Disruption of Gastrulation and Oral-Aboral Ectoderm Differentiation in the Lytechinus Pictus Embryo by a Dominant/Negative PDGF Receptor

Development 124, 2355-2364 (1997) 2355 Printed in Great Britain © The Company of Biologists Limited 1997 DEV3516

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

Ravi K. Ramachandran1, Athula H. Wikramanayake2, J. Akif Uzman1, Venkatesh Govindarajan1, and Craig R. Tomlinson1,3,* 1Department of Biology, 3The Institute for Molecular Biology, The University of Houston, Houston, TX 77204-5513, USA 2Department of Biochemistry and Molecular Biology, University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA *Author for correspondence (e-mail: [email protected])

SUMMARY

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

INTRODUCTION ferentiate to give rise to well-described tissues that express specific genes, some of which have been isolated and serve as The major events in the establishment of the sea urchin useful markers. For example, lithium causes ectodermal cells embryo body plan begin with the specification of the animal- to express marker genes normally restricted to the gut and the vegetal axis during oogenesis, and in some species, including primary mesenchyme cells (Livingston and Wilt, 1989), which those of the genus Lytechinus, the specification of the oral- produce the spicules; and induces the expression of aboral aboral axis before first cleavage (Schroeder, 1980; Cameron ectoderm markers in isolated animal caps (Wikramanayake et et al., 1989, Henry et al., 1992). Five major polyclonal cell al., 1995). Later embryonic and morphogenetic processes that territories; the small micromeres, skeletogenic mesenchyme, give rise to the pluteus larva also seem to require signal trans- vegetal plate, oral ectoderm, and aboral ectoderm are estab- duction events. Cameron et al. (1994) showed that the over- lished by sixth cleavage (Cameron et al., 1987). Specification stimulation of serotonin receptor- and muscarinic acetylcholine of these territories requires cell signaling and cell-cell inter- receptor-mediated signal transduction pathways caused defects actions. Evidence for the involvement of cell signaling and in gastrulation and spiculogenesis and in the differentiation of interactions rests mainly on blastomere transplantation studies cells along the oral-aboral axis. and animal cap assays (Hörstadius, 1973; Ransick and We are interested in platelet-derived growth factor (PDGF) Davidson, 1993; Wikramanayake et al., 1995) and the inhibi- receptor- and epidermal growth factor (EGF) receptor- tion of the phosphotidylinositol (PI) signaling pathway (Liv- mediated signaling during sea urchin embryogenesis and mor- ingston and Wilt, 1989). The lithium ion is thought to inhibit phogenesis. This interest began with the observation that the phosphatases involved in the PI regeneration cycle human PDGF-BB and TGF-α (which binds the EGF receptor) (Berridge et al., 1989) and is known to be a ‘vegetalizing’ rescued gut and spicule formation and expression of the aboral agent that causes the respecification of ectodermal cells to ectoderm-specific LpS1 genes in extracellular matrix-disrupted form gut and spicules (Hörstadius, 1973). The addition of a embryos (Ramachandran et al., 1993). This work suggested phorbol ester, an activator of protein kinase C, a downstream that these developmental events depended on PDGF- and EGF- player in the PI pathway, alters the fate of cells to form gut like signaling pathways in the embryo. This hypothesis was and spicules (Livingston and Wilt, 1992). supported by studies showing that antibodies against human As embryogenesis proceeds, the five cellular territories dif- PDGF-B, TGF-α, and their respective receptors inhibited gut 2356 R. K. Ramachandran and others and spicule differentiation and morphogenesis (Ramachandran Daniel F. Bowen-Pope (University of Washington; Seattle, WA). The et al., 1995; Govindarajan et al., 1995). 5.57-kb insert DNA was excised with EcoRI, inserted into pBluescript In this report, we present data that support the hypothesis SK+ (Stratagene; La Jolla, CA), and named hPR-β. The ∆hPR-β that a PDGF-like signaling pathway plays a major role in the construct, which lacks the region encoding the cytoplasmic portion of β early events of gastrulation and spiculogenesis. In vertebrates, PDGF receptor- , was constructed by digesting the 5.57-kb DNA it has been shown that the binding of the PDGF ligand induces insert with EcoRI and SacI, followed by ligation of the resulting 2-kb fragment into the EcoRI-SacI sites of pBluescript SK+. CAT assays dimerization and autophosphorylation of the PDGF receptor were carried out using a DNA construct consisting of LpS1-β (Seifert et al., 1989; Ek et al., 1982). The phosphorylated promoter region −762 to +17 fused to the CAT gene (Xiang et al., PDGF receptor acts as a ligand-activated transducer in the 1991) a gift from Dr William H. Klein, M. D. Anderson Center, Uni- initiation of several secondary cytoplasmic signaling pathways. versity of Texas, Houston, TX. These secondary pathways in turn initiate a cascade of events causing a variety of cellular responses including mitogenic In vitro transcription activity, cytoskeletal and extracellular matrix changes, and Capped mRNAs were prepared from RNA transcribed in vitro RNA and protein synthesis (Fantl et al., 1989). according to the manufacturer’s instructions (mCAP mRNA synthesis kit; Stratagene; La Jolla, CA) using as templates the hPR-β and ∆hPR- The role of specific growth factor receptors can be investi- β β gated using a dominant/negative strategy, in which mRNA DNA constructs. The hPR- construct was linearized with EcoRV, and sense mRNA was synthesized using T3 polymerase. The encoding a defective form of the receptor is injected into truncated sense mRNA was synthesized from ∆hPR-β linearized with embryos. The defective forms of the receptor encoded by the SacII using T7 polymerase, and the truncated antisense mRNA was injected mRNA dimerize with the intact endogenous forms synthesized from ∆hPR-β linearized with EcoRV using T3 poly- rendering the dimerized receptor nonfunctional (Herskowitz, merase. 