Spatial patterns of metallothionein mRNA expression in the sea urchin embryo

DEVELOPMENTAL BIOLOGY 116,543-547(1986)

Spatial Patterns of Metallothionein mRNA Expression in the Sea Urchin Embryo LYNNE M. ANGERER,**'GARY *Department

KAWCZYNSKI,* DAVID G. WILKINSON,I.MARTINNEMER,~AND

of Biology,

for Cancer Research,

University of Rochester, Rochester, New York 14627, and flnstitute Fox Chase Cancer Center, Philadelphia, Penns$vania 19111

Received January

14, 1986; accepted in revised form Felrmary

ROBERTC. ANGERER*

18, 1986

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. 0 1986Academicpress,IX INTRODUCTION

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals. This property and the fact that expression of MT genes is induced by heavy metals suggest that MTs function in detoxification and in the regulation of trace heavy-metal metabolism (Cherian and Goyer, 1978; Kagi and Nordberg, 1979). In the latter case MTs could be involved in a variety of processes that involve enzymes requiring heavy metals. Other suggested functions of MTs in mammaliam cells focus on their capacity to scavenge free radicals produced during certain stress responses (for review, see Karin, 1985). Although some of these functions may be basic cellular properties, others may be executed primarily by specific tissues during particular periods of development. Spatial and temporal regulation of MT mRNA levels have been demonstrated during development of both sea urchins (Nemer et aZ., 1984) and mice (Andrews et ab, 1984). Through tissue fractionation of sea urchin embryos at the pluteus stage, Nemer et ah (1984) demonstrated that MT mRNAs are expressed at high levels only in ectoderm, while response to induction by exogenous Zn is primarily a property of tissues in the endoderm + mesoderm fraction. Because each of these two tissue fractions is composed of a variety of cell types, we have used in situ hybridization to identify the cells that express MT mRNAs in normal and Zn-induced embryos. MATERIALS

AND

METHODS

Embryo culture and jixation. Embryos of Strongylocentrotus purpuratus (Pacific Biomarine Supply Co., 1 To whom correspondence

should be addressed. 543

Venice, Calif.) were cultured at 15°C in artificial seawater (Nemer et al., 1984) prepared from reagent-grade chemicals with deionized water and adjusted to pH 8.1 and specific gravity 1.028. Under these standard conditions levels of expression will be referred to as “basal,” but note that MT mRNA expression is highly sensitive to low levels of Zn (Nemer et aL, 1984). For analysis of induction of MT mRNA synthesis, plutei were transferred for 4 hr (68 to 72 hr of development) to seawater containing 500 PM ZnSOl (at the resulting pH of approximately 7.4). Embryos were fixed with 1% glutaraldehyde, embedded in paraffin, and sectioned as described previously (Angerer and Angerer, 1981). Sections were mounted on polylysine-coated slides (Cox et al., 1986). In situ hybridization. A single-strand RNA probe was transcribed from a recombinant DNA template containing approximately 600 nucleotides of the MT mRNA sequence (the PstI-HinfI fragment of the “MTa” cDNA clone; Nemer et aL, 1985). This probe includes amino acid coding sequence and most of the 3’ untranslated region. The most downstream portion of the S’untranslated sequence was deleted to eliminate an SP6 transcription termination site. The PstI-HinfI fragment was inserted at the PstI site of pSp64 (Melton et al, 1984) by sequential cohesive- and blunt-end ligation. The template sequence was excised from a recombinant plasmid yielding mRNA strand transcripts (pMTaR-) at the bordering EcoI and Hind111 sites, and was recloned between the corresponding restriction sites of pSp65 (Melton et al., 1984) to yield a template encoding antisense transcripts (pMTaR+). Methods for synthesis of 3H-labeled probes and in situ hybridization have been described previously 0012-1606/86 $3.00 Copyright 0 1986by AcademicPress,Inc. All rights af reproductionin any form reserved.

