The influence of the extracellular matrix (ECM) on differential gene expression during sea urchin develop­ment was explored using cell-type-specific cDNA probes. The ECM of three species of sea urchins, Strongylocen- trotus purpuratus, Lytechinus variegatus and Lytechinus pictus, was disrupted with the lathrytic agent /5-amino- propionitrile (BAPN), which inhibits collagen deposition in the ECM and arrests gastrulation (Wessel & McClay, Devi Biol. 121: 149, 1987). The levels of several mRNAs (Spec 1, Spec 2, Cylla actin, Cyllla actin and collagen in S. purpuratus, and metallothionine, ubiquitin and LpS3 in L. pictus and L. variegatus) were compared in BAPN- treated and control embryos. These mRNAs accumu­lated normally during BAPN treatment, even though the embryos did not gastrulate. To determine if the ex­pression of any gene product is sensitive to ECM disruption, a differential cDNA screen compared poly (A+) RNA from BAPN-arrested and control embryos in Lytechinus. A cDNA clone was isolated from this screen that represented a 2Ί kb mRNA that did not accumulate during BAPN treatment. Removal of BAPN resulted in the accumulation of this transcript coincident with the onset of gastrulation. This cDNA clone encodes a L. variegatus homologue of LpSl, recently demonstrated to be an ancestral homologue of the aboral ectoderm- specific Spec 1-Spec 2 gene family in S. purpuratus. Nuclear run-on assays in L. pictus suggested that transcriptional activity of LpSl was selectively inhibited by BAPN treatment. Thus, although the accumulation of many gene products occurred independently of the embryonic collagenous matrix, the accumulation of LpSl and LvSl appeared to be mediated by the ECM.

Cell interactions with the extracellular matrix (ECM) are known to be important for the differentiation of a variety of cell types (Hay, 1981; Trelstad, 1984; Wessells, 1977). Recent experiments with a variety of cell types in vitro have demonstrated that ECM components have a selective influence on the phenotype and differ­ential gene expression of these cells (Grover et al. 1983; Hadley et al. 1985; Medina et al. 1987). For instance, culturing mouse mammary epithelial cells on laminin and heparin sulfate proteoglycan induced the formation of glandular structures resembling their in vivo counter­parts and increased the level of β-casein mRNA 4- to 8­fold over the level in cells cultured on plastic, fibronec- tin, or type I collagen (Li et al. 1987). It is also now evident that transforming growth factor β (TGF-β) alters cell differentiation via a modification in the ECM (Massague, 1987). In response to TGF-/S, chick embryo fibroblasts alter the synthesis of ECM components and of complementary cell surface receptors (Ignotz & Massague, 1986,1987). The new cell-ECM interactions then bring about the differential gene expression and the phenotypic conversion that was initiated by TGF-β treatment.

Many events of embryogenesis in vivo also appear to be mediated by cell-ECM interactions (Boucaut et al. 1984; Naidet et al. 1987; reviewed in McClay & Etten- sohn, 1987). For example, in the sea urchin embryo, inhibition of either sulfated glycosaminoglycan syn­thesis (Karp & Solursh, 1974), laminin expression (McCarthy & Burger, 1987), or collagen deposition (Mintz et al. 1981; Blankenship & Benson, 1984; Wessel & McClay; Butler et al. 1987) result in the inhibition of gastrulation and spiculogenesis (Mintz et al. 1981; Wessel & McClay, 1987; Butler et al. 1987). In many types of cells, including those in the sea urchin embryo, the lathrytic agent /S-aminopropionitrile (BAPN) has been shown to reduce the cross-linking of collagen molecules by inhibition of lysyl oxidase activity (Pinnell & Martin, 1968; Kleinman et al. 1981; Butler et al. 1987) . This lack of cross-linking leads to their insta­bility, proteolytic digestion and rapid removal from the matrix (Martin et al. 1963; Kleinman etal. 1982; Wessel & McClay, 1987). BAPN treatment does not alter the developmental events prior to gastrulation in the sea urchin, and the inhibitory effect on gastrulation is reversible, i.e. removal of BAPN permits the reac­cumulation of collagen within the blastocoel and the restoration of normal development. The most obvious result of BAPN treatment of embryos is the inhibition of endoderm cell differentiation. This results in the absence of invagination of the archenteron and in a block of Endo 1 expression, a cell surface glycoprotein associated with differentiating endoderm (Wessel & McClay, 1985, 1987). Differentiation in the primary mesenchyme cells is also affected by BAPN treatment, because in the presence of this drug, these cells cannot synthesize spicules (Mintz et al. 1981; Wessel & McClay, 1987; Butler et al. 1987).

The basis for the BAPN-induced inhibition in em- bryogenesis might be explained as a general arrest in cellular differentiation in the early embryo. To test this hypothesis, the level of accumulation of several cell- type-specific and ubiquitously expressed mRNAs were compared in BAPN-treated embryos and control em­bryos of the same age. Most non-endodermal cell type- specific mRNAs continued to accumulate in BAPN- treated embryos, even though these embryos were developmentally arrested. However, the transcriptional activity of one mRNA of non-endodermal origin was inhibited by disruption of the collagenous ECM. Unex­pectedly, this mRNA turned out to be the Lytechinus counterpart to the well-described aboral ectoderm- specific Spec 1 and Spec 2 mRNAs of Strongylocentro- tus purpuratus. Our results suggest that cell-ECM interactions can mediate differential gene expression during embryogenesis in the sea urchin.