1987). Dominant/negative strategies have been used to examine the role of the FGF and activin receptors in Xenopus Microinjections and CAT assays laevis (Amaya et al., 1991, Hemmati-Brivanlou and Melton, Two picolitier volumes of the mRNAs and/or DNAs were pressure- 1992) and the role of the torso gene product in Drosophila injected into unfertilized L. pictus eggs. Stock RNA solutions of 10 µ µ melanogaster (Sprenger and Nüsslein-Volhard, 1992). g/ l in 0.2 M KCl were diluted in 0.2 M KCl to the proper concen- Truncated human PDGF-β receptors missing the cytoplasmic tration to inject the desired amount. Microinjection needles were pulled from Omega Dot glass capillaries (1 mm O.D., FHC, domains are known to dimerize and bind ligands in Chinese Brunswick, ME), which were backfilled with the injection solution. hamster ovary cells, yet are incapable of transducing signals The CAT assays and CAT DNA determinations were perfomed (Ueno et al., 1991). Recent experiments in sea urchins and exactly as described by Xiang et al. (1991) using L. pictus embryos starfish (Cameron et al., 1994; Shilling et al., 1994) showed at the gastrula stage. that injecting eggs with RNA for receptor tyrosine kinases led to the production of functional proteins that transduce signals, Immunoprecipitations and western blotting demonstrating the feasibility of a dominant/negative strategy Proteins were isolated from L. pictus embryos and immunoprecipi- to study the role of a receptor tyrosine kinase in echinoderm tated as described previously (Ramachandran et al., 1995). Immuno- precipitations were carried out using antibodies raised against the embryos. In this study, we have carried out the first reported β use in sea urchins of a dominant/negative experimental strategy extracellular or cytoplasmic domains of the PDGF receptor- (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA). The extracellular to examine the role of a putative PDGF-like pathway in sea antibody was a rabbit polyclonal anti-mouse PDGF receptor-β urchin embryogenesis. We found that expression of a truncated antibody to amino acids 425-446, and the intracellular antibody was human PDGF receptor-β consisting of only the transmembrane a rabbit polyclonal anti-PDGF receptor-β antibody to amino acids and extracellular domains in the Lytechinus pictus sea urchin 1082-1101. The isolated proteins were precleared with Protein A- embryo caused a dose-dependent range of morphological Sepharose CL-4B beads (Sigma; St. Louis, MO). The precleared deformities affecting most prominently gut and skeleton lysate was incubated with 10 µg/ml of the anti-receptor antibody, and formation and oral-aboral ectoderm differentiation. the antigen-antibody complex was immunoprecipitated with Protein A-agarose. Immunoprecipitates were resolved on an 8% SDS-polyacrylamide gel (Laemmli, 1970) and blotted onto nitrocellulose (Towbin et al., 1979). The blots were incubated with 1:5000 dilution MATERIALS AND METHODS of anti-PDGF receptor-β antibody (extracellular antibody) in Tris- buffered saline (pH 7.6) containing 0.1% Tween-20 for 1 hour at room Embryo cultures temperature. The blots were washed in the same buffer three times, Lytechinus pictus were obtained from Marinus, Inc. (Long Beach, 15 minutes each, at room temperature. An anti-rabbit IgG antibody- CA). L. pictus gametes were collected by intracoelomic injection of horseradish peroxidase conjugate/chemiluminescent assay (the ECL 0.5 M KCl. In preparation for microinjection, eggs were incubated kit from Amersham) was used to detect the bound secondary anti- briefly in 1 mM citric acid and neutralized in 1 mM Tris-HCl, pH 8.0 PDGF receptor-β antibody (Ramachandran et al., 1995). to remove the jelly coat followed by four or five washes in artificial sea water (ASW). The eggs were aligned on 60-mm Petri dish lids Reverse transcriptase-polymerase chain reaction (RT- treated with 1% protamine sulfate for 5 minutes and washed five times PCR) assays with distilled water. After microinjection, the eggs were fertilized with Approximately 200 injected larva stage embryos were harvested 48 a dilute sperm suspension and allowed to develop at 18¡C. Hatched hours postfertilization by gentle centrifugation and resuspended and embryos were transferred to fresh ASW and allowed to develop for lysed in an acid guanidinium buffer, from which RNA was obtained 48-72 hours. by the acid guanidinium thiocyanate method (Chomcynski and Sacchi, 1987). RNA was digested with 20 units of RQ-DNase Plasmid constructs (Promega; Madison, WI) for 30 minutes at 37¡C in a 25-µl reaction A human PDGF receptor-β cDNA clone in pUC13 was a gift from Dr containing 40 mM Tris, pH 8.0, 1 mM dithiothreitol (DTT), 20 units PDGF signaling in early sea urchin development 2357

