Structure and tissue-specific developmental expression of a sea urchin arylsulfatase gene

I)~VELOPMENTAI,

135,53-65 (1989)

BIOLOGY

Structure and Tissue-Specific Developmental Expression of a Sea Urchin Arylsulfatase Gene QING

YANG,

LYNNE

M. ANGERER, AND ROBERT

C. ANGERER

Department of Biology, University of Rochester, Rochester Nexl York 14627 Accepted May J+,1989 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 pw-puratus. 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 1’701 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. prpuratus 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. 8 198s .Aeademir

Press, Inc

INTRODUCTION

morphological consequences of this specification begin to be detectable only at gastrula stage, when aboral ectoderm cells begin to flatten and spread. However, assays of spatial patterns of mRNA accumulation in different territories of the early embryo have clearly shown that this morphological differentiation is the denouement of events at early blastula stage which establish specific patterns of gene expression in presumptive aboral ectoderm cells and other regions of the early blastula (Lynn et ah, 1983; Angerer and Davidson, 1984; reviewed by Davidson, 1986). Both results of classical experimental embryology that were analyzed by morphological criteria (reviewed by Horstadius, 1973) and contemporary criteria of expression of tissue-specific genes (Hurley et ab, 1989) indicate that differentiation of aboral ectoderm requires inductive signals from cells at the vegetal pole. Several practical considerations make aboral ectoderm an especially amenable model for the differentiation of a cell type. The cells comprise a large fraction of the embryo; they can be separated with good purity from endoderm and mesenthyme and, to a lesser extent, from oral ectoderm (reviewed by M&lay, 1986); and recent efforts of several laboratories have identified a number of mRNAs expressed exclusively in these cells (Lynn et ad.,1983; Cox et al., 1986; Angerer et al., 1986; Hardin et al., 1988;

During development of the sea urchin embryo cells derived from approximately three-quarters of the egg volume differentiate to form ectoderm (reviewed by Horstadius, 1973). Ectoderm includes two distinguishable regions, oral and aboral, which, in the unfed pluteus, contain similar numbers of cells. Oral ectoderm includes the ciliated band that forms the perimeter of the oral face of the embryo and a second major histologically distinguishable set of cells that forms an epithelium around the mouth. Aboral ectoderm forms a cone of squamous epithelium that covers the aboral surface of the pluteus larva. This tissue appears to contain a single cell type based both on histology and on the fact that, except for a gene containing a homeobox sequence (Angerer et al., 1989), each aboral ectoderm-specific mRNA examined thus far accumulates to similar concentration in different regions (for example, see Lynn et al., 1983; Cox et al., 1986; Angerer et al., 1986; Hardin et al., 1988). Differentiation of aboral ectoderm is an exhibition of the corresponding specification of the oral-aboral axis that is imposed on the radially symmetric blastula. The Sequence data from this article have been deposited with EMBL/GenBank Data Libraries under Accession No. M25815.

the

53

0012-1606/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

54

DEVELOPMENTAL BIOLOGY

Angerer et al., 1989) whose protein products must make a major contribution to the aboral ectoderm phenotype. To date, all tissue-specific mRNAs expressed in the sea urchin embryo are among the minority of messages that are essentially absent from maternal RNA and increase significantly in abundance during embryogenesis. The earliest aboral ectoderm-specific mRNAs begin this increase at early blastula stage (Nemer, 1986; Lee et al., 1986; Hickey et al., 1987; Hardin et al., 1988; Angerer et al., 1989). In an effort to identify additional tissue-specific mRNAs we screened a library of cDNAs, representing mRNAs expressed in gastrula stage embryos of Strongylocentrotus purpuratus, for sequences much less abundant in maternal RNA of the egg and then used in situ hybridization to identify tissue-specific sequences. One of these was found to be an abundant mRNA expressed exclusively in aboral ectoderm. Sequence analysis of this cDNA showed that it is closely related to a cDNA clone from a different urchin species, Hemicentrotus pulcherrimus, identified by Shimada’s group (Sasaki et al., 1988) that encodes arylsulfatase. The proteins encoded by the sea urchin genes also have significant sequence similarity to human arylsulfatase A (Stein et al., 1989) and arylsulfatase C (steroid sulfatase; Yen et al., 1987, 1988). In this work we have sequenced cDNAs encompassing the complete coding region and located exon borders and the transcription initiation site on a recombinant genomic DNA that includes the complete gene and about 10 kb of upstream sequence. Using RNase protection and in situ hybridization methods we have characterized the temporal and spatial patterns of expression of this gene throughout development. MATERIALS

Extraction

AND

METHODS

of RNA from Embryos

Total RNA was isolated from embryos of various developmental stages as described (Nemer et al, 1984). Poly(A)+RNA was selected by oligo(dT)-cellulose chromatography as described by Maniatis et al. (1982). Diflerential Screening for Genes Encoding Abundant mRNAs at Gastrula and Pluteus Stages Inserts from a hgtl0 gastrula cDNA library constructed by Dr. Terry Thomas (Texas A&M University) were transferred to the transcription vector, pGEM4Z, as follows. Phage DNA was digested by EcoRI and the inserts were separated on a 1% agarose gel, purified with Gene Clean (BiolOl, Inc.), and ligated to EcoRI-cut plasmid vector. Plasmid DNA containing inserts were electrophoresed in duplicate slot lysis gels according to

VOLUME 135,1989

the method of Sekar (1987) and transferred to Gene Screen Plus membranes (DuPont-NEN Research Products). These filters were hybridized with either egg or pluteus poly(A)+ RNA that had been hydrolyzed to about 300 nt (Cox et al., 1984) and end-labeled with [T-~“P]ATP and T4 polynucleotide kinase to a specific activity of approximately 1 X lo7 dpm/pg. Hybridization was carried out in 2~ SSC (1X SSC = 0.15 MNaCl, 0.015 M Na-citrate), 5X Denhardt’s, 25 mM sodium phosphate, 1% SDS, 1% sodium pyrophosphate, 10% dextran sulfate, and 12.5 yg/ml denatured calf thymus DNA at 60°C. Filters were washed at the hybridization temperature in a series of solutions containing 1% SDS:4X SSC, 2X SSC, 1X SSC, and 0.1X SSC. RNA Blots Two micrograms of RNA isolated from embryos of various stages were electrophoresed through a 1% agarose gel containing 2.2 Mformaldehyde (Maniatis et al., 1982) and blotted onto Gene Screen Plus membrane. Hybridization was carried out at 65°C in the same solution as described above using as probe the 1.2-kb cDNA (positions 1093-1387 in Fig. 5) that had been labeled with 32Pto 5 X lo7 dpm/yg by random oligonucleotide priming (United States Biochemical Corp.). Final washing of the filter was in 0.1X SSC, 1% SDS at 65°C. Genomic DNA Blots DNA isolated from sperm of a single male sea urchin was digested with various restriction enzymes and the resulting fragments were separated in a 1% agarose gel and transferred to Gene Screen Plus membrane. The blot was then hybridized with random primed probe (0.25-kb KpnI-Hind111 fragment) representing the protein coding region of the 3’ exon (shown on Fig. 9 and corresponding to amino acid residues 429-512 in Fig. 6) and washed as described above for RNA blot hybridization. Preparation

and Screening of a Genomic Library

DNA isolated from the sperm of a single male urchin was digested to different extents with Sau3A and DNA fragments averaging 15 kb were selected by sucrose density gradient centrifugation, pooled, ligated to BamHI-digested EMBLS vector (Promega), and packaged (Packagene, Promega). Approximately 3 X lo5 plaques, equivalent to about five genomes, were transferred to Colony/Plaque Screen filters (DuPont-NEN Research Products) and screened by hybridization as described above with gastrula cDNA clones, again labeled with 32P by random priming.