Gametes of Strongylocentrotus purpuratus, Lytechinus pictus and Lytechinus variegatus were obtained from gravid adults by injection with 0-5 m-KCI. Eggs were washed five times in artificial sea water (ASW; Dawson, 1969), fertilized with a dilute sperm suspension and cultured at 14°C. β- Aminopro- pionitrile (BAPN) (Sigma, St Louis, MO) was prepared as a stock solution in ASW and diluted in embryo cultures (4000-6000 embryos ml-1).

RNA extraction and Northern blots

Total RNA was recovered from embryos by a guanidine-HCl extraction essentially as described (Bruskin et al. 1981). Resulting RNA samples (10 jug/lane) were suspended in l× MOPS buffer (20 mM-3-(iV-morpholino)-propanosulfonic acid; 5mM-sodium acetate; ImM-EDTA, pH 7-0), 6% for­maldehyde, 50% formamide, heated for 5min at 65 °C and electrophoresed in a 1% agarose gel, l× MOPS buffer at 100 V. After being stained with ethidium bromide and photo­graphed, the gels were equilibrated with 20× SSC (1.5 m- NaCl, 0-15M-sodium citrate, pH7-4) and blotted to Hybond- N (Amersham, Arlington Heights, IL) overnight in 10× SSC.

Preparation of probes

The cDNA probes used in this study were as follows: a 0-38 kb £coRI fragment of the AgtlO Spec lb cDNA clone [Spec lb is one of the three alleles of the Spec 1 gene (Hardin et al. 1985); it contains 128 of the 151 codons of the Spec 1 protein (Carpenter etal. 1984)] was subcloned into M13mpl8; a 0-2 kb Taql fragment from a AgtlO Spec 2a cDNA clone was subcloned into M13mpl8; and the Cylla actin clone, a gift of Drs Andrew Cameron and Eric Davidson, consisting of a segment of a genomic clone encoding a portion of the 3' untranslated region of the Cylla actin mRNA (Lee et al. 1986) was subsequently cloned into the pGEM vector. A 0-5 kb EcoRI fragment of a AgtlO-mitochondrial DNA clone en­coding a portion of the 5. purpuratus 16S mitochondrial rRNA (Wells et al. 1982) was subcloned into M13mpl9; labeled single-strand sequences complementary to RNA tran­scripts for Spec 1, Spec 2a and mitochondrial 16S rRNA were generated from the M13 vectors by annealing ‘plus’ strand M13 DNA with the hybridization primer (New England Biolabs, Beverly, MA) and elongated with the Klenow fragment of DNA polymerase I and dTTP, dATP, dGTP, and [32P]dCTP essentially as described (Hardin & Klein, 1987). Complementary RNA transcripts to Cylla actin were gener­ated from the pGEM vector by in vitro transcription with T7 polymerase and CTP, GTP, ATP and [32P]UTP as described (Melton et al. 1984). DNA sequences complementary to Cyllla actin (Lee et al. 1986), collagen (gift of Drs Scott Chambers and Robert Simpson; Venkatesan et al. 1986), LpSl (Xiang et al. 1988), ubiquitin and metallothionine (Conlon et al. 1987) and LpS3 (gifts of Dr Bruce Brandhorst) were radiolabeled with [32P]CTP by a random oligonucleotide primer reaction (Feinberg & Vogelstein, 1983). [LpS3 is a L. pictus homologue of the ectoderm-specific Spec 3 gene of 5. purpuratus (Bruce Brandhorst, unpublished observations; Eldon et al. 1987).] The probes generated by these techniques had a specific activity of greater than 107 cts min-1 μg-1 DNA.

Hybridization and quantification

RNA blot hybridizations were performed at 42 °C as described (Bruskin et al. 1981). Hybridization signals were detected by autoradiography using Kodak XRP film. For quantification, autoradiographs exposed within the linear range of the film were scanned by densitometry (Beckman). Relative amounts of RNA between samples were calculated by determining the area under each peak. In some cases, hybridization signals were quantified directly by cutting out the appropriate regions of the blot and quantifying the hybridization signal by liquid scintillation counting (Beckman). RNA samples were normal­ized with mitochondrial rRNA which is known to be constant through development (Wells et al. 1982; Devlin, 1976).

In situ hybridization

L. pictus embryos were fixed in 2% glutaraldehyde and embedded in paraffin as described (Angerer et al. 1987). Sections cut at 5 μτη thickness were mounted on poly-L-lysine- coated slides. After deparaffinization and hydration, the slides were treated with proteinase K (μg ml-1, 30min, 21 °C) and acetic anhydride as described (Angerer etal. 1987).

3H-labeled antisense and sense transcripts were synthesized from the 900 bp EcoRI fragment of the LvSl or LpSl in Bluescript with T7 and T3 RNA polymerase as described (Cox et al. 1986) to achieve a specific activity of 1 × 108 disints min-1 μg∼1. Sections were hybridized with the labeled probe, washed and prepared for autoradiography as described (Angerer et al. 1987).

Embryo measurements

For cell number calculations, samples of known numbers of embryos were dissociated to single cells, quantified with a hemocytometer and the resulting number of cells per embryo calculated (Stephens et al. 1986).

Measurements of embryo dimensions were made on photo­graphs of several fields of embryos from each sample. At least 20 embryos were measured for each point.