Fig. 1. A diagram of the DNA templates used to make the in vitro transcribed human PDGF receptor-β RNA transcripts. The upper bar shows the relative positions of the different functional domains on human PDGF receptor-β, which is aligned with the corresponding coding regions of the hPR-β and ∆hPR-β cDNA constructs shown below. The hPR-β cDNA construct was used to transcribe the full- length sense (FL-S) 5.6-kb RNA transcript. The ∆hPR-β cDNA construct was used to transcribe the truncated ligand-binding domain sense (LBD-S) and anti-sense (LBD-AS) 2.0-kb RNA transcripts. T3 and T7, the respective T3 and T7 promoter regions on the cloning vector Bluescript SK+.

RNasin (Promega), 10 mM NaCl, 6 mM MgCl2, and 1 mM CaCl2. DNase was removed by phenol-chloroform extraction. One-half of the RNA was used for reverse transcription. A 20-µl reverse transcription reaction contained 5× MMLV reaction buffer (BRL-Gibco; Gaithersburg, MD), 1 mM DTT, 10 units RNasin, 1.2 mM of each nucleoside triphosphate, and 10 units of MMLV reverse transcriptase (BRL-Gibco). The reaction was incubated at 42¡C for 30 minutes. One-tenth volume of the reverse transcription reaction was used for each PCR reaction. PCR reactions contained 5 µl 10× buffer (Perkin Elmer; Norwalk, CT), 10 mM of each deoxynucleotide triphosphate, Fig. 2. Injection of LBD-S RNA into L. pictus eggs disrupts 32 1.5 mM MgCl2, 100 ng of each primer, and 5 µCi [α- P]dCTP. The development. Eggs of the same batch were uninjected (A), injected oligonucleotides used as primers were 5′-TACGGTGGCTTGGGA- with 2.0 pg of control LBD-AS RNA (B), or injected with 0.1 (C), CTTTG-3′ and 5′-ACCATCGCCATCCTTGTCTGCTTCT-3′ for 0.5 (D), 1.0 (E), 1.5 (F) and 2.0 (G,H) pg of LBD-S RNA, fertilized, LpS1 to produce a 349-bp PCR product, 5′-TGTATCACGGATTG- and cultured for 48 hours at 18¡C until the control embryos reached GCTCAAAG-3′ and 5′-TCCTGAACCTGCCTCAACTTGG-3′ for the pluteus larva stage. The arrowheads in D point to the multiple LpC2 to produce a 198-bp PCR product, and 5′-TTCCCACAC- spicules. CGTTCCCATCT-3′ and 5′-AGGCAGCAGTCAGCATCTCC-3′ for LpC to produce a 200-bp PCR product. After incubation of the reaction tube for 3 minutes at 94¡C, one unit of AmpliTaq DNA polymerase (Perkin Elmer) was added to the reactions. PCR RESULTS reactions were carried out at 94¡C for 1 minute, 52¡C for 1 minute, and 72¡C for 1 minute. The reactions were carried out for 20, 25, Our earlier work suggested that a PDGF-like signaling and 30 cycles to determine whether DNA amplification was linear. pathway plays an important role in diverse developmental One-tenth volume of the PCR was analyzed by gel electrophoresis events in sea urchin embryogenesis. We further examined the in a 5% polyacrylamide gel. Radioactive PCR products were visu- role of the PDGF-like signaling pathway in L. pictus embryos alized by autoradiography after an overnight exposure to Kodak by testing the effects of a truncated human PDGF receptor-β XAR X-ray film. on sea urchin development with a dominant/negative mutation. β Indirect immunofluorescence The truncated form of the PDGF receptor- lacking the kinase Indirect immunofluorescence was carried out on methanol-fixed L. domains can form a nonfunctional dimer upon ligand binding, pictus embryos using the Ecto-V monoclonal antibody, a gift from Dr leading to a dominant/negative phenotype in mammalian cells David McClay, Duke University. Staining was carried out exactly as (Ueno et al., 1991). A DNA template with such a mutation was described by Wikramanayake et al. (1995). Embryos were observed constructed by deleting the DNA region encoding the kinase on a Zeiss Axioskop microscope equipped with Nomarski differential domains from a DNA construct encoding a complete human interference contrast and epifluorescence optics. PDGF receptor-β (hPR-β) to produce the truncated construct 2358 R. K. Ramachandran and others

Table 1. Expression of the ligand-binding domain of human PDGF receptor-β RNA in L. pictus embryos

Amount Number 2 RNA1 of RNA of scored Morphology (number of embryos) Exp. type injected (pg) embryos normal3 dominant/negative4 dead 1 LBD-S 0.001 19 17 (89%) 1 (6%) 1 (6%) 1 LBD-S 0.01 19 12 (63%) 7 (37%) 0 1 LBD-S 0.1 22 19 (86%) 2 (9%) 1 (5%) 2 LBD-S 0.1 88 76 (86%) 8 (9%) 4 (5%) 3 LBD-S 0.1 138 122 (88%) 14 (10%) 2 (1%) 2 LBD-S 0.5 106 79 (75%) 12 (11%) 15 (14%) 3 LBD-S 0.5 90 73 (81%) 11 (12%) 6 (7%) 1 LBD-S 1.0 21 2 (10%) 19 (90%) 0 2 LBD-S 1.0 117 64 (55%) 35 (30%) 18 (15%) 3 LBD-S 1.0 114 62 (54%) 49 (43%) 3 (3%) 2 LBD-S 1.5 142 0 114 (80%) 28 (20%) 3 LBD-S 1.5 122 18 (15%) 97 (80%) 7 (6%) 2 LBD-S 2.0 100 0 54 (54%) 46 (46%) 3 LBD-S 2.0 124 0 121 (98%) 3 (2%) 1 LBD-S 9.0 15 0 0 15 (100%)

2 LBD-AS 2.0 101 99 (98%) 0 2 (2%) 3 LBD-AS 2.0 108 97 (90%) 10 (9%) 1 (1%) 1 LBD-AS 12.0 17 14 (82%) 2 (12%) 1 (6%)

2 UC 0 98 82 (84%) 2 (2%) 14 (14%) 3 UC 0 101 92 (92%) 6 (6%) 3 (3%)

1The human PDGF receptor-β RNA transcripts injected into L. pictus eggs were LBD-S, ligand-binding domain-sense strand and LBD-AS, ligand-binding domain-antisense strand. UC, uninjected control embryos. 2Embryos were cultured for 48 hours until untreated control embryos reached the pluteus larva stage. 3A normal morphology was deﬁned as that of a swimming pluteus larva. 4The dominant/negative morphology included reduced gut and spicules, highly reduced gut and spicules, multiple radialized reduced spicules, arrested development at the mesenchyme blastula stage, and occluded blastulae. Representative morphologies are presented in Fig. 2.

