Identification and Localization of a Sea Urchin

Development 124, 3363-3374 (1997) 3363 Printed in Great Britain © The Company of Biologists Limited 1997 DEV3707

Identiﬁcation and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation

David R. Sherwood and David R. McClay* Developmental, Cell and Molecular Biology Group, Box 91000, Duke University, Durham, NC 27708, USA *Author for correspondence (e-mail: [email protected])

SUMMARY

The specifications of cell types and germ-layers that arise plate. Experimental perturbations and quantitative from the vegetal plate of the sea urchin embryo are thought analysis of LvNotch expression demonstrate that the mes- to be regulated by cell-cell interactions, the molecular basis enchyme blastula vegetal plate contains both of which are unknown. The Notch intercellular signaling animal/vegetal and dorsoventral molecular organization pathway mediates the specification of numerous cell fates even before this territory invaginates to form the archen- in both invertebrate and vertebrate development. To gain teron. Furthermore, these experiments suggest roles for the insights into mechanisms underlying the diversification of Notch pathway in secondary mesoderm and endoderm vegetal plate cell types, we have identified and made anti- lineage segregation, and in the establishment of dorsoven- bodies to a sea urchin homolog of Notch (LvNotch). We tral polarity in the endoderm. Finally, the specific and dif- show that in the early blastula embryo, LvNotch is absent ferential subcellular expression of LvNotch in apical and from the vegetal pole and concentrated in basolateral basolateral membrane domains provides compelling membranes of cells in the animal half of the embryo. evidence that changes in membrane domain localization of However, in the mesenchyme blastula embryo LvNotch LvNotch are an important aspect of Notch receptor shifts strikingly in subcellular localization into a ring of function. cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has Keywords: sea urchin, Notch, phylogeny, vegetal plate, subcellular an asymmetric bias towards the dorsal side of the vegetal localization