YANG, ANGEREK, AND ANCERER

In Situ Hybridization and probe preparation were In situ hybridization carried out as described by Cox et al. (1984). Embryos from different stages were fixed in 1% glutaraldehyde, embedded in paraffin, and sectioned to a thickness of 5 pm as described previously (Angerer and Angerer, 1981). 3H-labeled antisense (1.2 kb representing the 3’ untranslated sequence and approximately one-third of the translated region) and control heterologous RNA probes were synthesized in vitro using [3H]UTP and C3H]CTP (Amersham) at a specific activity of 1 X lo8 dpm/yg using Sp6 RNA polymerase, and the fragment size was reduced to approximately 200 nts by limited alkaline hydrolysis. Autoradiographic exposures were for 7 weeks. RNase Protection To determine the timing of SPARS mRNA accumulation relative to that of other mRNAs expressed in aboral ectoderm, RNase protection assays were carried out exactly as described previously with the same RNA preparations used to measure accumulation of mRNAs encoding Specl, actin CyIIIa, and SpHboxl proteins (Angerer et al., 1989). =P-labeled antisense RNA was purified on a 5% polyacryamide gel containing 8 M urea. A gel slice containing the full-length runoff transcript was excised and the RNA was eluted overnight at room temperature in 300 ~1 of a solution containing 300 mM ammonium acetate, 1 mM EDTA, 5 kg yeast tRNA, and 0.1% sodium dodecyl sulfate. The eluate was collected by sedimentation through glass wool and the RNA was purified by organic extraction and ethanol precipitation. Ten micrograms of RNA isolated from embryos of various stages was incubated with a sequence excess of the labeled probe in a solution containing 50% formamide, 40 mM Pipes, pH 6.7, 0.5 M NaCl, and 1 mM EDTA at 50°C. Ten micrograms of yeast tRNA was also incubated as a negative control. Unhybridized probe was removed by digestion with 40 pg RNase A/ml and 0.75 U RNase Tl/ml at 37°C for 30 min. RNase-resistant hybrids were purified by digestion with proteinase K (145 pg/ml, 25°C 30 min), extraction with phenol:chloroform, and ethanol precipitation. The pellets were resuspended in a solution containing 80% formamide, 0.089 M Tris-borate, 0.089 M boric acid 2 mM EDTA, and 0.02% bromphenol blue and electrophoresed on the same denaturing polyacrylamide gel. The dried gel was exposed for l-3 days to Kodak X-Omat film using two intensifying screens. DNA Sequencing DNA sequencing was carried out according to the manufacturer’s instructions (United States Biochemi-

Sea Urchin

Arylsu(fntase

55

Gene

cal Corp.), using [35S]dATP and Sequenase on either single-stranded Ml3 DNA using universal primer (United States Biochemical Corp.) or double-stranded plasmid DNA using T7 or Sp6 primers (Promega). DNA sequences and deduced amino acid sequences were analyzed by the computer programs developed by Pustell and Kafatos (Pustell and Kafatos, 1982, 1984) and by Genetics Computer Group’s Sequence Analysis Software Package. Primer

Extension

A synthetic 21-base oligonucleotide was labeled at the 5’ end with [y-‘“P]ATP using T4 polynucleotide kinase. One nanogram of labeled oligomer was hybridized with 20 pg of total pluteus RNA in 0.25 M KCl, 10 mM TrisHCl, pH 8.0, 1 mM EDTA at 50°C for 3 hr. The primer was extended using M-MLV reverse transcriptase (Bethesda Research Laboratories) in the presence of 1 mM dNTPs, 12.5 mM DTT, 3 mM MgClz at 37°C for 1 hr. The reaction mixture was phenol:chloroform extracted and ethanol precipitated. The pellet was resuspended in the sequencing dye mix and electrophoresed on a 6% polyacrylamide gel. RESULTS

AND

Accumulation of ArylsuJfatase dewing Development

DISCIJSSION

mRNA

We screened 200 randomly selected recombinant clones from a library of S. purpuratus gastrula stage cDNAs by hybridization on blots with ‘“P-end-labeled poly(A)+RNA from unfertilized eggs or from pluteus stage embryos. One of the clones selected, G196, hybridized to an mRNA that was very abundant at pluteus, but undetectable in the egg. Furthermore, the G196 sequence did not hybridize to cloned DNAs representing other identified abundant mRNAs having a similar temporal pattern of expression. As shown below, G196 was subsequently found to be homologous to a gene from a different sea urchin species, H. pulcherrimus, that encodes arylsulfatase, and we refer to these genes as SPARS and HpARS. RNA blot analysis shows that the SPARS probe detects a single transcript, approximately 2.8 kb in length (Fig. 1). This message is undetectable in RNA from eggs and early blastulae (15 hr). Low levels are found at mesenchyme blastula stage (24 hr) and the abundance increases greatly by late gastrula and is maintained at pluteus stage. This is quite similar to the pattern of accumulation reported for HpARS mRNA (Sasaki et al., 1988). To establish the time of onset of accumulation of SPARS mRNA more precisely, we performed RNase protection assays using the antisense transcript of the

56

DEVELOPMENTALBIOLOGY egg

3.8

9h 15h 24h 48h 72h

kb -

1.8 kb -

FIG. 1. Accumulation of SPARS mRNA during development. Total RNAs from eggs and embryos at the indicated times of development were analyzed by blotting and hybridization with the G196 cDNA probe. Developmental times of 9, 15, 24, 48, and 72 hr correspond to late cleavage (about 100 cells), early blastula (about 225 cells), mesenthyme blastula, late gastrula, and pluteus stages, respectively. Size calibration, shown at left, was provided by positions of the ribosomal RNAs.

0.3-kb EcoRI-Hind111 fragment of G196-1 (positions 1093-1384, Fig. 5) as probe. As shown in Fig. 2A, SPARS message does not begin to accumulate until around 15 hr, shortly before hatching which occurs at approximately 18 hr. The relative levels of SPARS mRNA in 24-hr vs 4%hr embryos are variable among cultures (cf. Figs. 1 and 2a), as a result of the extremely rapid accumulation of this message during mesenchyme blastula stage. The results from RNase protection assays confirm the conclusion from RNA blot data that the mature S. purpuratus egg lacks ARS message; from measurements of the sensitivity of this assay we estimate that the egg contains fewer than 50 ARS transcripts (an abundance equivalent to less than one molecule in 20-40 pluteus cells). Relative intensities of signals compared to those obtained with probes for mRNAs of known prevalence indicate that SPARS mRNA is one of the most abundant messages so far identified in the pluteus larva and is present at a level similar to that of actin CyIIIa mRNA, several hundred copies per cell. SPARS mRNA Accumulates

only in Aboral Ectoderm

In a first effort to determine whether SPARS gene expression is spatially restricted in the embryo, we used RNase protection assays to measure the relative abundance of SPARS mRNA in tissue fractions of pluteus larvae enriched for ectoderm or endoderm + mesen-