∆hPR-β, containing the transmembrane and ligand-binding Not surprisingly, the human PDGF receptor antisense RNA domains (Fig. 1). hPR-β was used as the DNA template for in apparently was too long and lacked sufficient complementary vitro transcription of the full-length sense strand transcript (FL- sequence to disrupt expression of endogenous L. pictus S) and ∆hPR-β was the template for the sense and antisense PDGF receptor via RNA-RNA duplex formation, which RNA transcripts of the ligand-binding domain (LBD-S and allowed the LBD-AS RNA to serve as a useful negative LBD-AS, respectively). control. The ability of the human PDGF receptor-β to dimerize with Injection of human PDGF receptor-β RNA lacking the the endogenous L. pictus PDGF receptor is inferred from the template for the kinase domains causes inhibition of dominant/negative morphologies produced by the LBD-S RNA gut and spicule formation and differentiation along (Table 2). The dominant/negative morphologies included the oral-aboral axis embryos with a nearly complete gut and a thickened ectoderm, A range of truncated PDGF receptor mRNA concentrations either with several very small spicules (Fig. 2C) or lacking was injected into L. pictus eggs in order to determine an detectable spicules (Fig. 2F). The most interesting morphology effective concentration that might produce an abnormal, i.e., is shown in Fig. 2D and classified under multiple dominant/negative, morphology. The eggs were fertilized and spicules/reduced gut in Table 2. These embryos lacked the cultured until control embryos reached the prism or pluteus morphological features that distinguished the oral from aboral larva stage at approximately 48 hours (Fig. 2A,B). Table 1 ectoderm. The embryos formed a reduced and flaccid gut, three shows that injected doses of LBD-S RNA below 1.0 pg/egg to seven small radialized spicules, a spherical rather than the (9×105 transcripts) had relatively little effect on development, normal prism-like shape, no ciliary band nor defined and the morphology of most of the embryos appeared normal. stomadeum, and a thickened ectoderm in many embryos. A At 1.0, 1.5, and 2.0 pg/egg, the major effect of the LBD-S more pronounced effect is shown in Fig. 2E in which there are RNA was a dominant/negative morphology or lethality, no detectable spicules. The most severe effects are shown in depending on the egg batch. The effect was always lethal Fig. 2G and H, in which development is arrested at a mes- when 9.0 pg/egg of LBD-S RNA was injected, in which the enchyme blastula-like morphology, and embryos either contain eggs were fertilized, the zygote underwent several unequal an inordinate number of cells in the blastocoel (Fig. 2G), or and misoriented cleavages, and the cells ultimately dissoci- they display a normal mesenchyme blastula stage morphology ated and lysed. As much as 12 pg/egg of the antisense tran- (Fig. 2H). Another common morphology, not shown, was clas- script, LBD-AS, had no detectable effect on development. sified as occluded blastulae (Ramachandran et al., 1993) in PDGF signaling in early sea urchin development 2359

Table 2. A classiﬁcation of the dominant/negative morphologies from expression of the ligand-binding domain of human PDGF receptor-β RNA in L. pictus embryos 2 Amount of Morphology (number of embryos) injected LBD-S1 reduced spicules/ multiple spicules/ no spicules/ arrested occluded RNA per egg (pg) reduced gut reduced gut reduced gut MB stage3 blastulae 0.1 7 (29%) − − 2 (8%) 15 (63%) 0.5 7 (30%) − − − 16 (70%) 1.0 34 (33%) 29 (28%) − 28 (27%) 12 (7%) 1.5 141 (67%) − 42 (20%) − 28 (13%) 2.0 − − 9 (5%) 38 (22%) 128 (73%)

1LBD-S, ligand-binding domain-sense strand. 2Embryos were cultured for 48 hr until untreated control embryos reached the pluteus larva stage. 3MB, mesenchyme blastula.

Table 2, in which cells filled the blastocoel, and the embryos more of the intact endogenous PDGF receptors are progres- died within 24 hours. sively inactivated by dimerization to the truncated form. The A dose-response effect dependent on the amount of results suggest also that the Lytechinus and human PDGF truncated PDGF receptor-β RNA injected into eggs was receptors are structurally and functionally similar. evident (Table 1). As the amount of injected RNA increased, the percentage of embryos with a dominant/negative morphology increased from approximately 10% at 0.1 pg/egg to greater than 80% at 2.0 pg/egg. Some embryos arrested at the mesenchyme blastula stage or formed occluded blastulae as a result of nearly all the injected amounts of RNA. These results suggested that embryos may be especially sensitive to an interruption of the PDGF-like pathway at the blastula stage to produce a developmental bottleneck. The dose-response was reflected by a general increase in the severity of the dominant/negative effect as more truncated RNA was injected (Table 2); although the effects were variable, and nearly the entire range of morphologies could be produced by a given amount of injected RNA, e.g., 1.0 pg/egg. We attribute this variability to Fig. 3. The injected RNA for variation within and among egg batches. human PDGF receptor-β is Each experiment used an egg population expressed in L. pictus embryos collected from a different artifically and coimmunoprecipitates with spawned female or females. A similar the endogenous PDGF-like variability of morphologies was observed receptor. LBD-S (S) and LBD-AS (AS) RNA (1.0 pg/egg) were injected into L. pictus eggs, which were fertilized and when serotonin receptor mRNA was cultured until LBD-AS RNA-injected and uninjected (0) control embryos reached the injected into sea urchin eggs (Cameron et pluteus larva stage. Total proteins from 100 embryos (1X) or 200 embryos (2X) were al., 1994). Alternatively, in addition to the incubated with rabbit polyclonal anti-mouse PDGF receptor-β antibody directed against possibility that PDGF signaling is amino acids 425-446 of the extracellular domain (A,C) or rabbit polyclonal anti-human affecting many developmental events, it PDGF receptor-β antibody directed against amino acids 1082-1101 of the intracellular may be that the embryos are highly domain (B,D). For controls to detect background bands, the antibody against the sensitive to the amount of PDGF receptor extracellular domain (A,C, Ab lane) and intracellular domain (B, Ab lane) were carried expressed, which may be a consequence through the immunoprecipitation process without the addition of Lytechinus proteins; and µ of small variations in the amount of RNA with proteins from uninjected embryos (A,B, 2X and 100 g). The immunoprecipitated injected into each egg. The dose-response proteins were resolved on an 8% SDS-polyacrylamide gel, blotted onto nitrocellulose filter membranes, and incubated with the antibody against the extracellular domain. An anti- effect exerted by the truncated PDGF rabbit IgG antibody horseradish peroxidase conjugate-chemiluminescent assay was used to 3 receptor is consistent with a detect the bound secondary antibody. The band at 55×10 Mr represents the H chain from dominant/negative mechanism. These the primary antibody. The blots were exposed to Kodak X-Omat AR X-ray film for experiments indicate that as more of the 5 minutes. Ab, antibody; IP, immunoprecitation; 0, no RNA injected; numbers between 3 truncated PDGF receptor is expressed, lanes, Mr ×10 . 2360 R. K. Ramachandran and others

