-hair cell type. The CPC, TRY, and ETC1 proteins arestructurally-related small MYB transcription factors that appear tomove between cells and inhibit the formation of the WER-GL3/EGL3-TTG complex; this negative regulation represents a type of lateralinhibition mechanism. The position-dependent pattern relies on aleucine-rich-repeat receptor-like kinase (SCRAMBLED (SCM)), thatappears to influence WER gene expression causing an unequaldistribution of the transcriptional regulators in the N and H cellpositions. Current research is focused on using genomics, bioinfor-matics, and math modeling to further dissect the regulatory network.These studies are likely to provide new insights into the basicmechanisms of regulatory gene networks and cell-type specificationduring development.doi:10.1016/j.ydbio.2011.05.056Program/Abstract # 40Neurons develop in situ in foregut endoderm of seaurchin embryosZheng Wei

Many proteins containing domains related to epidermal growth factor (EGF) function in intercellular interactions that mediate specification of cell fate. We have used in situ hybridization to show that the expression of two EGF-related genes (SpEGF I and SpEGF II) is restricted to the same subset of ectodermal cells in sea urchin pluteus larvae. However, the concentration of EGF I mRNA in different epithelial cells of aboral ectoderm and postoral facial epithelium is constant while that of EGF II mRNA is highly modulated. RNase protection assays show that both genes are activated during the period when ectoderm founder cells are established, i.e., between fourth and fifth and between fifth and sixth cleavages for EGF I and EGF II, respectively. By mesenchyme blastula stage EGF I mRNA reaches maximum abundance (800–1000 copies/expressing cell) as a result of a high transcription rate, while EGF II mRNA peaks at about half that concentration by gastrula stage. EGF I expression begins at early stages of oogenesis while EGF II expression appears to be confined to embryogenesis.

The mechanism that establishes the maternally determined animal–vegetal axis of sea urchin embryos is unknown. We have analyzed the cis -regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a β-galactosidase reporter gene (Wei, Z., Angerer, L. M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195–211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic β-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis -acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957–1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early embryo.

In order to investigate how the maternally specified animal-vegetal axis of the sea urchin embryo is established, we have examined the molecular basis of regulation of several genes transcribed differentially in nonvegetal and vegetal domains of the very early blastula. Here we present an initial characterization of the regulatory region of one of these, SpAN, which encodes a protease in the astacin family related to Drosophila tolloid and vertebrate BMP-1 (Reynolds et al., Development 114, 769-786). Tests of SpAN promoter function in vivo show that high-level activity and correct not-vegetal expression are mediated by sequences within 300 bp upstream of the basal promoter. In vitro studies have identified six protein binding sites serviced by at least five different proteins. Comparison of the structure of the SpAN promoter to that of SpHE, whose expression pattern is identical, shows that both promoters contain multiple positively acting upstream elements close to the basal promoter. We show that two elements are critical for high-level transcription of SpAN, since exact replacement of either results in 10- to 20-fold reduction in promoter strength. These shared elements are, however, not essential for spatially correct SpHE gene transcription. We conclude that the coordinate strong activities of the SpAN and SpHE promoters in the nonvegetal domain of the embryo rely primarily on different transcription factor activities.

We have identified a gene in the sea urchin Strongylocentrotus purpuratus that encodes a member of the transforming growth factor β (TGF-β) gene superfamily. We have named the gene univin, and it is the first member of this superfamily to be reported in echinoderms. The cDNA sequence predicts a 383-amino-acid residue protein with 7 cysteine residues characteristic of members of this superfamily and with a cluster of basic residues appropriately situated to signal proteolytic cleavage. Sequence comparisons place univin in the bone morphogenetic protein (BMP) group of the TGF-β superfamily along with the vertebrate BMPs, decapentaplegic protein from Drosophila, and Vg-1 from Xenopus. Analyses of univin expression in early embryos by RNA blots and in situ hybridization revealed the highest levels of expression in the egg and prehatching blastula. During late cleavage stages, univin mRNA accumulation is progressively restricted to a circumequatorial band. Expression is further restricted during gastrulation when univin transcripts are detected primarily in the presumptive foregut and ciliated band. By pluteus stage, signals are detectable only in these cell types. The restricted temporal and spatial patterns of expression of univin during early blastula stages parallel those of SpAN , which encodes an astacin-like protease related to tolloid and BMP-1 (Reynolds et al., 1992). The fact that these proteases are thought to function in the proteolytic activation of TGF-β-related proteins that, respectively, regulate Drosophila embryonic dorsal-ventral patterning and vertebrate bone development suggests that SpAN and univin could also have critical roles in early developmental decisions in the sea urchin embryo.