VOLUME 135,1989

thyme. As shown in Fig. 2B, SPARS mRNA is highly enriched in the ectoderm fraction. The ratio of signals in ectoderm to endoderm + mesenchyme is similar to that for other mRNAs, such as Specl (Lynn et ah, 1983) and actin CyIIIa (Cox et al., 1986), previously shown to be abundant only in aboral ectoderm (data not shown), indicating that the signal obtained for the endoderm + mesenchyme fraction is attributable to contamination of this fraction with a small proportion of ectoderm cells. To determine the distribution of SPARS message within the ectoderm of plutei and in embryos at earlier stages, we used in situ hybridization with a 3H-labeled RNA probe corresponding to the 3’ 1.2 kb of the cDNA, including the untranslated region and about one-third of the protein coding sequence. As shown in Fig. 3, the temporal pattern of accumulation evident from hybridization to sections of embryos at different stages is consistent with that demonstrated by RNA blot and RNase protection: It is undetectable in unfertilized eggs (Fig. 3a) and in embryos at the 16-cell stage (Fig. 3b) and very early blastula stage (12 hr; Fig. 3c), accumulates to moderate abundance at mesenchyme blastula stage (Figs. 3d and 3e), and to high levels in gastrulae and plutei (Figs. 3f and 3g, respectively). In embryos of all stages at which it is detectable in this analysis, SPARS mRNA is distributed in the stereotyped pattern for an aboral ectoderm-specific mRNA, as described by Lynn et al. (1983). In the 20- and 23-hr blastula sections (Figs. 3d and 3e, respectively), which pass through the animal-vegetal axis and perpendicular to the oralaboral axis, there are two labeled regions corresponding to the future left and right sides of the aboral ectoderm.

FIG. 2. Assays of SPARS message accumulation and tissue specificity by RNase protection. The SPARS mRNA content of different RNAs was analyzed by hybridization with an excess of antisense transcript of the 0.3-kb EcoRI-Hind111 cDNA fragment, digestion with RNase, and gel electrophoresis. (a) Time course of accumulation of SPARS message during development. Embryos at 15 hr are at early blastula stage (200-250 cells), about 3 hr before hatching. Stages for the other times of development indicated are described in the legend to Fig. 1 (ytRNA) An equal amount of yeast tRNA was substituted for sea urchin RNA. (b) RNA was isolated from tissue fractions enriched for endoderm + mesenchyme (endo/meso) and from ectoderm (ecto). (probe) Position of migration of the probe transcript.

57

FIG. 3. SpARS mRNA accumulates only in aboral ectoderm. A “H-labeled antisense SPARS RNA probe corresponding to the protein coding sequence of the fifth exon was hybridized in sits to sections of S. yur;vu~rtzt.s embryos of the following stages of development: (a) unfertilized egg; (b) 16-cell stage; (c) early blastula, about 180 cells at 12 hr; (d) early mesenchyme blastula, 20 hr; (e) mesenchyme blastula, 23 hr (f) gastrula, 43 hr; and (9) pluteus larva, 73 hr. The same sections are shown photographed under phase-contrast (upper rows) and dark-field (lower rows) illumination. Sections in b, d, e, and f are shown with the animal pole at the top. Sections in f and g pass through the plane of bilateral symmetry with the aboral sides on the right in the gastrula section f and on left in the pluteus section (g). Arrowheads in f and g mark the position of the supraanal ectoderm. Sections in d and e are cut approximately perpendicularly to the oral-aboral axis and labeled regions correspond to left and right sides of the aboral ectoderm; the unlabeled region at the animal pole is the region that will form oral ectoderm above the mouth (oral lobe). All sections are shown at the same magnification; bar in a =lO gm. aoe, aboral ectoderm; oe, oral ectoderm.

Grain densities over presumptive oral ectoderm at the animal pole (top) and endoderm and mesenchyme at the vegetal pole (bottom) are not distinguishable from background (Figs. 3d-3f). In embryos of later stages, when aboral ectoderm can be identified by histology, all aboral ectoderm cells are uniformly labeled, as illustrated by the 44-hr gastrula and 73-hr pluteus stages in Figs. 3f and 3g, respectively. The arrowheads in Figs. 3f and 3g mark the position of supraanal ectoderm which is a portion of aboral ectoderm tissue. The SPARS signals at all stages indicate that during the differentiation of aboral ectoderm this mRNA accumulates uniformly in different cells as do all other aboral ecto-

derm-specific mRNAs examined to date (Lynn et ul., 1983; Cox et al., 1986; Angerer et al., 1986; Hardin et al., 1988), except one encoding a protein with a homeobox domain (Angerer et al., 1989). We conclude that, within the limits of sensitivity of detection, SPARS expression is confined to aboral ectoderm cells and their precursors throughout development. Although i?z sifu hybridization patterns reveal uniform distribution of SPARS mRNA in aboral ectoderm tissue, we have noticed polarity of SPARS mRNA distribution zoitlh these cells at early stages. Figure 4 shows that, in both mesenchyme blastulae (a and b; also see Fig. 3e) and early gastrulae (d and e; 50% gut in-

58

DEVELOPMENTAL BIOLOGY

vagination), grain densities are clearly higher over basal sides of the blastomeres than they are over apical cytoplasm. Unlabeled apical regions do not correspond to nuclei, which are basal by this stage and sometimes appear as unlabeled spots in heavily labeled sections. Furthermore, it is unlikely that this grain distribution results from an artifact of fixation or hybridization, because similar sections of the same embryos in the same experiment show that Specl mRNA is not localized within the cytoplasm (Figs. 4c and 4f). We cannot determine whether SPARS mRNA is similarly localized in cells at later stages because the resolution of 3H autoradiography is low relative to the thickness of these flattened cells. Similar subcellular localization is not observed for other aboral ectoderm-specific mRNAs that encode intracellular proteins, and this mRNA distribution is undoubtedly related to the fact that the SPARS protein is secreted into, and accumulates at high concentration within, the blastocoel from blastula through pluteus stages (Yang et al., manuscript in preparation). Nucleotide Sequence and Deduced Amino Acid Sequence of SPARS cDNA Longer cDNA clones were obtained by screening the gastrula cDNA library with the original G196 sequence (positions 1093-2300 in Fig. 5) as probe. The nucleotide sequence of the longest cDNA (Fig. 5) has an open

VOLUME 135.1989

reading frame of 1701 bp and includes 15 bases of untranslated sequence at the 5’ end. The sequence shown for the remainder of the 5’ leader in Fig. 5 was obtained from the corresponding genomic sequence after determination of the transcription initiation site by primer extension as described below. The 3’ untranslated region includes at least 575 nt. None of the cDNA clones recovered included sequence corresponding to the 3’ poly(A) tail or the AATAAA polyadenylation signal (Proudfoot and Brownlee, 1976). However, two potential poly(A) addition signals are present in the genomic clone just 11 bases downstream of the 3’ end of the cDNA and the length of the mRNA estimated from RNA blots indicates that it cannot extend far beyond this region. Although many genes contain more than one polyadenylation signal (reviewed by Leff et al., 1986), the overlapping arrangement found here, AAUAAAAUAAA, is unusual; whether either or both of these sequences is functional remains to be examined. The protein sequence deduced from the open reading frame is also shown in Fig. 5 and predicts a protein with a molecular mass of about 61 kDa. Antibodies raised against a trpE fusion protein detect a major peptide of this size on Western blots as well as several minor species of higher apparent molecular weight (data not shown) that may result from incomplete denaturation of the protein, which is thought to be a decamer of identical subunits (Sasaki et al., 1987). Consistent with the fact that arylsulfatase is an extracellular protein

FIG. 4. Concentration of SPARS mRNA at the basal end of cells in the blastula-gastrula period. Sections of 20-hr mesenchyme blastulae (a-c) or early gastrulae (d-f) were hybridized with the SPARS probe (a, b, d, e) or with a probe for the Specl mRNA aboral ectoderm marker (c, f). Pairs (a and b; d and e) show the same sections photographed in phase-contrast (left) and dark-field (right) illumination. Bar in a = 10 Grn.