The truncated human PDGF receptor-β RNA is 95×103 in the Ab and AS lanes of Fig. 3C shows an undisso- expressed in the Lytechinus embryo and disrupts ciated form of the antibody. development by a dominant/negative mechanism To demonstrate by another method that the truncated and Although no direct evidence is shown demonstrating that the full-length PDGF receptors formed dimers, lysates from LBD- anti-human PDGF receptor-β antibodies crossreact with the S RNA-injected embryos were immunoprecipitated with a Lytechinus PDGF-like receptor, immunoprecipitation experi- polyclonal anti-human PDGF receptor-β antibody that recog- ments were carried out that indicate that the truncated RNA nizes the intracellular domain (Fig. 3B,D). The epitope for this was translated and that the truncated protein dimerized with antibody is not present on the truncated form and cannot be the endogenous receptor (Fig. 3). LBD-S and LBD-AS RNA directly immunoprecipitated by the intracellular domain- from the ∆hPR-β construct were injected into L. pictus eggs directed antibody. If the Lytechinus and human PDGF (1.0 pg/egg), and the injected embryos were cultured until the receptors dimerize, then the immunoprecipitate should contain 3 3 LBD-AS-injected control embryos reached the pluteus larva both the 95×10 and 180×10 Mr forms of the receptor. stage. Pluteus larva stage embryo lysates from the injected Analysis of L. pictus precipitates using the intracellular embryos were incubated under nondenaturing conditions with domain-directed antibody (Fig. 3D) revealed both bands. There 3 a polyclonal anti-mouse PDGF receptor-β antibody specific for is no band at 95×10 Mr in proteins from uninjected embryos the extracellular domain (Fig. 3A,C) or the intracellular (Fig. 3B, lane 2X). The simplest explanation for the presence domain (Fig. 3B,D). The immunoprecipitated proteins were of the truncated PDGF-like receptor in LBD-S RNA-injected resolved on an SDS-polyacrylamide gel and blotted. Immuno- embryos is that the truncated and endogenous receptors existed precipitates were detected by incubating the blot with the anti- in vivo as a dimer and coprecipitated. The crossreactivity extracellular domain antibody and an anti-IgG antibody exhibited by the anti-human PDGF receptor antibody indicates conjugate. that the cytoplasmic domains of the human and Lytechinus The results indicate that the truncated RNA was translated PDGF receptors are similar. These results also indicate that the into protein and that the truncated and full-length PDGF truncated form acts via a dominant/negative mechanism. receptors dimerized. Proteins isolated from 200 (2×) uninjected embryos (Fig. 3A) and 200 embryos injected with LBD-AS Coinjection of full-length human PDGF receptor-β RNA (Fig. 3C, AS lane) and incubated with an antibody to the RNA rescues development extracellular domain of the human PDGF receptor, showed no The rescue of dominant/negative embryos by full-length detectable amounts of an immunoprecipitate representing the human PDGF receptor-β RNA would be strong corroborative 3 endogenous PDGF-like receptor of Mr, 180×10 (there is evidence that an endogenous PDGF-like-mediated pathway is approximately 2.6 µg of total proteins per 200 embryos). As involved in the specification and differentiation along the oral- shown in Fig. 3A and by Ramachandran et al. (1993), when aboral axis and that the truncated form exerts its effect by a 100 µg of total proteins was used, the antibody crossreacted dominant/negative mechanism. The expression of sufficient 3 specifically to an endogenous protein of 180×10 Mr. These amounts of FL-S RNA should reverse the dominant/negative results demonstrated that the antibody did not immunoprecip- effect by out-competing the truncated form to allow dimeriza- itate the L. pictus PDGF-like receptor from the embryos tion of full-length receptors. This is a reasonable prediction injected with LBD-AS RNA. In contrast, immunoprecipitation based on the fact that the PDGF receptor-β readily homod- of proteins from embryos injected with LBD-S RNA using the imerizes (Seifert et al., 1989), and the reasonable assumption same extracellular-domain antibody resulted in the one protein that the intact and truncated human forms would have a greater 3 corresponding to the truncated human receptor of 95×10 Mr affinity for each other than either does for the Lytechinus 3 and another protein of 180×10 Mr (Fig. 3C, S lanes). This PDGF-like receptor. experiment indicated that the truncated receptor was synthe- L. pictus eggs were coinjected with a constant amount of sized in the embryo and that the full-length Lytechinus PDGF- LBD-S RNA (1.5 pg/egg; 1.45×106 transcripts) and increasing 5 like receptor dimerized to it. The faint band of approximate Mr amounts (0 to 10.0 pg; where 1 pg=3.2×10 transcripts) of FL-