Cloning and sequencing of Tripneustes gratilla genomic DNA and cDNA encoding a developmentally regulated, embryonic messenger RNA, referred to as Tg616, revealed an actin-encoding gene orthologous to the CyI actin gene described from Strongylocentrotus purpuratus. Tg616 and SpCyI share: (1) 150 nucleotides of highly conserved sequence 5′ of the transcription start site, (2) 95% nucleotide sequence identity in the protein encoding regions, which specify identical amino acid residues in 375 of 377 positions, and (3) extensive nucleotide sequence identity in the 3′ untranslated region of their messenger RNAs. Tg616 was therefore designated TgCyI. In situ hybridization shows sequential activation of TgCyI in various cells of the embryo. TgCyI mRNA becomes abundant in primary and secondary mesenchyme cells as they prepare to enter the blastocoel, in prospective aboral ectoderm cells at blastula stage, in gut cells during gut differentiation, and in oral ectoderm at pluteus stage. This pattern of embryonic gene expression is more complex than any of the major patterns of developmentally upregulated genes observed in S. purpuratus embryos and is distinct from SpCyI expression which is progressively restricted to the gut and oral ectoderm.

We have investigated the temporal and spatial patterns of accumulation of mRNAs randomly selected from the sea urchin gastrula polyadenylated RNA population. Three different assays show that the predominant temporal pattern of expression, exhibited by about three-fourths of these messages, consists of a large (mean 80-fold) increase in mRNA abundance between egg and gastrula stages. Most mRNAs are present in the maternal population and are detectable on blots as single mature-sized messages; however, a large number of high-molecular-weight, heterodisperse transcripts containing these same sequences also exist in the egg cytoplasm. The majority of gastrula messages are not embryo specific but are present in total adult urchin RNA at concentrations similar to those in embryos. Fine-scale RNA blot analysis indicates that the majority of mRNAs begin to accumulate at very early blastula stages, although there is considerable diversity in the time when these messages reach peak abundance. Most gastrula mRNAs are also spatially regulated during development. The observed distributions can be categorized into three major functional or regulatory classes: (1) Forty percent of mRNAs accumulate in cells which are cycling or preparing for growth. (2) About one-third of the messages accumulate in one or more differentiating cell types. (3) Only slightly more than one-fourth of the messages are present in all cell types throughout development. Most tissue-specific messages are relatively abundant, indicating that the differentiated functions of cells are executed through mRNAs operating at the level of hundreds of copies per cell. In contrast, most rare messages are expressed in most or all cell types, in which they function at only a few copies per cell. All messages which begin to accumulate before hatching blastula stage are initially distributed broadly, and their distribution becomes progressively restricted during embryogenesis. In contrast, all messages which begin to accumulate after the onset of gastrulation accumulate only in discrete subsets of cells. The results presented here illustrate much more extensive temporal regulation of gene expression during sea urchin embryogenesis than previously detected. This is accompanied by spatial regulation of expression of most genes which is itself temporally modulated as the cellular requirements for cell division and differentiation change during development.