YAN(:,

ANGEKER,

AND

SPU Urch iv Arylsulfatu~se

ANCERER

59

Gene

ctcttctctcctccctcttttcttcttctttcatacgcatttttttcccctcctttttcttttttccctccttttttctacttttctccctttttcttttccccccttccccctttcccc

-241

cctcttttcctttttttttacgagacgtgccccctgtgtccccctgaatccqccactqactatgtcqacgtqcaaatctaaaaqtccctaatgaagtattttgatqactqatcqctqqca

-121 -1

acatcgctgtctccttctgcatqaagaaqacGTTTATG~T~~TG~GC~CGTTGTTTGC~GTCTCTTCCTCTTT~CGTTCTTG~CTGGTAG~~CC~G~~C~CACT -M-9-R--T-L-rP-S-4.-F-b-F--L-y-h,-G-L-y-4

G Q G

D

D

T

120 25

GACGGPCCAGATGCCGAAAGTCTGGCCAGCCTTGATCGCA~~TAC~~~TACG~~T~T~~CCTGCTGCATCTCCTG~TCA~CT~CAGCA~~CCGC~T~CG DGEDLLHLLGQTGQHRTAMT DGPDAESLASLDRTATRRYG

240 65

AAGCCGAATGTCATCCTGCTCCTCGCTGACGACATGGGAGTC KPNVILLLADDMGVG

360 105

DLSVYGHPTQEPGFIDQMANQGLRF

1.

ACCCAAGGATACTCAGGAGACTCGGTTTTGTACCCC~~A~TC~C~TAG~ACA~TCGT~CCTATTCGTACC~GTCTAC~~G~GCGTATCTTCCTACCAT~CTACC TPSRSAIVTGRQPIRTGVYGEERIFLPWTT TQGYSGDSVC

480 145

1. ACGGGGCTGCCGCTGTATGAAGTGACGATCGCTGAAGCGA TGLPLYEVTIAEAMKGAGYTTGMVGK

600 185

WHLGINE)JSSSDGA .I .

CATCTTCCCGCCAATCGCGTTTGATTTTGTAGGACATAGCTTGTTTCCTG HLPANRGFDFVGHNLPFGN

720 225

SWRCDDTGLHQDFPDTNACFL

840 265 .

&

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TCCTTCGCTCACATGCACACCAGTCTCTTCTCCA~~T~TTTCTCTT~~TCCC~CGC~TC~TATG~~T~CCTCCGT~~T~CCA~~TC~~A~TCGT~~ SFAHMHTSLFSSDDFSCTSRRGRYGDNLREMDQAIEQIVT ACACTCGPCGACAACGACATCGATGACAACACAGTCATCTTCTT~CCTCC~T~C~TCC~TCGC~GTACT~~~~T~CGC~CGTCTTCA~~T~~~ FFTSDHGPHREYCGEGGDANVFRGGKG TLVDNDIDDNTVI

960 305 I

1080 345

CAGTCATGGGAAGGAGGGCACCGTATCCCGTACATCGTCTATTG~C~GTAC~TTAGCCCT~TGTCTCTCATGA~TCGT~~TC~T~TAT~TC~CACC~CGTC~TCTC PYIVYWPGTISPGVSHEIVTSMDIIATAVNL QSWEGGHRI

1200 385

GGCGGTTCACAGCTACCAACAGATCGTATCTAC~T~C~T~CT~~GTGTTCTCTTA~G~CGCTTCATCTC~CAC~T~CTTCTTCTACTACTGC~~~CCCT~T~ YDGKCLKSVLLEGASSPHDDFFYYCKDTLM GGSQLPTDRI

1320 425

GCCGTCCGAGTTGW;AAGTACAAGGCGCACTTCAAGACCC AVRVGKYKAHFKTQTD

1440 465

SSQMKLGERCDGGFPLDDYFLCSD

TGCGAGGGTGACTGCGTGACAGAGCACAACCCCCCTATTATGTTC~CCTC~~CCCT~T~CTATCCTCTGG~CCTT~G~TAC~CATGTTCTC~~CGT~~ IMFDLEKDPGENYPLGPcGYEHvLLHvK CEGDCVTEHNPP

1560 505

GACGCCATCGCAAGGCACGACGCGGAGATGGTGATAG~CCCCTGTCCT~T~CTTC~TTTCTC~TTATTCCTT~T~~CCC~CC~CT~GTCTGT~CTA~C~AT DAIARHDAEMVIGTPVLDNFDFSI IPCCNPETNCVCFYTH

1680 545 1800 1920 2040 2160 2280 2400

FIG:. 5. Sequence of the SPARS cDNA, surrounding genomic DNA and the deduced polypeptide. The transcription initiation site, +l, was determined by primer extension (see text and Fig. 8); (*) the first and last nucleotides of the primer used. The longest cDNA identified contained the sequence from +-3% to 2322 and is given in capital letters. The remainder of the sequence in small letters was derived from adjacent regions of genomic DNA clones. The 3’ terminus of the mRNA has not been defined, but probably lies lo-40 nt downstream of the putative ,4ATAAA polgadenylation signal(s) shown underlined. Potential glycosylation sites are underlined doubly, and the putative hydrophobic leader peptide region is indicated by a broken underline. Sequences upstream of the transcription initiation site include homologies to TATA (open box), CC’AAT (shaded box), and an inverted SPl (GGGCGG) site shown by the arrow.

(Rapraeger and Epel, 1981; Yang ef al., manuscript in preparation), the predicted polypeptide contains a highly hydrophobic region about 20 residues long at the amino terminus which could serve as a signal peptide. According to the “(-3,-l) rule” (von Heijne, 1984), the 20th amino acid (Gly) could be the amino terminal residue of the mature protein. Computer analysis also reveals three potential sites (Asn-X-Thr) for addition of

N-linked carbohydrate indicating likely to be a glycoprotein.

that

the protein

is

Sequence Homology with Arylsulfatase of Hemicentrotus pulcherrimus and with Two Human Arylsulfutases The sequence presented here for SPARS is very similar to that recently reported for the arylsulfatase cDNA

60

DEVELOPMENTAL BIOLOGY

isolated from H. pulcherrimus by Sasaki et al. (1988). A computer search of the GenBank data bases also revealed significant sequence similarity to human steroid sulfatase (STS; Yen et al, 1987) and these sequences are all related to that of a recently reported cDNA encoding human arylsulfatase A (Stein et al., 1989). The deduced

a HpARS SPARS Human ASA Human STS

HpARS SPARS Human ASA Human STS

HpARS SPARS Human ASA Human STS

HpARS SPARS Human ASA Human STS

sequences of these four polypeptides are compared in Figs. 6a and ‘7. (In calculation of the percentages of similarity given here, regions not represented in all four proteins have been omitted.) The two urchin proteins are very similar, having about 75% identity. The sequences of the sea urchin polypeptides are as closely