Table 3. Injection of full-length human PDGF receptor-β RNA reverses the dominant/negative effect Amount of injected Amount of injected Ratio of Morphology (number of embryos)2 LBD-S RNA per FL-S RNA per LBD-S RNA egg (pg)1 egg (pg)1 to FL-S RNA normal3 dominant/negative4 dead 1.5 0 ∞ 0 0 147 (100%) 1.5 0.01 400/1 0 7 (5%) 131 (95%) 1.5 0.1 40/1 0 120 (85%) 21 (15%) 1.5 1.0 4/1 113 (84%) 9 (7%) 12 (9%) 1.5 2.0 2/1 123 (75%) 8 (5%) 40 (25%) 1.5 10.0 2/5 101 (74%) 1 (1%) 35 (26%) 0 10.0 0 131 (87%) 4 (3%) 15 (10%)

1The human PDGF receptor-β RNA transcripts injected into L. pictus eggs were LBD-S, ligand-binding domain-sense strand and FL-S, full-length sequence- sense strand. 2Embryos were cultured for 48 hr until untreated control embryos reached the pluteus larva stage. 3A normal morphology was deﬁned as that of a prism or pluteus larva. 4The dominant/negative morphology included reduced gut and spicules, highly reduced gut and spicules, multiple radialized reduced spicules, arrested development at the mesenchyme blastula stage, and occluded blastulae. PDGF signaling in early sea urchin development 2361