We have identified an mRNA that encodes a protein, SpS24, of the small ribosomal subunit in the sea urchin, Strongylocentrotus purpuratus. RNA blot and in situ hybridization analyses show that the SpS24 gene is active during early oogenesis, downregulated in the mature egg and during cleavage, and reactivated in the early blastula. The mRNA then increases in abundance at least 100-fold. Later in development, expression of SpS24 mRNA becomes restricted primarily to cells in the oral ectoderm and endoderm of the pluteus larva, and the message is undetectable in aboral ectoderm cells and most mesenchyme cells. To determine whether transcription of the ribosomal RNA genes occurs at a higher rate in oral ectoderm and endoderm tissues, a probe for the transcribed spacer was used in RNase protection and in situ hybridization assays. High concentrations of rRNA-processing intermediates were observed in unfertilized eggs and shown to reside primarily, if not exclusively, in the cytoplasm. The spatial and temporal distributions of these sequences strongly suggest that they are associated with heavy bodies. New embryonic rRNA transcripts are first detectable at the very early blastula stage. In later embryos, the content of this transcribed spacer sequence is similar in all but a few cells, which implies that they synthesize rRNA at a similar low rate. Comparison of available estimates of rRNA transcription rate with the potential rate of SpS24 protein synthesis, calculated from SpS24 mRNA prevalence, shows that oral ectoderm and endoderm cells have the capacity to synthesize 15- to 30-fold more SpS24 protein than is required to keep pace with rRNA synthesis in these cells. Because the sea urchin embryo develops from an egg to a pluteus larva in the absence of growth, this stockpiling of SpS24 mRNA anticipates rather than accompanies the onset of growth, which does not begin until after feeding. Upregulation of this gene is therefore part of the developmental program, rather than a physiological response to nutrient availability.

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals which induce their synthesis. Tissue fractionation of embryos at pluteus stage previously demonstrated that in the absence of added zinc, basal expression of MT mRNA is confined to ectoderm, whereas induction by zinc results in increased expression in the endoderm + mesoderm tissue fraction. Using in situ hybridization we now show that expression in the pluteus larva is restricted almost exclusively to the single cell type comprising the aboral ectoderm. Induction by Zn results in a marked accumulation of MT mRNA in gut and oral ectoderm to levels at least as high as that in aboral ectoderm. MT mRNA is also expressed in presumptive aboral ectoderm at earlier stages of normal development. In addition it is transiently expressed at variable levels in oral ectoderm and, to a lesser extent, in presumptive gut.

Asymmetric RNA probes, which contain only the mRNA coding strand, provide a large increase in hybridization efficiency in situ over that observed with either symmetric (both strands represented) RNA or DNA probes. Asymmetric RNA probes are synthesized in vitro by transcription from recombinants formed between sequences encoding sea urchin mRNAs and the transcription vector R7 delta 7. Using a probe representing early variant histone mRNA sequences we have characterized hybridization to sections of sea urchin embryos with respect to thermal stability of the hybrids formed, optimum temperature, effect of sequence divergence on hybrid thermal stability, and dependence of the hybridization signals on probe concentration and hybridization time. Estimates from the observed signals indicate that a large fraction of target RNAs is both retained in sections and hybridized with probe at saturation. Coupled with measurements of nonspecific background binding of heterologous probes, these data indicate that the method has sufficient sensitivity to detect many moderately abundant mRNAs (20-75 molecules per cell in the 1500-cell pluteus). In situ hybridizations to embryos at different developmental stages show that while histone mRNAs are uniformly distributed in cleaving embryos, different cell lineages of older embryos show large differences in accumulation of these mRNAs.

We have used in situ hybridization and RNA blotting analysis to compare the timing of accumulation of poly(A) and α-subtype histone mRNA during oogenesis in the sea urchin Strongylocentrotus purpuratus . In situ hybridization with 3 H-poly(U) shows that the content of poly(A) in the developing oocyte increases four- to sixfold during vitellogenesis, implying a similar increase for polyadenylated maternal RNAs. In contrast, both RNA blotting and in situ hybridization demonstrate that there is little, if any, α-subtype histone mRNA in large oocytes. These results suggest that these maternal mRNAs accumulate in the pronucleus of the haploid egg after completion of meiotic maturation where they are stored until their release during the breakdown of the pronucleus during prophase.