In vitro nuclear transcription reactions

Nuclei were isolated as described by Morris & Marzluff (1983). The nuclear transcription (run-on) reactions (Marzluff & Huang, 1984) contained 1-OmCi [o'-32?] CTP (Amersham), 1-25 mM each of ATP, GTP, and UTP; 0-05mM-CTP, 90 itim- KC1, 5mM-MgCl2, 0-1 mM-S-adenosylmethionine, 0-5πιμ- spermidine, 80-100 units RNasin (Promega, Madison, WI), and 100μΐ of approximately 1 × 108 nuclei ml-1 in a total volume of 200μΐ. The run-on reactions were performed at 18°C. After a 5 min incubation, 1-25 mM-cold CTP was added, and the sample was incubated an additional 15 min. The nuclear transcription reactions were terminated by the ad­dition of 200 μΐ of 1% SDS and 10mM-EDTA. Radiolabeled incorporation into run-on transcripts were assessed by TCA precipitation (Marzluff & Huang, 1984).

In vitro transcript hybridization

To assay the in vitro nuclear transcription reactions, radio­actively labeled run-on transcripts were hybridized to DNA immobilized on nylon filters (Hybond-N, Amersham). Two picomoles of each DNA insert, calculated to be in excess over transcript abundance, were spotted onto the filters. The prewash, hybridization and wash solutions were the same as those described for RNA blotting. Hybridization reactions were performed at 37°C for 72h using equivalent incorpor­ated radioactive counts for each sample.

Developmental inhibition with BAPN treatment

The effect of BAPN on collagen deposition and devel­opment has been demonstrated previously (Mintz et al. 1981; Wessel & McClay, 1987; Butler et al. 1987). Embryos of S. purpuratus, L. pictus and L. variegatus cultivated in the presence of 100-125 μgmΓ1 BAPN developed to the mesenchyme blastula stage, and up to this point appeared indistinguishable from control em­bryos (Fig. 1, and data not shown). However, the treated embryos neither gastrulated nor formed spic-ules. Instead, these embryos remained mesenchyme blastulae, even up to the time when control embryos had reached the larval stage. This inhibition in develop­ment did not appear to be due to a toxic effect, since the embryos remained viable and, upon removal of the agent, the embryos gastrulated, formed spicules and developed into normal larvae. Consistent with the fact that BAPN treatment blocked normal endoderm differ­entiation, it has been shown that treated embryos do not express the Endo 1 antigen (Wessel & McClay, 1987; and data not shown for L. pictus, and S. purpur­atus) which is specific for endoderm differentiation.

Fig. 1.

Developmental inhibition of S. purpuratus by BAPN treatment. (A) Control embryos gastrulate 36 h postfertilization. (B) Embryos fertilized at the same time, but cultivated in the presence of BAPN, do not gastrulate. Instead, these embryos remain as apparent mesenchyme blastulae. Note the primary mesenchyme cells clustered at the vegetal plate in the blastocoel. (C) Removal of BAPN, however, permits these embryos to gastrulate normally.

Fig. 1.

Developmental inhibition of S. purpuratus by BAPN treatment. (A) Control embryos gastrulate 36 h postfertilization. (B) Embryos fertilized at the same time, but cultivated in the presence of BAPN, do not gastrulate. Instead, these embryos remain as apparent mesenchyme blastulae. Note the primary mesenchyme cells clustered at the vegetal plate in the blastocoel. (C) Removal of BAPN, however, permits these embryos to gastrulate normally.

Fig. 2.

RNA gel blot analysis of S. purpuratus control embryos compared to embryos treated with BAPN. In each analysis, RNA levels increased from early blastula (12 h) through mesenchyme blastula (24 h) to the gastrula stage (48 h) in both control and BAPN-treated embryos (denoted by *). Note the accumulation of each mRNA in BAPN-treated embryos from 24 h* to 48 h*, even though the embryos remained morphologically arrested as mesenchyme blastulae (see Fig. 1). Removal of BAPN, which permits recovery of gastrulation in the treated embryos, results in continued message accumulation (60R). There is no apparent difference in size of transcripts through development between control and treated embryos. A, C, E, G, I = RNA gel blot hybridization; B, D, F, H, J = plot of average densitometric scans of gel blot hybridizations from either two (Cylla actin) or three (Spec 1, Spec 2a, Cyllla actin and collagen) separate RNA preparations, ×——×, control embryos; ○ - - - - ○, embryos treated with BAPN; ○——○ treated embryos allowed to recover. The values plotted are relative to the control gastrula 48 h value and thus no standard deviations are computed for these points. Error bars represent ± 1 standard deviation. The internal standards used for RNA quantification are not shown.

Fig. 2.

RNA gel blot analysis of S. purpuratus control embryos compared to embryos treated with BAPN. In each analysis, RNA levels increased from early blastula (12 h) through mesenchyme blastula (24 h) to the gastrula stage (48 h) in both control and BAPN-treated embryos (denoted by *). Note the accumulation of each mRNA in BAPN-treated embryos from 24 h* to 48 h*, even though the embryos remained morphologically arrested as mesenchyme blastulae (see Fig. 1). Removal of BAPN, which permits recovery of gastrulation in the treated embryos, results in continued message accumulation (60R). There is no apparent difference in size of transcripts through development between control and treated embryos. A, C, E, G, I = RNA gel blot hybridization; B, D, F, H, J = plot of average densitometric scans of gel blot hybridizations from either two (Cylla actin) or three (Spec 1, Spec 2a, Cyllla actin and collagen) separate RNA preparations, ×——×, control embryos; ○ - - - - ○, embryos treated with BAPN; ○——○ treated embryos allowed to recover. The values plotted are relative to the control gastrula 48 h value and thus no standard deviations are computed for these points. Error bars represent ± 1 standard deviation. The internal standards used for RNA quantification are not shown.