differentiation, then disruption of this pathway should cause abnormal expression of these ectoderm-specific genes. Aboral ectoderm expression was examined first (Fig. 5) using two available markers, the LpS1 and LpC2 genes. The LpS1 genes are activated prior to the hatching blastula stage, and expression is restricted to the aboral ectoderm (Xiang et al., 1988; Tomlinson and Klein, 1990). The LpC2 cytoplasmic actin gene is expressed primarily in the aboral ectoderm, and RNA transcripts accumulate at detectable levels by mesenchyme blastula stage (Fang and Brandhorst, 1996). L. pictus eggs were injected with 1.0 pg of LBD-S or LBD- AS RNA transcripts, fertilized, and cultured until uninjected Fig. 4. L. pictus embryonic development is rescued with sufficient control embryos reached the late prism stage. Total RNA was amounts of coinjected FL-S RNA. Eggs were coinjected with 1.5 pg isolated from the uninjected control, from sense RNA-injected, of LBD-S RNA and 0.1 pg of FL-S RNA (A) and 1.0 pg of FL-S and from antisense RNA-injected embryos and used as the RNA (B), fertilized, and cultured for 48 hours. template in reverse transcriptase-polymerase chain reaction (RT-PCR) assays (Fig. 5A). Embryos cultured from eggs injected with the truncated RNA for the human PDGF receptor S RNA (Table 3). Due to egg batch variation, these embryos showed an inhibition of aboral ectoderm-specific LpS1 and showed a heightened sensitivity to the LBD-S RNA in the form LpC2 RNA expression relative to uninjected and antisense of lethality. Injection of 1.5 pg of the LBD-S RNA was lethal RNA control embryos. Similar results were obtained when the when no or very little FL-S RNA was coinjected. There was PCR was continued for 20, 25, and 30 cycles, demonstrating no developmental rescue by the FL-S RNA at 0.01 and 0.1 pg that the absence of expression in the dominant/negative of RNA/egg, although at 0.1 pg/egg, 85% of the embryos were embryos was not due to a rate-limiting component in the PCR. rescued from the lethal effects of the truncated receptor and Oligonucleotide primers for the protein coding region of L. manifested a dominant/negative morphology. Injection of 1.0, pictus actin genes (LpC) were used as a control for RNA 2.0, and 10.0 pg of FL-S RNA/egg rescued 74-84% of the embryos in that they developed normally to the pluteus larva stage. Table 3 shows that as the molar ratio of coinjected LBD-S to FL-S RNA decreased, the effect went from lethality (400:1) to a partial rescue seen as a dominant/negative morphology (40:1) to full developmental rescue (4:1 and less). A rescued embryo is shown (Fig. 4B) in which 1.0 pg of FL-S RNA (1.3×105 transcripts) was coinjected with 1.5 pg of LBD-S RNA (5.25×105 transcripts). The rescued embryos formed a full gut, properly oriented triradiate spicules, and normal ectodermal features. This is in contrast to an embryo (Fig. 4A) which had received a coinjection of 0.1 pg of FL-S RNA (1.3×104 transcripts), where a dominant/negative morphology predomi- nated. These results show that the rescued morphology is dose dependent on the ratio of injected LBD-S to FL-S RNA Fig. 5. Aboral ectoderm and support the earlier conclusion that the truncated PDGF differentiation is inhibited by receptor is operating by a dominant/negative mechanism to expression of the disrupt a PDGF-like signaling pathway. dominant/negative PDGF receptor. (A) Total RNA was The truncated human PDGF receptor-β inhibits isolated from 48-hour ectoderm differentiation embryos cultured from uninjected eggs (lane U), Injection of LBD-S RNA into L. pictus eggs caused the eggs injected with 1.5 pg LBD-S RNA (lane S), and eggs injected with loss of ectodermal features (Fig. 2). The shape of the 1.5 pg of LBD-AS RNA (lane AS) for RT-PCR assays. Shown are the embryo was spherical rather than trapezoidal, the oral expected amplified DNA products of 349 bp for LpS1, 198 bp for LpC2, ectoderm did not form a recognizable stomadeum, and no and 200 bp for LpC. As a control (lane C) to check for contaminating ciliary band was evident. The gut, when one did form, DNA, an equal amount of RNA from uninjected embryos was subjected appeared flaccid and without direction, and multiple to RT-PCR analysis, in which the reverse transcriptase was omittted from the reaction. The PCR was continued for 20 cycles. (B) CAT spicules often formed and were arranged in a radialized β fashion. These morphological characteristics suggested assays were carried out to show that the LpS1- gene is regulated by a PDGF signaling pathway. Approximately 2,000 copies of −762 LpS1- that specification of positional identity along the oral- CAT DNA and 1.0 pg of LBD-AS or LBD-S RNA were injected per aboral axis had not occurred in the affected embryos. egg. The eggs were fertilized and the embryos cultured to the prism Establishment of the oral-aboral axis was assayed by larva stage. Each CAT assay was carried out with approximately 200 examining the expression of ectoderm markers. If the embryos. Std., CAT standards. Relative CAT DNA amounts are shown PDGF-like signaling pathway is required for ectoderm below the CAT assays. 2362 R. K. Ramachandran and others recovery. The LpC bands show that nearly equivalent amounts DISCUSSION of RNA were used in the RT-PCRs. Additional experiments were carried out using a different The introduction into Lytechinus eggs of human PDGF experimental approach to determine whether the LpS1 receptor-β RNA lacking the region encoding the tyrosine promoter is regulated by a PDGF-like signaling pathway kinase domains caused specific developmental defects. A dose (Fig. 5B). Equal amounts of the truncated sense and antisense response was observed in which expression of the truncated RNA species for the human PDGF receptor and a −762 to RNA caused a gradation of morphologies: from a mild +17 LpS1-β promoter-CAT gene reporter fusion construct reduction of gut formation and spiculogenesis, to a radializa- (Xiang et al., 1988) were coinjected into Lytechinus eggs, tion of the embryo and loss of oral structures, to an arrest at which were fertilized and cultured to the pluteus larva stage. the mesenchyme blastula stage and loss of ectoderm differen- Embryos cultured from eggs injected with the truncated tiation. Our data are consistent with a hypothesis in which a LBD-S RNA inactivated CAT expression while embryos dominant/negative mechanism caused the observed develop- coinjected with the control LBD-AS RNA had no effect on mental defects, in which the exogenous truncated form CAT activity. The combined results in Fig. 5 indicate strongly dimerized with the intact endogenous form to interrupt signal that LpS1 gene activity is dependent on PDGF-like signaling. transduction. The coinjection of the full-length transcript with The near absence of LpS1 and LpC2 expression in the truncated transcript for the PDGF receptor-β prevented the dominant/negative embryos suggested that the differentiation developmental defects induced by the truncated receptor alone. of those cells normally destined to be aboral ectoderm was These results support our contention that a PDGF-like blocked by disruption of a PDGF-like signaling pathway. signaling pathway is involved in key early developmental These cells would have two alternatives: form a cell type decisions. other than aboral ectoderm or remain undifferentiated. To determine the state of ectoderm differentiation in A PDGF-like pathway in sea urchin embryos dominant/negative embryos, the spatial expression of the oral transduces signals for gastrulation and ectoderm marker Ecto-V was examined in embryos injected spiculogenesis with LBD-S or LBD-AS RNA (Fig. 6). Ecto-V is a cell Several lines of evidence support the hypothesis that a PDGF- surface antigen expressed at nearly undetectable levels like receptor in Lytechinus sea urchin embryos transmits throughout the embryo until the midgastrula stage (Wikra- signals for proper gut and spicule differentiation and morpho- manayake et al., 1995). Later, it accumulates to relatively genesis. ECM disruption arrests development at the mes- high levels in the oral ectoderm and foregut of the pluteus larva (Coffman et al., 1990). Control embryos injected with LBD-AS RNA showed normal patterns of Ecto-V expression with nearly undetectable signal at the mesenchyme blastula stage (Fig. 6A,B); a weak signal in the thicker