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(Angerer and Angerer, 1981; Cox et al., 1984). Probe specific activity was 1.2 X lo8 dpm/pg and probe concentration for hybridization was 0.2 pg/ml which is estimated to be saturating (Cox et ak, 1984). The autoradiographic exposure time was 14 days. RESULTS

AND

DISCUSSION

The pattern of expression of MT mRNA in the sea urchin pluteus was analyzed by in situ hybridization with

VOLUME 116, 1986

an MT probe containing the coding sequence and most of the 3’ untranslated sequence of the sea urchin MTa cDNA clone (Nemer et aZ., 1985). In the pluteus larva (Fig. la, right) basal expression of complementary mRNA is strikingly localized in the single cell type comprising the aboral ectoderm. Examination of sections of a large number of embryos from four different cultures indicates that all regions of the aboral ectoderm contain MT mRNA. The concentration is essentially uniform

FIG. 1. Expression of MT mRNA in developing sea urchin embryos. Single-strand RNA transcripts of the MTa sequence were hybridized to sections of embryos allowed to develop in synthetic seawater prepared from reagent-grade chemicals. All sections shown are from the same slide, so that grain densities are directly comparable. All sections are shown with the vegetal pole at the bottom. (a) left, unfertilized egg and right, pluteus (73 hr). The “a” marks the aboral side, and “0” the oral side of the pluteus; the arrowheads point to several labeled cells in the oral ectoderm. For both stages the same section is shown in phase-contrast and dark-field illumination. (b), Sections of early mesenchyme blastula (20 hr) are shown in dark-field illumination to illustrate the observed range in grain density. (c), shows the sections adjacent to those in row b, demonstrating the reproducibility of the signals. (d) sections of three different gastrulae (44 hr). In (b-d) each left panel shows a phase-contrast micrograph of the section shown in the middle dark-field panel of each row; these are most typical in extent and pattern of labeling. The bar represents 10 pm.

BRIEF NOTES

545

within any one embryo section, although the signals vary the labeling of preectoderm is uniform, with no indias much as fivefold among different embryos (see below). cation of a distinction between the presumptive oral and No labeling is observed over gut, mesenchyme cells or aboral regions. The unlabeled cells always include prithe majority of oral ectoderm. In some sections labeling mary mesenchyme cells that have ingressed into the is observed over very small groups of cells in the oral blastocoel and a small region of the blastula wall. The ectoderm, adjacent to the oral lobe and/or developing latter corresponds in size and position to presumptive anal arms (arrows in Fig. la, right). Except for this mi- secondary mesenchyme cells which are distinguishable nor signal in the oral region, MT mRNA represents the from the surrounding cells at this stage by their content fourth example of a message restricted to the aboral of a specific actin mRNA, CyIIa (Angerer and Davidson, ectoderm cell type, the others being Specl (Lynn et al., 1984; Cox et aZ., 1986). In some embryos the unlabeled 1983) and the cytoplasmic actin CyIIIa and CyIIIb mes- area is large enough to include most, if not all, of the sages (Angerer and Davidson, 1984; Cox et al., 1986). precursors to the larval endoderm as well. To determine whether MT mRNAs are restricted to During gastrulation there is a transition toward the the aboral ectoderm lineage throughout development, highly tissue-specific pattern exhibited by plutei (Fig. we examined their distribution at earlier stages. The Id). Primary mesenchyme cells along the wall of the grain densities indicate that the average level of MT blastocoel and secondary mesenchyme cells at the tip of mRNA rises during development from egg (Fig. la, left) the archenteron remain unlabeled. Although signals to blastula (Figs. lb and c), falls during gastrulation above background are observed over the presumptive (Fig. Id), and rises again in the pluteus (Fig. la, right), gut of some embryos (e.g., Fig. Id, middle panel), most in agreement with results obtained from RNA blots MT mRNA is restricted to the ectoderm. Labeling of the (Nemer et al, 1984). Moreover, this analysis of individual ectoderm varies from rather high uniform levels to those embryos revealed variability among them both in ab- close to background. Some embryos show differences in solute amounts of MT mRNA and, at earlier stages, in MT mRNA abundance between aboral and oral lineages its cellular distribution (Figs. lb and c). Three obser- (Fig. Id, right panel). Thus the transient decrease in vations indicate that these differences are real. First, average MT mRNA content of whole embryos of gastrula similar variability was observed for several separate stage (Nemer et al., 1984) is due both to the progressive cultures. Second, the pattern and overall intensity of elimination of these messages from presumptive oral labeling were quite reproducible in adjacent sections of ectoderm and gut, and to a temporary decrease in MT individual embryos whose MT mRNA content differed message content in the presumptive aboral ectoderm of significantly (compare each section in Fig. lb with its most embryos. Flytzanis et al. (1982) have shown that adjacent section shown in Fig. lc). Third, variation in at gastrula stage most moderately abundant mRNAs grain density among different embryos was considerably exhibit a transient two- to threefold decrease in congreater (for example, about fivefold for plutei) than that centration per embryo that seems to reflect a basic modobserved for sections of embryos from the same culture ulation of synthesis and decay rates that occurs at this hybridized with probes for other mRNAs (data not stage. The data presented here show that, at stages before shown). The sections shown in Fig. 1 have been chosen to illustrate both the range observed at several stages the pluteus, expression of MT mRNA is not restricted (Figs. lb-d) and the levels and patterns of labeling most to aboral ectoderm. We note that, in individual embryos, typical for each stage (middle dark-field micrograph of major cell lineages can be ranked with regard to relative each row). Since cultures consisted of the mixed progeny abundance of MT mRNA: aboral ectoderm > oral ectoof three to six different females, the variations among derm > endoderm > mesenchyme. As development proembryos may reflect different levels of, and/or suscep- ceeds, expression of MT message within individual emtibilities to, heavy metals acquired during oogenesis. bryos becomes progressively restricted to fewer lineages At the mesenchyme blastula stage (Figs. lb and c) MT according to this same hierarchy. The progress of this mRNA is clearly distributed throughout a much larger restriction varies among different embryos and its regfraction of the embryo than that which comprises pre- ulation may therefore depend on cues other than time cursors to aboral ectoderm (about one-third of the em- of development. Although MT, Specl and CyIII actin bryo volume). Individual sections passing through the mRNAs are all highly localized in the aboral ectoderm animal-vegetal axis contain a large labeled region and of pluteus larvae, their distributions at earlier stages a small region at the vegetal pole with grain densities differ. Specl and CyIII actin mRNAs are confined to the at, or only slightly above, background. The labeled region precursors of aboral ectoderm from the time that these always includes both oral and aboral portions of the messages begin to accumulate at blastula stage, while presumptive ectoderm. Whereas the overall intensity of MT mRNA is expressed much more widely. labeling varies among embryos, within any one section Nemer et al. (1984) previously showed that treatment