-cLLGLvTAoT-------l-----QDPALLDLLRENPDLLS MARTLFASLFLFLVLGLVAGQGDDTDGPDAESLASLDRTATRRYGDGEDLLHLLGQTGQHRT~TKP~ILLLADDMGVGDLSVYGHPTQEPGFIDQMAN MGAPRSLLLALAAGLAVARP-------------------------------------------------PNI"LI~ADDLGYGDLGCYGHPSSTTPNLDQL~ MPLRKMKIPFLLLFFLWEAESH-------------------------------------------EASRPK~~L”~DDL~~GDPG~~~~~T~RTP~~D~LAS ..* . * *. * *** * **. **=

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TQLF--TDDALGFIEDNHADPFFLYVAFAHMLTSLFSSDDFSCTS~GRYGDNLLE~DAVQKI~KLEE~~ENTIIFFISDHGPH~YCE-----EG TQLL--RDDTVGFIEDNVNKPFFMYVSFAHMLTSLFSSDDFSCTSRRGRYGDNL~~QAIEQIVTTL~~IDDNTVIFFTSDHGPHREYCG-----EG EARYMAFAHDLMAoAQRQDRFFLYYASHHTHYPQFSGQSFAERSGRGPFGDS~LD~VGT~TAIGDLGLLEETLVIFTADNGPET~S-----RG TQRL--TVEAAQFIQRNTETPFLLVLSYLHVHTALFSSKDFAGKSQHG~GDAVEE~WSVGQILNLLDELR~~LIYFTSDQGAHVEEVSSKGEIHG SE **.o. * . .**. .* * .* ,** .. . * . *o.* . l .o .O . . ..

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GDASIFRGGKSHSWEGGHRIPYIVYWPGTISPG-ISNEIVTSMDIIATAADLGGTTLPTDRIYDGKSIKDVLLEGS-ASPHSSFFYYCKDN~VRVGKY GDANVFRGGKGQSWEGGHRIPYIVYWPGTISPG-VSHEIVTSMDIIATAVNLGGSQLPTDRIYDGKCLKSVLLEGA-SSPHDDFFYYCKDTL~VRVGKY GCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGHIAPG-VTHELASSLDLLPT~~GAPLP-~LDGFDLRPP~GHRQEPSAVSLLLPVLPRR-------GSNGIYKGGKANNWEGGIRVPGILRWPRVIQAGQKIDEPTSNMDIFPTVAKLAGAPLPEDRIIDGRDLMPLLEGKSQRSDHEFLFHYCNAYLNAVRWH-..** .*** * l . **. * .* * ..*. * 0 * * ** . . . ** 0 . . . . . . *

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KAHFRTQRVRSQDEYGLECAGGFPLEDYFDCNDCEGDCVTEHDPPLLFDLHRDPGEAYPLEACGHEDVFLTVKSTVEEHKAALVKGTPLLDSFDHSIVPC KAHFKTQTDSSQMKLGERCDGGFPLDDYFLCSDCEGDCVTEHNPPIMFDLE~PGENYPLGPCGYEHVLLQVKDAIA~DAE~IGTPVLDNFDFSIIPC -GPWGFCCADWKVQGSLLHPGSAHSDTTRDPACHASSSSLTAHEPPLLYDLS~PGENYNL-LGGVAGATPEVLQALKQLQLL~QLD~VTFGPSQVARG -PQNSTSIWKAFFFTPNFNPWPTDCFATHVCFCFGSYVTHHDPPLLFDISKDPRERNPL-TPASEPRFYEILKVMQEAADRHTQTLPEVPDQFSWNNFL . . . .* * **oo.*. o**.* ..* . . . . .

HpARS SPARS

CNPANGCICNYVHEPGMPECYQDPVATAARHYRP CNPETNCVCNYTHEPGVAECYQDLINIALRNGVPK EDPALQICCHPGCTPRPACCHCPDPHA WKPWLQLCCPSTGLSCQCDREKQDKRLSR * * . .

ASA Human STS

.*

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HpARS SPARS Human ASA Human STS

Human

l

EGLRFTNGYVGDAVCTPSRSAIMTGRLPVRIGTFGETRVFL---PWTKTGLPKSELTIAE~KEAGYATG~GKWHLGINENSSTDGAHLPFNHGFDFVG QGLRFTQGYSGDS"CTPSRSAIVTGRQPIRTGVYGEERIFL---PWTTTGLPLYEVTIAEAMKGAGYTTGMVGKWHLGINE~SDGAHLPANRGFDFVG GGLRFTDFYVPVSLCTPSRLLTGRLPVRMGMYPGVLV-----PSSRGGLPL~EVTVA~V~RGYLTG~GKWHLGVGP~FLPPHQGFH~LGIPY GGVKLTQHLAASPLCTPSRFMTGRYPVRSGMASWSRTGHLGMSCHSKTDFCHHPLHHGFNYFY *...*

HpARS SPARS Human ASA Human STS

VOLUME 135, 1989

. . .

600

b I I

I

I

I I

I

I I

I I

I

SPARS

I

I

Human t---d

STS

50 aa

FIG. 6. (a) Comparison of the deduced amino sequences of HpARS, SPARS, human STS (arylsulfatase C), and human arylsulfatase A. The sequence presented in Fig. 5 was visually aligned with those for arylsulfatase from H. pulcherrirrcus (HpARS; Sasaki et aZ., 1988), human steroid sulfatase (STS; Yen et al, 1987; corrected in Yen et al., 1988). and human arylsulfatase A (ASA; Stein et al., 1989). Three levels of similarity are indicated in decreasing order of significance: (*) Positions identical in the four polypeptides; (0) Residues that are the same in both human genes as in one of the urchin sequences; (0) Residues that are the same in both sea urchin genes as in one of the human genes. Putative hydrophobic leader peptides are underlined. Potential sites of glycosylation are doubly underlined. The sequence in human STS indicated by the wavy underline is the hydrophobic region proposed to be involved in localization of this polypeptide to microsomes (Yen et al., 1987). (b) Division into exons is shown for SPARS (this work) and human STS (Yen et al., 1988). Open-ended boxes indicate that the corresponding exons extend to include untranslated sequence. The shaded portions correspond to a major region in each of these proteins that is missing in the other.