To determine whether the differentiation of other cell types of the embryo was also arrested, we asked whether transcripts that normally accumulate during gastrulation either in specific non-endodermal cell types or in all cells were also arrested by BAPN treatment. The levels of several cell type mRNAs were compared by RNA gel blot analysis between embryos that had been treated with BAPN and control embryos that were fertilized at the same time. The three species of sea urchins used in this study, S. purpuratus, L. pictus and L. variegatus, each exhibited the same morphological inhibition in response to BAPN treatment. TTie probes used in this study are described in Table 1 and were chosen largely because the profiles of transcript ac­cumulations showed steep slopes from the blastula to the gastrula stages and hence could be readily moni­tored.

Table 1.

Characteristics of mRNAs used to monitor BAPN effects

Characteristics of mRNAs used to monitor BAPN effects
Characteristics of mRNAs used to monitor BAPN effects

The effects of BAPN on aboral ectoderm and mesenchyme cell markers of S. purpuratus

The S. purpuratus aboral ectoderm markers Spec 1 and Spec 2a are members of a small gene family whose products encode troponin C-related, calcium-binding proteins expressed strictly in aboral ectoderm cell lineages (Carpenter etal. 1984; Lynn etal. 1983; Hardin et al. 1988). In control S. purpuratus embryos, the Spec 1 mRNA was detected in low levels at the early blastula stage (12 h) and increased 20-fold to the mesenchyme blastula stage and then an additional 2-fold to the gastrula stage (Fig. 2A, B, lanes 24h and 48h). In BAPN-treated embryos from three independent exper­iments, accumulation of Spec 1 mRNA followed a similar time course of expression and proceeded to levels close or identical to those seen in control em­bryos. In the experiment shown in Fig. 2A, a decrease in Spec 1 mRNA levels was seen when 24 h control embryos were compared with 24h-treated embryos. While this may represent a slight negative effect of BAPN on Spec 1 expression, averaging three separate experiments failed to convincingly demonstrate this affect (Fig. 2B). We have observed that BAPN-treated embryos are sometimes delayed in their development relative to controls which could also explain this result. We conclude that embryos treated with BAPN for the first 12 or 24 h in development showed little or no differences from control embryos in morphology and in Spec 1 transcript accumulation. Between 24 and 48h, embryos cultivated in BAPN showed a striking inhi­bition in development (Fig. 1), but Spec 1 mRNA accumulated to similar levels as in control embryos (Fig. 2A, B, lanes 24* and 48*). Embryos treated for 48 h with BAPN and then allowed to recover for 12 h after removal of the agent contained Spec 1 mRNA at the level expected for normal 60 h prism stage embryos (Fig. 2A, lane 60R; Bruskin et al. 1981; Hardin et al. 1988).

Similar results were seen with the related aboral ectoderm probe, Spec 2a. In this case, the pattern of Spec 2 transcript accumulation in BAPN-treated em­bryos closely paralleled that of control embryos (Fig, 2C, D; compare lanes 12, 24, 48 h with lanes 12*, 24*, 48* h). As with the Spec 1 transcripts, after BAPN- treated embryos recovered their normal morphology, the level of Spec 2 transcripts was as expected for 60 h prism-stage embryos (Fig. 2C, lane 60R).

Cylla actin mRNAs are restricted to primary and secondary mesenchyme cells during the mesenchyme blastula-gastrula stages, but expression shifts entirely to endoderm at the prism stage (Cox et al. 1986). In control blastulae (12 h), low levels of Cylla actin mRNA were seen (Fig. 2E, F, lane 12 h) and these levels remained low over the next 12 h (mesenchyme blastula) (Fig. 2E, F, lane 24 h). Gastrula-stage em­bryos showed a 30-fold increase in Cylla actin mRNA (Fig. 2, lane 48 h). These data are very similar to those of Lee et al. (1986) and Shott et al. (1984). BAPN- treated embryos showed message levels similar to those of gastrula controls, but an increase in Cylla actin transcript levels at the mesenchyme blastula stage (24 h) was relative to that seen in mesenchyme blastula controls (Fig. 2E, lane 24 h versus 24*h). This apparent enhancement of Cylla actin mRNA was observed in two experiments but not in a third; the average of the two experiments with the elevated levels of mRNA is shown in Fig. 2F. While not definitive, it may be that the Cylla mRNA begins accumulating earlier in BAPN-treated embryos. The accumulation profile of the Cyllla actin transcript, which normally accumulates with the same temporal and spatial characteristics as Spec 1, also was essentially unaltered in embryos whose gastrulation was inhibited by BAPN (Fig. 2G, H). As with Spec 1, one experiment showed a decrease in Cyllla mRNA levels when 24 h control embryos were compared with 24h-treated embryos (Fig. 2G). How­ever, when this experiment was averaged with two other experiments, little effect could be seen (Fig. 2H). The 9 kb collagen transcript that accumulates from blastula to gastrula stages in the mesenchyme cell lineage (Angerer et al. 1988) was unaffected by the inhibition of collagen polypeptide deposition in the ECM (Fig. 2I, J). This latter finding also suggests that BAPN treatment did not cause a feedback alteration (neither positive nor negative) in levels of collagen transcript accumulation.