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FIG. 2. Zinc-induced expression of MT mRNA in plutei. Plutei were treated with 500 @M ZnSOl from 68 to ‘72 hr after fertilization mRNA was detected by in situ hybridization. The same sections are shown in phase-contrast (a) and dark-field (b) illumination. indicate unlabeled mesenchyme cells. The insets show a cluster of mesenchyme cells in a different section, that typically are observed the tips of the spicules at the vertex of the cone of aboral ectoderm (The spicules themselves are lost in processing of the tissue). represents 10 Wm.

of plutei with zinc induces a dramatic accumulation of MT message in the endoderm-mesoderm tissue fraction, but little or no increase in ectodermal fractions. To identify the responding cells we analyzed sections of plutei that had been treated with high levels of zinc (500 PM) for 4 hr before fixation, which results in maximal levels of MT message in RNA prepared from whole cultures. As shown by the grain densities in Fig. 2, this results in expression of MT message in most cells of the embryo at levels at least as high as those in aboral ectoderm. In comparison to uninduced embryos, grain densities over different sections were much more uniform, suggesting that all embryos can accumulate similar maximal induced levels of MT mRNA. The only unlabeled cells that we can consistently identify in zincinduced plutei are mesenchyme (see arrows, Fig. 2). Thus in the endoderm-mesoderm fraction the gut is the principal, if not exclusive, site of induction. Induction of MT mRNA accumulation in ectoderm is primarily a response of the oral region which, by morphological criteria, includes several different cell types. The induction in whole ectoderm (aboral and oral) involves a much smaller relative increase over basal levels than is observed for the endoderm-mesoderm fraction, and was not readily detectable using tissue fractionation techniques (Nemer et al., 1984). However, as oral ectoderm comprises about one-third of the embryo volume (Lee et ab, 1986), it accounts for a substantial fraction of inducible MT mRNA. MT mRNA expression in whole embryos responds in