YANG, ANGERER, AND ANGEREK

SPARS HpARS Human

ASA

Human

STS

FIG. ‘7. Matrix of sequence similarities among arglsulfatase genes. The percentage identity (upper numbers) and the percentage identity + conservative changes (lower numbers) are shown for the four arylsulfatase peptide sequences shown in Fig. 6. Regions not represented in all four polypeptides have been excluded from this calculation.

related to those of the two human arylsulfatases as the latter are to each other (Fig. 7), suggesting that the duplication producing the two human genes occurred at about the time of divergence of the lines leading to echinoderms and vertebrates, assuming equal rates of evolution. Limiting the sequences compared to regions more highly conserved increased each of the percentage similarities shown in Fig. 7 approximately 10% without altering their relative values. The region from positions 200 to 300 in the alignment shown in Fig. 6a includes both a large portion of the human STS polypeptide (residues 200-253) that is not represented in the other three proteins and an adjacent sequence (residues 253-300) that shows little or no similarity between human and sea urchin genes. Most of this region is occupied by a hydrophobic domain (Fig. 6a wavy underline) of the human STS protein that has been suggested to anchor the protein in the microsomal membrane (Yen et al., 1987). Consistent with this suggestion, the urchin proteins are extracellular (Rapraeger and Epel, 1981) and human ASA is lysosomal (Stein et al., 1989). Although the peptide sequences at the amino termini are not similar for the four proteins, they all are hydrophobic (Fig. 6a, underlined), consistent with their function as leader peptides. The positions of two of the three potential glycosylation sites (Fig. 6a, double underlines) are conserved in the sea urchin proteins and one of these is also found in human ASA. The S. purpuratus Genome Does Not Contain Other Sequences Close114Related to SPARS Most abundant mRNAs that have been shown to be expressed in aboral ectoderm are members of small

gene families. These include the cytoskeletal actins CyIIIa and CyIIIb (Cox et al., 1986), Specl and multiple Spec2 genes (Hardin et al., 1988), and genes encoding metallothioneins (Wilkinson and Nemer, 198’7). Genetic and biochemical data indicate that there are three different arylsulfatase genes (encoding arylsulfatases A, B, and C) in the human genome and, on the basis of biochemical properties, it has been suggested that an arylsulfatase isolated from sea urchin seminal plasma (Moriya and Hoshi, 1980) is a protein different than that found in embryos (Sasaki et al., 1987). We used blot analysis of genomic DNA digested with different restriction endonucleases to determine whether the ARS coding sequence might detect more than one gene. When blots of genomic DNA digested with KpnI were hybridized with a 0.25-kb KWI-Hind111 fragment from the protein coding region of the 3’ exon (shown in Fig. 9 and corresponding to amino acid residues 429-512 in Fig. 6) and washed at high stringency, a single fragment of about 4 kb was detected (Fig. 8) which corresponds to the size predicted from analysis of a genomic DNA clone (Fig. 9). Two bands are observed with SalIdigested DNA; a 2-kb fragment matches the genomic clone (Fig. 9) and a 3-kb fragment does not. The fact that the bands are of similar intensity, which is about

23.1

-

9.4

-

6.6

-

4.4

-

2.3 2.0

-

1.4

-

1.1 0.9

-

0.6

-

FIG. 8. Southern blots of genomic DNA. DNA purified from sperm of a single male sea urchin was digested with the indicated restriction endonucleases and hybridized with a 250-nt probe, the Kyr&HizdIII fragment from exon 5, representing protein coding sequence (see Fig. 9).

62

DEVELOPMENTAL BIOLOGY

VOLUME 135.1989

0.9 kb Sall-Kpnl

S

B

R

XH

I

I

II I

SH

H

SR

St

BS

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I St

H H

S

K

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I st

K

P

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I

I H

S

H

I H

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K

S

K

S

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s R

2.5 kb

1 kb I

0.65 kb

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R

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probe

the SPARS gene. Restriction maps are shown for two overlapping genomic clones, hG196-1 and XG196-7, which were with the G196 cDNA probe. Subfragments that hybridized with SPARS cDNA clones were identified and sequenced to borders, all of which contained canonical splice junction sequences. As noted in the text, the 5’ end of the first exon was extension because it is not represented in the longest cDNA, and the exact position of the 3’ end of the message is not for introns are approximate.

half that of the KpnI band, suggests that the 3-kb fragment results from a sequence polymorphism. A similar argument applies to the bands observed for DNA digested with PstI. The finding of several polymorphisms is not surprising since Britten et al. (1978) have estimated that the single copy nucleotide sequence divergence between any two individuals of S. purpuratus averages approximately 4%. Although it is possible that there are two genes, both of which have a 4-kb KpnI fragment, and both of which hybridize to the probe with equal efficiency, results of this and similar experiments with other probes (data not shown) are most simply interpreted as indicating that at this stringency the SPARS probe detects only a single gene. Rehybridization and washing of the blot shown in Fig. 8 at 15°C lower temperature did not detect additional bands (data not shown). We conclude that it is unlikely that the S. purpuratus genome contains other genes related closely to SPARS. However, multiple genes in the sea urchin would not cross-react under the conditions used in our analysis (most stringent wash at -T, -20°C) if they were as divergent as the human STS and arylsulfatase A sequences. Structure of the SPARS Gene Two partially overlapping genomic clones were isolated that hybridized with the G196 cDNA probe. The

restriction map of the reconstructed gene is shown in Fig. 9. The gene consists of six exons interrupted by five introns and spans approximately 10 kb. Sequencing of the exon regions revealed >99% positional identity in nucleotide sequence alignments between the genomic clones and the cDNA clones within the coding region (data not shown), indicating that the cloned genomic region is the active SPARS gene. As expected, all of the exon/intron junctions (indicated by arrows in Fig. 5) follow the GT-AG rule. The exon structure for SPARS is compared to that for human STS (Yen et al., 1988) in Fig. 6b. Only one exon junction corresponds exactly in these two genes, and most of the exons are of quite different lengths. In the human STS gene, four of nine introns interrupt glycine codons, always after the first nucleotide. This phenomenon is also observed for four of the five SPARS introns; in the remaining ease, the intron is adjacent to the 3’ side of a glycine codon. We note that the large hydrophobic region of human STS, not represented in SPARS, does not correspond closely to a single exon in human STS, suggesting that the difference in the two polypeptides is not simply the result of deletion of an exon or inactivation of a splice site. To determine precisely the 5’ end of the SPARS gene, a synthetic primer was hybridized to pluteus RNA and extended using reverse transcriptase. The primer ex-

CTAGP

+I

A C A T C

longer than the longest cDNA clone). Alignment of the extension product with the sequencing lanes shows a single major cap site at an A residue 45 base pairs upstream of the ATG codon. Sequence upstream of the SPARS gene includes a sequence at -25 to -31, TATATAA, whose sequence and position agree well with those of known TATA boxes. Immediately adjacent and upstream of the TATA homology is a sequence, GGCCAAT, which matches identified CCAAT box elements of several promoters (MeKnight and Tjian, 1986) but which is usually found 40-45 bases upstream of TATA. A CCAAT box found upstream of the sea urchin sperm-specific H2B gene has been shown to function in tissue-specific regulation through the binding of a negative regulator, found only in somatic tissues, which displaces stimulatory CCAAT binding factors (Barberis et al., 1987). The extreme proximity of CCAAT and TATA elements in the SPARS gene suggests that this could be a site of similar negative regulation by competition for binding of positive regulatory factors. A GC-rich region at -90 to -110 contains an Spl binding site homology (GGCGGG) in inverted orientation (CCCGCC). This sequence has also been shown to be important in regulation of histone gene expression in S. purpuratus (Lai et al., 1988). Finally, the sequence for more than 100 base pairs upstream of -220 is unusually rich in T and C. Comments 011D$erentiafion qf’ilborul nw~?the Role of Ar&xlfa~tase

FIG. 10. Determination of the initiation site for SPARS transcription. (lane P) The sequence indicated in Fig. 5 was used as a primer to reverse transcribe RNA isolated from plutei. The same primer was used in sequencing reactions of a genomic DNA fragment spanning the initiation site. The arrow indicates the major initiation site at an A residue taken as fl in Fig. 5.