The effects of BAPN treatment on specific mRNA accumulation in L. variegatus

In embryos of L. variegatus, the effects of BAPN were tested on the mRNA accumulation profiles of ubiquitin, metallothionine and LpS3 (cf. Table 1).

The cluster of 3 kb ubiquitin mRNAs are present in all cell types at similar levels throughout development (Bruce Brandhorst, unpublished observation). As expected, the levels of ubiquitin mRNA among the developmental stages in these experiments were also similar, and no effect of BAPN on mRNA accumu­lation was observed (Fig. 3A, B). Note that develop­ment in this species proceeds more rapidly than in S. purpuratus. Thus, hatched blastula stage occurred at 8h, mesenchyme blastula stage at 12h and gastrula stage at 24 h.

Fig. 3.

RNA gel blot analysis of L. variegatus control embryos compared to embryos treated with BAPN. Levels of ubiquitin mRNA are similar through development, and between the control and BAPN-treated (*) embryos. Metallothionine (MT) mRNA levels increase significantly between hatched blastula (8h) and gastrula (24 h) stages in both control and BAPN-treated (*) embryos. LpS3 levels peak in hatched blastula-staged embryos (8h), decline at mesenchyme blastula stage (12 h) and reaccumulate during gastrulation (24 h). This bimodai distribution is present in control and BAPN-treated (*) embryos. A, C, E = RNA gel blot hybridization; B, D, F = plot of average densitometric scans of gel blot hybridizations from two separate RNA preparations; ×——×, control embryos; ○ - - - ○, embryos treated with BAPN; ○——○, treated embryos allowed to recover. The values plotted are relative to the control gastrula (24h) point. Each point represents the average of at least two experiments and error bars represent ± standard deviation. The internal standards used for RNA quantification are not shown.

Fig. 3.

RNA gel blot analysis of L. variegatus control embryos compared to embryos treated with BAPN. Levels of ubiquitin mRNA are similar through development, and between the control and BAPN-treated (*) embryos. Metallothionine (MT) mRNA levels increase significantly between hatched blastula (8h) and gastrula (24 h) stages in both control and BAPN-treated (*) embryos. LpS3 levels peak in hatched blastula-staged embryos (8h), decline at mesenchyme blastula stage (12 h) and reaccumulate during gastrulation (24 h). This bimodai distribution is present in control and BAPN-treated (*) embryos. A, C, E = RNA gel blot hybridization; B, D, F = plot of average densitometric scans of gel blot hybridizations from two separate RNA preparations; ×——×, control embryos; ○ - - - ○, embryos treated with BAPN; ○——○, treated embryos allowed to recover. The values plotted are relative to the control gastrula (24h) point. Each point represents the average of at least two experiments and error bars represent ± standard deviation. The internal standards used for RNA quantification are not shown.

The 800-b metallothionine mRNA was first detected at the mesenchyme blastula stage in samples both from control embryos and BAPN-arrested embryos (Fig. 3C, D). Metallothionine mRNA levels then increased two­fold between the 12 and 24 h samples in the control embryos, whereas in BAPN treated embryos this mRNA accumulated to somewhat lower levels (com­pare control 12 h to treated 12 h* and control 24 h to treated 24 h*). One explanation for this result is that the metallothionine mRNA did not accumulate in the arrested endoderm cells of treated embryos, yet total RNA remained the same between control and treated embryos. Thus, the metallothionine mRNA levels would be expected to be reduced approximately two­fold, since equal amounts of total RNA were tested in the Northern blots from control and treated embryos.

The ectoderm-specific LpS3 mRNA accumulated to detectable levels by the blastula stage in control and treated embryos (LpS3 was found only at low levels in the egg; data not shown). As previously reported, the mRNA levels of Spec 3, the S. purpuratus homolog of LpS3, peak at hatching blastula stage, decline at the mesenchyme blastula stage, and then increase after gastrulation (Eldon et al. 1987). This behavior is repro­duced in the experiments shown in Fig. 3E, F, both in control embryos and embryos treated with BAPN. As seen by comparing 8h* to 12 h*, and 12 h’ to 24 h*, a normal decrease and increase in LpS3 mRNA levels were observed in BAPN-treated embryos.

Morphogenesis in arrested embryos

Microscopic examination of BAPN-treated embryos revealed certain morphogenetic changes similar to those found in normal embryos. In normal embryos, the diameter of the blastocoel increased almost 50% through the 18-24 h of gastrulation (Fig. 4 and data not shown). Coincident with this volume change, the pre­sumptive aboral ectodermal cells changed from a cu- boidal shape at the mesenchyme blastula stage to a squamous morphology during gastrulation. At the same time, the mesenchyme cells, after having ingressed within the blastocoel and aggregated at the vegetal plate, dispersed by migration, extended filopodial pro­cesses and generated the embryonic skeleton. As shown in Fig. 1 and 4, BAPN-inhibited embryos proceeded with many of these morphological changes. Thus, in the developmentally arrested embryos, the blastocoel increased in diameter; and the cells of the ectoderm flattened. Mesenchyme cells of the BAPN-treated em­bryos underwent normal initial morphological changes in filopodial extension and migration (Fig. 1), although spicule formation in these cells was not detected. Therefore, in addition to the events of embryogenesis up to the mesenchyme blastula stage, at least some morphological changes in the embryo did proceed in the absence of collagen in the ECM. Cell division, however, did not continue in the BAPN-arrested em­bryos. As shown in Fig. 5 (and data not shown for L. variegatus), BAPN-arrested embryos did not appear to undergo significant cell division during the developmen­tal arrest, even though cell division was unaffected during earlier developmental stages. This apparent lack of cell division can be explained in large part by the absence of endoderm cells, which are known to divide over this period in normal development. Upon removal of BAPN, the arrested embryos did resume normal morphogenesis and cell division, eventually approxi­mating the number of cells found in control embryos.