cuboidal cells of the oral ectoderm at the early to mid-gastrula stage (Fig. 6E,F); and a strong signal in the oral ectoderm and foregut of a pluteus larva (Fig. 6I,J). Control experiments, in which the primary antibody was omitted from the antibody staining procedure, produced no signal (data not shown). Embryos injected with LBD-S RNA arrested at a mesenchyme blastula-like stage or formed a radialized, spherical body plan similar to those shown in Fig. 2D and H, respectively. Ecto-V expression in these embryos (Fig. 6C,D,G,H) was relatively strong in the entire single-cell layer of the presumptive ectoderm. The ectodermal cells were cuboidal in shape, which are more clearly seen with Nomarski optics (Fig. 6K,L), and morphologically resembled oral ectoderm rather the squamous cell shape characteristic of Fig. 6. The dominant negative PDGF receptor causes the oral ectoderm-specific aboral ectoderm. The cuboidal cell morphol- Ecto-V gene to be expressed in all ectoderm cells of the L. pictus embryo. Eggs ogy, the absence of expression by the aboral were injected with 1.5 pg of control LBD-AS RNA (A,B,E,F,I,J) or 1.5 pg LBD-S ectoderm-specific genes, and the expanded RNA (C,D,G,H,K,L) and cultured for 14 hours to the mesenchyme blastula stage (A,B), 20 hours to the gastrula stage (E,F) or 48 hours to the pluteus larva stage (I,J) expression of the oral ectoderm-specific Ecto- or equivalent 48 hour duration (C,D,G,H,K,L). The Nomarski differential V gene suggest that disruption of the PDGF- interference contrast images (A,C,E,G,I) are paired with the corresponding indirect like signaling pathway inhibits aboral ectoderm immunofluorescent images (B,D,F,H,J). The arrowheads in C,D,G,H delineate the differentiation, such that the entire ectoderm thickened outer cell layer. The thickened ectoderm is more clearly seen in the acquires an oral-like ectoderm morphology. embryos showin in K and L. Bar, 50 µm. PDGF signaling in early sea urchin development 2363 enchyme blastula stage and inactivates the LpS1 genes (Wessel One leading hypothesis regarding the specification of early et al., 1989). Ramachandran et al. (1993) demonstrated that cell lineages in the sea urchin embryo is that the factors which human PDGF-BB, in conjunction with human TGF-α, rescued give rise to the five cellular territories are globally but asym- mesenchyme blastula stage arrested/ECM-disrupted embryos metrically distributed in the egg and early embryo, and that so that they reactivated the LpS1 genes and resumed normal proper differentiation depends on the local activation of these development to gastrulate and form spicules. These results factors via cell to cell communication (Davidson 1989). Our suggested that there are receptors in Lytechinus similar to results are consistent with such a hypothesis. The PDGF-like human PDGF and EGF receptors that can bind human growth signaling pathway may be one of the locally activated compo- factors for the transduction of signals necessary for gastrula- nents required for induction of aboral ectoderm. When this tion and spiculogenesis. signaling pathway is disrupted, aboral ectoderm may differen- Later work showed that the addition of anti-human PDGF tiate as oral ectoderm. This signaling pathway may be inter- receptor-§ (1 µg/ml) and anti-human PDGF-BB (3 µg/ml) anti- rupted by disassembly of the ECM, possibly resulting in the bodies caused the embryos to arrest at the mesenchyme loss of concentrated ligand/receptor interactions; or by the use blastula stage (Ramachandran et al., 1995; Govindarajan et al., of a dominant/negative PDGF receptor. 1995), a morphology observed when the ECM is disrupted and The precursor cells that give rise to the oral and aboral when the truncated PDGF receptor is expressed. Treatment of ectoderm cell lineages are segregated by fifth cleavage Lytechinus embryos with the human PDGF receptor-§ antibody (Cameron et al., 1990), and the aboral ectoderm-specific LpS1 produced morphologies in a dose-response fashion similar to genes are activated near this time of development (Tomlinson those seen when the embryos express the truncated PDGF and Klein, 1990). Therefore, the putative PDGF-dependent cell receptor. communication necessary for aboral ectoderm specification probably occurs at or soon after fifth cleavage. Earlier results Formation of aboral ectoderm is dependent on a are consistent with this time period. Ramachandran et al. PDGF signaling pathway (1995) showed that a PDGF-like receptor is present in the Using molecular markers specific to each ectoderm lineage, the Lytechinus egg and early embryo, and that the period of time Ecto-V marker for oral ectoderm and the LpS1 and LpC2 during development that the embryo is sensitive to disruption markers for aboral ectoderm (Coffman and McClay, 1990; of the PDGF signaling begins sometime between the second Xiang et al., 1988; Fang and Brandhorst, 1996), we showed and third cleavage stages to the time when the embryo forms that the ectoderm develops abnormally in embryos injected a blastula (seventh and eighth cleavages). with the truncated PDGF receptor RNA. The embryos did not accumulate LpS1 and LpC2 RNA and expressed Ecto-V inap- The use of a dominant/negative strategy in sea propriately in all ectoderm cells. These results suggested that urchin embryos a PDGF-like signaling pathway is required for proper com- This is the first reported use of a dominant/negative strategy in mitment along the oral-aboral axis. This conclusion was further sea urchin embryos and opens a new in vivo approach to inves- supported by the CAT assays showing that the activation of tigate gene function in this system. Other methods have been exogenous LpS1 promoter required a PDGF-like signaling used recently to examine gene function during sea urchin pathway. The data suggest that a PDGF-like pathway is development including the injection of antisense normally involved in a cell to cell communication process that oligodeoxynucleotides (Char et al., 1994) and the activation of induces aboral ectoderm. Wikramanayake and Klein (1997) introduced receptors (Cameron et al., 1994). The disadvantages suggested that normal expression of the Ecto-V antigen in oral of these methods is that the former does not appear to work for ectoderm cells requires suppressive signals from vegetal blas- many mRNAs and does not allow the study of mutations at tomeres. Our results suggest that a PDGF-like signaling selected sites; while the latter method may be misleading if pathway may be involved in the generation of the suppressive activated inappropriately. The dominant/negative approach has signal in vegetal blastomeres. several advantages (Herskowitz, 1987): the removal rather than The morphologies produced by the truncated PDGF receptor the imposition of a cellular component on a system, the are similar in some ways to those produced by treatment with injection into selected blastomeres to study problems regarding nickel (Hardin et al. 1992) and the stimulation of an introduced spatial regulation, and the deletion or alteration of selected sites serotonin-mediated pathway (Cameron et al., 1994). Both to define precisely functional domains. A disadvantage is that treatments caused multiple foci of spicules to form in a radi- the dominant/negative strategy depends on protein-protein alized embryo. Nickel treatment caused a loss of commitment interactions, a qualification that does not fit all proteins. along the oral-aboral axis and a shift in cell fate to oral Nonetheless, many questions in sea urchin development can be ectoderm, in which Ecto-V expression rose several-fold and addressed by using a dominant/negative approach. LvS1 expression dropped relative to control embryos. In contrast, serotonin treatment did not alter the expression of oral We dedicate this work to the memory of Dr Richard D. Brodeur. and aboral markers. A distinct difference of the nickel We thank Drs E. Gangolli, Y. Sanchez and A. Sater for their helpful treatment to treatment with the dominant/negative PDGF comments about the manuscript. This work was supported in part by receptor was the gross exaggeration of later oral structures and Sigma Xi to R. K. R. and V. G., and the American Heart Association Grants 91G-441 and 93R-441 to C. R. T. the shift in the position of the ciliary band. 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