tension product and the products of a sequencing reaction using the same primer and an appropriate genomic template (the 1-kb SalI-KpnI fragment indicated in Fig. 9, subcloned into M13) were electrophoresed on the same gel, the autoradiograph of which is shown in Fig. 10. The extended cDNA consisted of major and minor products of 210 and 209 bases, respectively (31 bases

Ectoderm Cells

Arylsulfatase is a new member of a set of genes expressed exclusively or primarily in aboral ectoderm cells of the pluteus larva. Other members of this set include the calcium-binding proteins of the Spec family, which are related to the myosin light chain family (reviewed by Hardin et al., 1988), the cytoplasmic actins CyIIIa and CyIIIb (Cox et al., 1986), “constitutively expressed” metallothionein (Angerer et al., 1986), and a putative DNA binding protein, Hboxl, containing a homeodomain (Angerer et ul., 1989). Although they share the same tissue specificity, different members of this set accumulate with different temporal patterns during embryogenesis. Thus, RNase protection assays using the same RNA samples used here, and probes of the same specific activity, show that actin CyIIIa and Specl messages begin to accumulate between 9 and 12 hr of development (Angerer et al., 1989), whereas accumulation of SPARS and Hboxl mRNAs begins about 3 hr later. RNA blot analyses indicate that messages of the Spec gene family are also activated at different times during development (Hardin et crl., 1988). Thus, initial specification of aboral ectoderm does not result in the simultaneous activation of genes whose expression

64

DEVELOPMENTAL BIOLOGY

characterize this cell type. While these genes might share regulatory signals for tissue specificity, signals for temporal regulation must be diverse and complex. This is consistent with the large number of protein binding sites reported for the region upstream of actin CyIIIa (Calzone et al., 1988) and with the fact that (with the exception of TATA and CCAAT) we have not detected any sequences upstream of SPARS with obvious homology to those upstream of either the actin CyIIIa or Specl genes. The extracellular location of the majority of arylsulfatase enzymatic activity (Rapraeger and Epel, 1981) has led to the suggestion that this enzyme may be involved in metabolism of the extracellular matrix of the embryo which contains sulfated proteoglycans. However, the facts that arylsulfatase is a very abundant protein, comprising about 0.5% of total protein in H. pulcherrimus (Sasaki et al., 1987), that its enzymatic activity is markedly inhibited at the pH of seawater (Sasaki et al., 1987), and that the prevalent sulfated proteoglycan isolated from H. pdcherrimus embryos does not serve as a substrate for HpARS (Akasaka and Terayama, 1983) all make a solely enzymatic role less appealing. An alternative role suggested for arylsulfatase is that it serves as a structural component of the ECM by binding to (rather than hydrolysis of) sulfated proteoglycans (Sasaki et al., 1987). Consistent with this idea are the facts that the structure proposed for native HpARS (Sasaki et al., 1987) is a decamer of identical subunits and that the K, of the sea urchin arylsulfatase for p-nitrophenylsulfate is about loo-fold lower than that of the vertebrate enzymes (Sasaki et al., 1987). Previous work has suggested that sulfated macromolecules deposited in the blastocoel are important for the migration of primary mesenchyme cells and for invagination of endoderm at gastrulation (Solursh and Katow, 1982, and references cited therein). Embryos reared in sulfate-free seawater (Immers and RunnStrom, 1965, Karp and Solursh, 1974) or in the presence of sodium selenate, which inhibits the sulfation of proteoglycans (Kinoshita and Saiga, 1979), arrest at mesenchyme blastula stage. The developmental stage of arrest coincides with the period of rapid accumulation of SPARS mRNA. Our recent observation (Yang et al., manuscript in preparation) that aryl sulfatase is primarily located in the blastocoel during these stages supports the idea that this protein may interact with the sulfated proteoglycans and the glycoproteins already identified (Yamagata and Okazaki, 1974; Oguri and Yamagata, 1978; Solursh and Katow, 1982). Although the SPARS protein is broadly distributed in the blastocoel, it is the unique secretory product of differentiated aboral ectoderm cells. A similar situation is observed for an S. purpuratus collagen, which coats the

VOLUME 135,1989

entire wall of the blastocoel (G. Wessel, personal communication) despite the fact that it is translated from an mRNA detected by in situ hybridization only in mesenchyme cells (Angerer et al., 1988). Studies are currently in progress to examine possible structural and functional roles that aryl sulfatase may play in the developing embryo. This work was supported by Grant GM25553 from the National Institutes of Health. R.C.A. was the recipient of a Research Career Development Award from the Public Health Service (HD00601). We thank Dr. T. Thomas (Texas A & M) who provided the X gtl0 cDNA library. We are grateful to Yue Xiong and Dr. Tom Eickbush, who provided helpful consultation on the sequence comparisons, and Paul Kingsley, who assisted with the computer analysis. Dr. Julia Grimwade and Mike Gagnon of our laboratory contributed RNA samples. REFERENCES AKASAKA, K., and TERAYAMA, H. (1983). Sulfated glycan present in the EDTA extract of Hemicentrotus embryos (mid-gastrula). Expl. Cell Res. 146, 177-185. ANC;EKER, 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 Rea 9, 2819-2840. ANGERER, L. M., CHAMBERS, S. A., YANG, Q., VENKATESAN, M., ANGERER, R. C., and SIMPSON, R. T. (1988). Expression of a collagen gene in mesenchymal lineages of the Stron&ocentrotus ~rpumtus embryo. Gelles Dev. 2, 239-246. ANGERER, R. C., and DAVIIISON, E. H. (1984). Molecular indices of cell lineage specification in the sea urchin embryo. Science 226, 1153-1160. ANGERER, L. M., DOLECKI, G. J., GAGNON, M. L., LTJM, R., WANG, G., YANG, Q., HUMPHREYS, T., and ANC;ERER, R. C. (1989). Progressively restricted expression of a homeo box gene within the aboral ectoderm of developing sea urchin embryos. Genes De?). 3,370-383. ANGERER, L. M., KA~CZYNSKI, G., WILKINSON, D. G., NEMER, M., and ANGERER, R. C. (1986). Spatial patterns of metallothionein mRNA expression in the sea urchin embryo. Dm Biol. 116,543-547. BARBERIS, A., SUPERTI-FURGA, G. and BITSSLINGER, M. (1987). Mutually exclusive interaction of the CCAAT-binding factor and of a displacement protein with overlapping sequences of a histone gene promoter. Cell 50, 347-359. BRITTF,N, R. J., CETTA, A., and DAVIDSON, E. H. (1978). The single-copy DNA sequence polymorphism of the sea urchin Stror&ocentrofus purpmtus. Cd 15,1175-1186. CALZONE, F. J., THOZF:, N., THIEBAUD, P., HILI,, R. L., BRITTEN, R. J., and DA~I~ISON, E. H. (1988). Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo. Ge~cs Del: 2,1074-1088. Cox, K. H., AN(;EREK, L. M., LEF., J. J., DAVIUSON, E. H., and ANGERER, R. C. (1986). Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis. .I Mol. Biol. 188,159-172. Cox, K. H., DELEON, D. V., ANGERER, L. M., and ANGERER, R. C. (1984). Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101,485-502. DAVIDSON, E. H. (1986). “Gene Activity in Early Development,” 3rd ed. Academic Press, Orlando, FL. HARIXN, P. E., ANGERER, L. M., HARLAN, S. H., ANGERER, R. C., and KI.EIN, W. H. (1988). The Spec2 genes of Strongylocentrotus purpurafus: Structure and expression in embryonic aboral ectoderm cells. J Mol. Bid. 202, 417-431.