Fig. 4.

Morphometric analysis of S. purpuratus embryos inhibited with BAPN. During normal development the width of the blastocoer (measured at the equator) and the thickness of the epithelial layer (equatorial region) increase and decrease respectively. BAPN-inhibited embryos follow a similar pattern of morphological change, independent of gastrulation. Representative embryos are shown in Fig. 1.

Fig. 4.

Morphometric analysis of S. purpuratus embryos inhibited with BAPN. During normal development the width of the blastocoer (measured at the equator) and the thickness of the epithelial layer (equatorial region) increase and decrease respectively. BAPN-inhibited embryos follow a similar pattern of morphological change, independent of gastrulation. Representative embryos are shown in Fig. 1.

Fig. 5.

Quantification of cell number in 5. Purpuratus control and BAPN-inhibited embryos. The increase in cell number seen during normal development is not present in BAPN-treated embryos. Upon removal of BAPN, cell division resumes and approximates that of normal embryos.

Fig. 5.

Quantification of cell number in 5. Purpuratus control and BAPN-inhibited embryos. The increase in cell number seen during normal development is not present in BAPN-treated embryos. Upon removal of BAPN, cell division resumes and approximates that of normal embryos.

Gene products sensitive to disruption of the ECM

Two types of evidence above, mRNA accumulation and morphological measurements, suggest that differen­tiation of non-endodermal cell types proceeds normally in embryos that are developmentally arrested by BAPN treatment. To ask directly whether there were any genes whose expression was dependent on proper cell-ECM interactions, a differential cDNA screen was performed. Poly (A)+RNA was isolated both from gastrula-stage embryos and from embryos of the same age treated with BAPN; these embryos were develop­mentally arrested at the mesenchyme blastula stage. One hundred thousand plaques from an L. variegatus prism stage cDNA library were screened in duplicate with 32P-labeled cDNA from both control and BAPN- treated embryos. The primary screen supported the Northern blot analysis in that nearly all cDNA plaques were labeled at similar levels in the two samples. The screen did result in 47 plaques that selectively hybrid­ized to labeled control cDNA relative to labeled cDNA from BAPN-arrested embryos. Four cDNA clones of high abundance were rescreened by selective Northern blot hybridization; three of these four clones sub­sequently were shown to represent the same sequence (data not shown). A 2-1 kb mRNA was identified by these cDNAs. This mRNA accumulated an estimated 20-fold during embryogenesis from the hatched blastula (8h) to the gastrula stages (24 h, Fig. 6). However, embryos treated with BAPN did not accumulate sub­stantial levels of this mRNA, even when cultured for prolonged periods (Fig. 6). Except for the lack of collagen deposition in the blastocoel (Wessel & McClay, 1987), the inhibition in accumulation of this RNA is the earliest effect seen in BAPN-treated embryos. This inhibition occurred several hours before the morphological arrest seen at gastrulation (Fig. 1). Removal of embryos from the BAPN treatment resulted in an accumulation of the 2-1 kb transcript similar to the initial control levels (Fig. 6). This inhi­bition in accumulation of the transcript did not appear to be due to BAPN specifically, but rather to a disruption in the development of the ECM. This conclusion was derived by disrupting the ECM two different ways. Embryos cultured in the presence of the proline analog dehydroproline also exhibited no col­lagen deposition into the ECM and did not accumulate the 2-1 kb transcript (data not shown). In addition, embryos cultured in sulfate-free sea water, which dis­rupts formation of sulfated glycosaminoglycans (Karp & Solursh, 1974) and also inhibits gastrulation, similarly did not accumulate significant levels of 2-1 kb mRNA (data not shown). Thus, BAPN treatment probably disrupts the general organization of the ECM by disrupting a major, collagen constituent.

Fig. 6.

Accumulation profile of LvSl in L. variegatus control embryos and in embryos raised in the presence of BAPN. LvSl mRNA accumulates approximately 25-fold between the hatched blastula (8h) and the gastrula (24 h) stages. BAPN-treated embryos did not accumulate significant levels of LvSl (8*h hatched blastula, 12*h mesenchyme blastula, 24*h mesenchyme blastula), even with extended culture (36*h mesenchyme blastula). Removal of BAPN from the culture resulted in recovery in accumulation of LvSl (36Rh gastrula).

Fig. 6.

Accumulation profile of LvSl in L. variegatus control embryos and in embryos raised in the presence of BAPN. LvSl mRNA accumulates approximately 25-fold between the hatched blastula (8h) and the gastrula (24 h) stages. BAPN-treated embryos did not accumulate significant levels of LvSl (8*h hatched blastula, 12*h mesenchyme blastula, 24*h mesenchyme blastula), even with extended culture (36*h mesenchyme blastula). Removal of BAPN from the culture resulted in recovery in accumulation of LvSl (36Rh gastrula).