We present an initial characterization of a database that contains temporal expression profiles of sequences found in 35,282 gene predictions within the sea urchin genome. The relative RNA abundance for each sequence was determined at 5 key stages of development using high-density oligonucleotide microarrays that were hybridized with populations of polyA+ RNA sequence. These stages were two-cell, which represents maternal RNA, early blastula, the time at which major tissue territories are specified, early and late gastrula, during which important morphogenetic events occur, and the pluteus larva, which marks the culmination of pre-feeding embryogenesis. We provide evidence that the microarray reliably reports the temporal profiles for the large majority of predicted genes, as shown by comparison to data for many genes with known expression patterns. The sensitivity of this assay allows detection of mRNAs whose concentration is only several hundred copies/embryo. The temporal expression profiles indicate that 5% of the gene predictions encode mRNAs that are found only in the maternal population while 24% are embryo-specific. Further, we find that the concentration of >80% of different mRNAs is modulated by more than a factor of 3 during development. Along with the annotated sea urchin genome sequence and the whole-genome tiling array (the transcriptome, Samanta, M., Tongprasit, W., Istrrail, S., Cameron, R., Tu, Q., Davidson, E., Stolc, V., in press. A high-resolution transcriptome map of the sea urchin embryo. Science), this database proves a valuable resource for designing experiments to test the function of specific genes during development.

Measurements are presented of the number of transcripts in sea urchin embryos and in adult intestine cells complementary to two cloned genomic single copy sequences. These sequences apparently code for maternal mRNAs stored in the egg. About 1000 and 1400 transcripts are stored per egg, close to the average value for maternal mRNAs. RNAs complementary to the cloned sequences are also found in the polysomal message of early embryos. The number of polysomal transcripts representing each cloned sequence is typical of the “complex” or “rare” class of message in sea urchin embryos. Expression of these putative complex class structural genes is regulated developmentally. Thus, polysomal transcripts of both sequences essentially disappear by the blastula stage. One sequence is again represented at a low level in adult intestine cytoplasmic RNA while the other is absent. However, in all the developmental stages examined, the number of nuclear transcripts of the cloned sequences remains about the same as for any average single copy sequence transcript.

Arylsulfatases are a group of enzymes that remove sulfate moieties from a diverse set of substrates including glycoproteins, steroids, and cerebrosides. We have isolated recombinant cDNA clones corresponding to an arylsulfatase (SpARS) message that encodes an abundant protein of pluteus larvae of the sea urchin Strongylocentrotus purpuratus. Although vertebrate arylsulfatases have broad tissue distributions, in situ hybridization with a probe for SpARS shows that the sea urchin message accumulates in the embryo only in the single cell type of aboral ectoderm and its precursors. The message is first detectable by RNase protection assays around hatching blastula stage and accumulates through pluteus larva stage. The open reading frame of cDNA clones is 1701 nt long and encodes a deduced protein with a predicted molecular mass of 61 kDa. Analysis of corresponding genomic DNA clones reveals that the pre-mRNA contains six exons. Consistent with the fact that arylsulfatase enzyme activity is extracellular, this polypeptide has a hydrophobic leader sequence and three potential glycosylation sites. Furthermore, hybridization in situ shows that in blastulae arylsulfatase message is preferentially concentrated around nuclei at the basal sides of cells. The S. purpuratus sequence is very similar to that recently reported for the same enzyme from Hemicentrotus pulcherrimus and 30% of the amino acid residues are also identical to those of both human arylsulfatase C (steroid sulfatase) and arylsulfatase A. Sequence relationships among these four mRNAs suggest that, assuming equal rates of evolution, the duplication separating the human genes occurred at about the time of separation of the echinoderm and vertebrate lineages.

Previous studies demonstrated that the pronucleus of the unfertilized sea urchin egg contains a high concentration of transcripts complementary to the early histone repeat unit (D. L. Venezky, L. M. Angerer, and R. C. Angerer (1981). Cell 24, 385-391.) In this paper, in situ hybridization techniques of improved sensitivity are used to show that these nuclear RNAs include authentic histone mRNA but not spacer-complementary sequences. It is estimated that most early-variant histone mRNA contained in the egg is, in fact, restricted to the pronucleus. These mRNAs are released to the cytoplasm at the time of nuclear breakdown of first cleavage and rapidly distribute throughout the cytoplasm.