YANG, ANGERER, AND ANCERER HICKEY, R. J., BOSHAR, M. F., and CKAIN, W. R., JR. (1987). Transcription of three actin genes and a repeated sequence in isolated nuclei of sea urchin embryos. Dev. Biol. 124,215-227. H~RSTADIUS, S. (1973). “Experimental Embryology of Echinoderms.” Oxford Univ. Press (Clarendon), London/New York. HTJRLEY, D., ANGERER, L. M., and ANGERER, R. C. (1989). Altered expression of developmentally regulated genes in the progeny of separated sea urchin blastomeres. Development, in press. IMMERS, J., and RUNNSTROM, J. (1965). Further studies on the effects of deprivation of sulfate on the early development of the sea urchin Parucentrotus lividus. J Emhryol. Exp. Mwrph,ol. 14, 289-305. KARP, G. C., and SOLURSH, M. (1974). Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchgme cell activity in the sea urchin embryo. Dev. Biol. 41 110-123. KINOSHITA, S., and SAXA, H. (19’79). The role of proteoglycan in the development of sea urchins I. Abnormal development of sea urchin embryos caused by the disturbance of proteoglycan synthesis. Expl. Cell Res. 123, 229-236. LAI, Z-c., MAXSON, R., and CHILDS, G. (1988). Both basal and ontogenic promoter elements affect the timing and level of expression of a sea urchin Hl gene during early embryogenesis. Genes Dev. 2,173-183. LEE, J. J., CALZONE, F. J., BRITTEN, R. J., ANGERER, R. C., and DAVIDSON, E. H. (1986). Activation of sea urchin actin genes during embryogenesis: Measurement of transcript accumulation from five different genes in Strmtg!4Zo~Pntrotzr.s purpurufus. J Mol. Bid. 188, 173-183. LEFF, S. E., ROSENFELI), M. G., and EVANS, R. M. (1986). Complex transcriptional units: Diversity in gene expression by alternative RNA processing. Annu. Rev. Biochem. 55, 1091-1117. LYNN, D. A., ANGERER, L. M., BRIJSKIN, A. M., KLEIN, W. H., and ANGERER, R. C. (1983). Localization of a family of mRNAs in a single cell type and its precursors in sea urchin embryos. E’roc. Natl. Acatl. Sci. USA 80, 2656-2660. MANIATIS. T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning. A laboratory manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. M&LAY, D. R. (1986). Embryo dissociation, cell isolation, and cell reassociation. M&hods Cell Bid. 27, 309-323. MCKNIGI~T, S., and TJIAN, R. (1986). Transcriptional selectivity of viral genes in mammalian cells. Cell 46, 795-805. MOKIYA, T., and HOSHI, M. (1980). Characterization and partial puritication of arylsulfatase from the seminal plasma of the sea urchin, Str~~~lg~lloCe?~tr~tz~.~ intermdius. Arch. Biochem. Biophys. 201, 216-223. NEMEK, M. (1986). An altered series of ectodermal gene expressions accompanying the reversible suspension of differentiation in the zinc-animalized sea urchin embryo. Dnl. Biol. 114, 214-224. NEMER, M., TRAVAGLINI, E. C., RONDINELLI, E., and D’ALONZO, J. (1984). Developmental regulation, induction, and embryonic tissue specificity of sea urchin metallothionein gene expression. Dev. Bid. 102,471-482. OGITRI, K.. and YAMAGATA, T. (1978). Appearance of a proteoglycan in

Sea Urchin

A rylsulfu tase Gene

65

developing sea urchin embryos. Biochim. Biophys. Acta 541, 385-393. PRO~JDFOOT,N., and BROWNLEE, G. (1976). 3’ Non-coding region sequences in eukaryotic messenger RNA. Nuture (Londonj 263, 211-213. PXXTELL, J., and KAFATOS, F. C. (1982). A convenient and adaptable package of DNA sequence analysis programs for microcomputers. Nucleic Acids. Res. 10, 51-60. PUSTELL, J., and KAFATOS, F. C. (1984). A convenient and adaptable package of computer programs for DNA and protein sequence management, analysis and homology determination. Nucleic Acids. Re.9. 12.643-655. RAPRAEGER, A. C., and EPEI., D. (1981). The appearance of an extracellular arylsulfatase during morphogenesis of the sea urchin Stro~1~yloce~Lfmfus purpuratus. Dw. Biol. 88, 269-278. SASAKI, H., AKASAKA, K., SHIMADA, H., and SHIROYA, T. (1987). Purification and characterization of arylsulfatase from sea urchin (Hemicentrotus pulcherrimus) embryos. Camp Biochwm. Physiol. 88B, 147-152. SAUKI, H., YAMADA, K., AKAS.~KA, K., KAWASAKI, H., SUZIJKI, K., SAITO, A., SATO, M., and SHIMADA, H. (1988). cDNA cloning, nucleotide sequence and expression of the gene for arylsulfatase in the sea urchin (Hemicentrofus yulcherrimws) embryo. Eur. J Biochem. 177,9-13. SF.KAR, V. (1987). A rapid screening procedure for the identification of recombinant bacterial clones, Biotechwiques 5, 11-13. SOLURSH, M., and KATO~, H. (1982). Initial characterization of sulfated macromolecules in the blastocoels of mesenchyme blastulae of Strorqyylocentrotns pwpuratus and Lytechinus pi&s. DPI’. Biol. 94.326-336. STEIN, C., GIESELMANN, V., KREYSING, J., SCHMIDT, B., POHLMANN, R., WAHEED, A., MEYER, H. E., O.BRIEN, J. S., and VON FI~URA, K. (1989). Cloning and expression of human arylsulfatase A. .J Biol. Chem. 264, 1252-1259. VON HEIJNE, G. (1984). How signal sequences maintain cleavage specificity. J. Mol. Biol. 173, 243-251. WILKINSON, D. G., and NEMER, M. (1987). Metaliothionein genes MTa and MTb expressed under distinct quantitative and tissue-specific regulation in sea urchin embryos. Mol. Cell. Biol. 7, 48-58. YAiblilGATA, R., and OKAZAKI, K. (1974). Occurrence of a dermatan sulfate isomer in sea urchin larvae. Biochim. Biophys. Ada 372, 469-473. YEN, P. IL, ALLEN, E., MARSH, B., MOHANDAS, T., WANG, N., TAGGART, R. T., and SHAPIRO, L. J. (1987). Cloning and expression of

steroid sulfatase cDNA and the frequent occurrence of deletions in STS deficiency: Implications for X-Y interchange. Cell 49,443-454. YEN, P. H., MARSH, B., AILEE, E., Ts.41, S. P., ELLISON, J., CONNOLY, L., NEIS~~NGER, K., and SHAPIRO, L. J. (1988). The human X-linked steroid sulfatase gene and a Y-encoded pseudogene: Evidence for an inversion of the Y chromosome during primate evolution. (5,U 55, 1123-1135.