Our expectation was that cDNA clones selected in this differential screen would be associated with endo­derm cell types, since lack of endoderm differentiation was the most obvious morphological defect in the BAPN-treated embryos. However, in situ hybridization analysis demonstrated that during normal embryogen­esis this mRNA was selectively accumulated in aboral ectoderm cells (Fig. 7A). This restricted accumulation was apparent in plutei, as well as in the presumptive aboral ectoderm cells of gastrulae (Fig. 7D) and mesen­chyme blastulae (data not shown). Embryos treated with BAPN did not contain a detectable in situ hybridiz­ation signal for this transcript (Fig. 7B). Occasionally, single cells would contain a few localized grains (cf. Fig. 7B), but this was also seen when using the sense- probe control (data not shown). Upon recovery of the BAPN-arrested embryos by removal of the agent, the 21 kb transcript selectively accumulated in aboral ecto­dermal cell lineages, and the level of accumulated mRNA was approximately one-half the level seen in control gastrulae (compare Fig. 7C and D). This corre­sponds well to the Northern blot data (Fig. 6) and can be explained by the length of time that LvSl accumu­lated in the recovered embryos relative to the control embryos, ie. 12 h for the recovered embryos and 24 h for control embryos.

Fig. 7.

In situ hybridization analysis of LvSl in L.variegatus. Embryos shown correspond to samples taken in Fig. 6. Embryos in A, B, and C are all the same age, 36 h postfertilization. (A) Early pluteus, hybridization of LvSl is found only in aboral ectoderm. (B) Apparent mesenchyme blastula developmentally arrested by BAPN. No significant hybridization signals are detected in these embryos. (C) BAPN-treated embryos permitted to recover by removal of BAPN (24 h BAPN -»12 h recovery). LvSl begins to accumulate in aboral ectoderm cells, but levels of accumulation are about 2-fold less than control embryos of the same stage, shown in D control gastrula (24 h).

Fig. 7.

In situ hybridization analysis of LvSl in L.variegatus. Embryos shown correspond to samples taken in Fig. 6. Embryos in A, B, and C are all the same age, 36 h postfertilization. (A) Early pluteus, hybridization of LvSl is found only in aboral ectoderm. (B) Apparent mesenchyme blastula developmentally arrested by BAPN. No significant hybridization signals are detected in these embryos. (C) BAPN-treated embryos permitted to recover by removal of BAPN (24 h BAPN -»12 h recovery). LvSl begins to accumulate in aboral ectoderm cells, but levels of accumulation are about 2-fold less than control embryos of the same stage, shown in D control gastrula (24 h).

The clone isolated via the differential screen had temporal and spatial characteristics similar to the Spec 1 and Spec 2 genes found in S. purpuratus and to LpSl recently isolated in L. pictus and demonstrated to be a Spec l/Spec 2 ancestral homologue (Xiang et al. 1988). Sequencing the L. variegatus cDNA clone demon­strated great similarity to LpSl (88% match over 256bp; data not shown). Thus the 2-1 kb mRNA is a Spec homologue of L. variegatus, and we have desig­nated this clone LvSl (L. variegatus Spec l/Spec 2 homologue). Subsequently, the accumulation of LpSl mRNA was also measured by Northern blot analysis in L. pictus and was found not to accumulate during BAPN treatment, as was seen for LvSl in L. variegatus (data not shown). The isolation of a Spec 1 -like sequence by the differential screen was unusual not only because aboral ectoderm differentiation appears normal in BAPN-treated embryos but also because in S. purpuratus the accumulation of Spec 1 and Spec 2 were unaffected by BAPN treatment.

BAPN inhibits the transcription of LpSl

To determine whether the lack of accumulation of LpSl transcripts in BAPN-treated embryos was due to inhi­bition of transcription or to some post-transcriptional event, nuclear run-on experiments were performed. Nuclei were isolated from three batches of sibling embryos: (1) control gastrula, (2) embryos treated with BAPN and thus developmentally arrested at the mesen­chyme blastula stage, and (3) BAPN-arrested embryos that were removed from BAPN culture and that sub­sequently gastrulated. Although transcriptional activity of LpSl in control nuclei was comparable to that seen in other studies (C. Tomlinson and W. Klein, in prep­aration), there was no detectable transcriptional ac­tivity of LpSl in the nuclei isolated from embryos treated with BAPN. This transcriptional inhibition was not the result of BAPNs contaminating the nuclear run- on assays and directly interfering in LpSl transcription in the run-on reaction because the addition of 100 μg ml-1 BAPN (the same concentration used in the embryo cultures) to the nuclear run-on reaction mixture did not affect the transcriptional activity of LpSl and several other genes (data not shown). Embryos allowed to recover from BAPN treatment (12 h recovery after 24 h treatment) completely recovered the transcrip­tional activity of LpSl (Fig. 8). In contrast to LpSl, the transcriptional activity of ubiquitin, metallothionine and LpS3 appeared to be identical in nuclei isolated from each of the embryo cultures even during BAPN treatment, and supports the results obtained earlier by RNA gel blot analysis (Fig. 3).

Fig. 8.

Nuclear run-on transcripts are analyzed for ubiquitin, metallothionine, LpSl, and LpS3. (A) The three batches of nuclei isolated were from control gastrula (24 h, C), BAPN-treated embryos arrested at mesenchyme blastula stage (24h, B), and these same embryos allowed to recover for 12 h (24h BAPN—»⋅ 12h recovery, R). LpSl transcripts are not produced at detectable levels in nuclei isolated from BAPN-treated embryos. In contrast, run-on transcript levels are similar between the different nuclei for ubiquitin (Ub), metallothionine (MT), and LpS3. (B) Graphic representation of LpSl run-on transcript levels compared to LpS3 transcript levels in the three nuclear samples.