and MT Arrows around The bar

a dose-dependent manner to a range of low concentrations of zinc (5-25 PM; Nemer et al, 1984). It is clear that regulation of this expression is a complex process involving different cell lineages and probably different MT genes. As is the case in most higher organisms, metallothionein proteins of sea urchins are encoded by more than one gene, and the MTa probe cross-hybridizes with at least two different mRNAs (unpublished results of D. Wilkinson and M. Nemer). Resolution of the potential relationships of the expression of individual genes to specific tissues, and to basal or induced expression awaits characterization of different MT genes and development of specific probes for the mRNAs they encode. This work was supported by a Research Career Development Award to R.C.A., U.S. Public Health Service grants GM25553 (to L.M.A. and R.C.A.), HD-04367 (to M.N.), CA-06927 and RR-05539 (to Institute for Cancer Research), and an appropriation from the Commonwealth of Pennsylvania. We appreciate comments from E. Stephenson and J. B. Olmsted which improved the manuscript. REFERENCES ANDREWS, G. K., ADAMSON, E. D., and GEDAMU, L. (1984). The ontogeny of expression of murine metallothionein: Comparison with a-fetoprotein gene. Dev. Biol. 103,294-303. ANGERER, L. M., and ANGERER, R. C. (1981). Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res. 9,2819-2840. ANGERER, R. C., and DAVIDSON, E. H. (1984). Molecular indices of cell lineage specification in sea urchin embryos. Science (Washington, D.C.) 226,1153-1160.

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CHERIAN,M. G., and GOYER,R. A. (1978). Metallothioneins and their LEE, J. J., CALZONE,F. J., BRIXTEN,R. J., ANGERER,R. C., and DAVIDSON, role in metabolism and toxicity of metals. L$“e Sci. 23, l-10. E. H. (1986). Activation of sea urchin actin genes during embryogenesis: Measurement of transcript accumulation from five different Cox, K. H., DELEON,D. V., ANGERER,L. M., and ANGERER,R. C. (1984). genes. J. Mel Biol 188,173-183. Detection of mRNAs in sea urchin embryos by in situ hybridization LYNN, D. A., ANGERER,L. M., BRUSKIN,A. M., KLEIN, W. H., and ANusing asymmetric RNA probes. Dev. Bid 101,485-502. GERER,R. C. (1983). Localization of a family of mRNAs in a single Cox, K. H., ANGERER,L. M., LEE, J. J., DAVIDSON,E. H., and ANGERER, cell type and its precursors in sea urchin embryos. Proc. Natl. Accd R. C. (1986). Cell lineage-specific programs of expression of multiple Sci. USA 80,2656-2660. actin genes during sea urchin embryogenesis. J. Mel Biol. 188,159MELTON,D., KRIEG, P., REBAGLIALI, M., MANIATIS, T., ZINN, K., and 172. GREEN,M. (1984). Efficient in vitro synthesis of biologically active FLYTZANIS,C. N., BRANDHORST,B. P., BRI’ITEN, R. J., and DAVIDSON, RNA and RNA hybridization probes from plasmids containing a E. H. (1972). Developmental patterns of cytoplasmic transcript bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035-7056. prevalence in sea urchin embryos. Dew. Biol 91,2i’-35. NEMER,M., TRAVAGLINI,E. C., RONDINELLI,E., and D’ALONZO, J. (1984). KAGI, J. H. R., and NORDBE&G,M. (Eds.) (1979). “Metallothionein: ProDevelopmental regulation, induction and embryonic tissue specificity ceedings of the First International Meeting on Metallothionein and of sea urchin metallothionein gene expression. Dev. Biol 102, 471Other Low Molecular Weight Metal-Binding Proteins.” Experientia, 482. Suppl. 34. Bikhausen, Basel. NEMER, M., WILKINSON, D. G., TRAVAGLINI, E. C., STERNBERG, E. J., KARIN, M. (1985). Metallothioneins: Proteins in search of function. CeU and BUTT, T. R. (1985). Sea urchin metallothionein sequence: Key to 41.9-10. an evolutionary diversity. Proc. Natl. Acad Sci USA 82,4992-4994.