Fig. 8.

Nuclear run-on transcripts are analyzed for ubiquitin, metallothionine, LpSl, and LpS3. (A) The three batches of nuclei isolated were from control gastrula (24 h, C), BAPN-treated embryos arrested at mesenchyme blastula stage (24h, B), and these same embryos allowed to recover for 12 h (24h BAPN—»⋅ 12h recovery, R). LpSl transcripts are not produced at detectable levels in nuclei isolated from BAPN-treated embryos. In contrast, run-on transcript levels are similar between the different nuclei for ubiquitin (Ub), metallothionine (MT), and LpS3. (B) Graphic representation of LpSl run-on transcript levels compared to LpS3 transcript levels in the three nuclear samples.

Our results demonstrate that while disruption of the collagenous ECM by BAPN dramatically affects gastru­lation, many transcriptional and morphological events not specifically associated with endoderm differen­tiation proceed normally. This suggests that many of the events required for cell differentiation in embryos of S. purpuratus, L. pictus and L. variegatus can take place independently of normal gastrulation and collagen deposition in the ECM. The unexpected result was that in a search for L. variegatus genes whose transcript levels are sensitive to ECM formation, the aboral ectoderm gene LvSl was isolated. This was particularly surprising since the accumulation of the LvSl counter­parts in S. purpuratus, the Spec 1 and Spec 2 genes, were not sensitive to ECM disruption. Since the two genera have diverged over an estimated 50 million years, this contrast may result from a divergence in any one of the steps of signal transduction from the extra­cellular environment to the nucleus.

The lack of LvSl/LpSl mRNA accumulation in BAPN-treated embryos appears to be a specific tran­scriptional response of the LvSl/LpSl genes to an improper ECM. Evidently collagen participates either directly by binding to cell surface receptors, or in­directly via structuring, stabilizing or organizing other components of the ECM to participate in the signal transduction events that ultimately lead to the transcriptional activation of select genes. An alternative explanation for the observed inhibition of LvSl/LpSl transcription by BAPN treatment may be that the aboral ectoderm per se does not differentiate. That is, a set of genes associated with the aboral ectoderm pro­gram could be generally affected by ECM disruption. If true, this explanation suggests that the blockage of ectoderm differentiation is highly restricted to the aboral ectoderm gene set, since LpS3, a gene associated with ectodermal ciliogenesis and believed to be acti­vated in all ectodermal cells (but not in endodermal or mesenchymal cells), is not affected by BAPN. Unfortu­nately, distinguishing between a specific inhibition of LvSl/LpSl or a more general effect on the aboral ectoderm set of genes is not possible at present since other mRNA markers for the aboral ectoderm cell type have not been isolated in Lytechinus. With either explanation, though, there is a rough temporal corre­lation in the initiation in transcription of LpSl (C. Tomlinson and W. Klein, in preparation) and the deposition of collagen into the newly forming blastocoel (Wessel et al. 1984). Since many of the ECM molecules of the embryo are maternally derived (Wessel et al. 1984), perhaps the transport of these molecules to the basal aspect of the cleaving blastomeres and the depo­sition of these molecules into the blastocoel is function­ally related to the initiation of differentiation of this cell type.

Besides LpSl/LvSl, all mRNAs tested in the three species studied were not significantly affected by BAPN treatment. A possible exception was the high levels of Cylla actin mRNA observed at earlier times than controls in S. purpuratus embryos. Recently, Hurley et al. (1989) have shown a very similar elevation in Cylla actin mRNA levels when S. purpuratus embryos were dissociated at the 16-cell stage and the cells cultured without being allowed to reaggregate. It is possible that both proper cell-cell and cell-ECM contacts are required to prevent the Cylla actin mRNA from being overexpressed.

Several morphological changes also were unaffected by BAPN treatment. For instance, the increase in the diameter of the blastocoel during development appears to be independent of a collagenous ECM matrix and of the volume displacement by invagination of the archen- teron into the blastocoel. However, cell division of presumptive endoderm and secondary mesenchyme cells at the mesenchyme blastula stage was effectively inhibited. This inhibition does not, however, appear to be a sufficient explanation for the inhibition seen in gastrulation. This was previously demonstrated by use of the DNA polymerase inhibitor aphidicolin, which prevents both DNA replication and cell division: sea urchin embryos treated with aphidicolon at the mesen­chyme blastula stage do gastrulate even in the absence of significant cell division (Stephens et al. 1986).

The first indication of morphogenetic arrest by BAPN treatment in these embryos was at gastrulation. Yet the accumulation of LvSl/LpSl mRNAs was in­hibited well before gastrulation. Since no LvSl/LpSl mRNA was detectable in our analysis, it is likely that the inhibition takes place when the LvSl/LpSl gene is initially activated. It is likely this occurs at the seventh or eighth cleavage (Davidson, 1989). Furthermore, preliminary evidence suggests that disruption of the ECM by BAPN treatment, even during gastrulation, can inhibit additional accumulation of LvSl. This suggests that the transcriptional activity of this gene requires the continued presence of the signal provided by the ECM.

This work was supported by a National Institutes of Health grant HD22619 to WHK, HD18590 to WJL and a Robert A. Welch grant (G-1088) to WJL. As a Robert A. Welch Professor of Chemistry, Dr Lennarz gratefully acknowledges the Robert A. Welch Foundation. GMW is the recipient of an NIH postdoctoral fellowship award (HD-06899).

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