INTRODUCTION segregation that must occur in cells derived from the vegetal plate is the differential specification of SMCs and endoderm The vegetal plate of the sea urchin mesenchyme blastula cells. Lineage analysis has shown that the presumptive SMCs embryo is a structurally simple, yet developmentally dynamic, lie in the central region of the mesenchyme blastula vegetal region. This concave single cell-layer epithelium contains all plate and are clonally distinct from the presumptive endoderm three future germ-layers of the embryo concentrically arranged cells, which are positioned in a ring around the SMCs (Ruffins within a few cell diameters (Ruffins and Ettensohn, 1996). and Ettensohn, 1996). The clonal isolation of presumptive During gastrulation, vegetal plate cells undergo dramatic SMCs from endoderm cells is consistent with these germ- movements and shape changes to invaginate and form the layers being differentially specified before vegetal plate invagi- archenteron (reviewed in Hardin, 1996). Cells at tip of the nation. To date, however, molecular evidence has suggested archenteron segregate and become secondary mesenchyme otherwise. The vegetal plate marker Endo16 and sea urchin cells (SMCs). Behind the SMCs, the archenteron gives rise to homologs of brachyury and forkhead are expressed throughout the endoderm, which subdivides into several regions. Finally, the vegetal plate prior to invagination; only after invagination the ectoderm is excluded from the archenteron. These changes has begun do these and other identified molecular markers that occur in cells derived from the vegetal plate during gas- become differentially expressed between SMCs and endoderm trulation offer a relatively simple cellular model for studying lineages (Ransick et al., 1993; Harada et al., 1995, 1996; the integration of mechanisms underlying cell fate diversifica- reviewed in Davidson, 1993). Therefore, it remains unclear tion and morphogenesis. when these germ-layers are specified. Given the evidence for Cellular studies of vegetal plate regionalization suggest that cell-cell interactions specifying cell-types in the vegetal plate, local cell-cell interactions play an essential role in the diversi- an analysis of evolutionarily conserved intercellular signaling fication of cell-types that arise from the vegetal plate (McClay pathways may shed light into the time and mechanism by and Logan, 1996; reviewed in Davidson, 1993). The molecular which these two germ-layers become distinct. basis of these cell-cell interactions, however, is unknown. One The Notch signaling pathway mediates cell-cell interactions 3364 D. R. Sherwood and D. R. McClay leading to the specification and patterning of a wide array of LvNotch gave no evidence for differential processing of the LvNotch cell types in both invertebrate and vertebrate development gene. (Conlon et al., 1995; de Celis et al., 1996; reviewed in Artavanis-Tsakonas et al., 1995). The conserved role and Northern analysis µ + pleiotropic function of this pathway made it a likely candidate A 1.0% agarose/formaldehyde gel was loaded with 3 g/lane poly(A) for involvement in the diversification of cell-types arising from RNA (isolated with QuickPrep; Pharmacia), fractioned by elec- trophoresis, transferred onto a nylon membrane (Gene Screen, NEN the vegetal plate. The Drosophila Notch gene and two related Research Products), and hybridized with a 948 bp fragment of genes, lin-12 and glp-1 in C. elegans, have been identified as LvNotch corresponding to a portion of the extracellular domain receptors in the Notch pathway. These genes, and four identi- (amino acids 798-1114). The blot was then probed and washed as fied vertebrate homologs of Notch, all encode single-spanning described (Bachman and McClay, 1995). Identical results to those transmembrane proteins (Uyttendaele et al., 1996; reviewed in shown in Fig. 2 were also obtained with a probe corresponding to a Artavanis-Tsakonas et al., 1995). Studies on the localization of portion of the intracellular domain (data not shown). both Drosophila Notch and C. elegans GLP-1 have led to the proposal that the activity of this signaling pathway may in part Antibody production be controlled by the precise distribution of the receptor (Fehon Eight fusion proteins were expressed using subcloned or PCR- et al., 1991; Crittenden et al., 1994). Therefore, knowledge of amplified fragments of LvNotch in pGex1, 2 and 3 vectors (Glu- tathione S-transferase (GST) expression system; Smith and Johnson, the localization of Notch proteins is a likely indicator of where 1988). The fusion proteins and corresponding LvNotch amino acids this pathway functions. they encompass are as follows: Pet1, 29-287; Pet2, 198-464; 2s, 488- We have isolated and generated antibodies to the first echin- 799; Bam3, 715-878; Bam4, 1002-1130; Bam1, 1128-1472; Ank, oderm Notch homolog (LvNotch). Analysis of LvNotch 1751-2095; CloneB, 1886-2531. All fusion proteins were expressed expression demonstrates that the presumptive SMCs and and purified as described (Smith and Johnson, 1988), with the endoderm are indeed differentially specified in the mes- exception of 2s and CloneB. The fusion protein 2s was insoluble and enchyme blastula embryo, earlier than any previous markers purified as outlined for Dmoesin (McCartney and Fehon, 1996). have shown. In addition, there is a dorsal bias in the expression Isolated fusion proteins were injected into animals to generate poly- of LvNotch in the mesenchyme blastula vegetal plate that is clonal antibodies (pAb; Harlow and Lane, 1988); clone B, which maintained in the presumptive endoderm during invagination. could not be eluted from the glutathione-agarose beads, was injected coupled to these beads. Animals injected with fusion proteins were as These results, together with several experimental manipula- follows: mice, all fusion proteins; guinea pigs, Bam1 and Ank; tions and a quantitative analysis of LvNotch expression, Rabbits, Ank. Whole serum from mice was prepared by ammonium suggest that the Notch signaling pathway is involved in the seg- sulfate precipitation (Harlow and Lane, 1988). Serum from guinea regation of SMCs from endoderm cells, and in the establish- pigs and rabbits was affinity purified as described (McCartney and ment of a dorsoventral axis in the endoderm. Finally, we Fehon, 1996). Specificity of antibodies to LvNotch was determined present compelling evidence that shifts in membrane domain by staining gastrula-stage embryos and comparing immunofluores- localization are an important component of Notch receptor cence patterns. Controls for specificity included staining with pre- function. immune serum, staining of embryos with antibodies to GST, and staining after pre-incubation of pAb with the appropriate fusion protein. MATERIALS AND METHODS Western analysis Protein extracts were prepared by pelleting embryos, adding 10 µl of Animals pelleted embryos to ice-cold SDS Lysis buffer (200 µl; 100 mM Tris, Sea urchins (Lytechinus variegatus) were obtained from Susan Decker pH 6.8; 4% SDS; 20% glycerol; 1 mM PMSF; 1 µg/mL Leupeptin), (Hollywood, FL) and Tracy Andacht (Duke University Marine Labo- douncing 4× with a pestle, and then immediately boiling for 5 ratory). Gametes were harvested and cultured as described by Hardin minutes. Samples were then spun at 16,000 relative centrifugal force et al. (1992). (RCF) for 15 minutes, and the supernatant frozen at −70¡C until use. The number of embryos homogenized in the 10 µl sample was deter- Cloning of LvNotch mined by serial dilutions taken from the original pellet of embryos. Poly(A)+ RNA was prepared and cDNA made from various stages of Samples were run on 3%-15% gradient gels, transferred to nitrocel- embryos as described (Bachman and McClay, 1995). Degenerate lulose and probed with affinity-purified guinea pig α-Bam1 (1:1000; oligonucleotide primers were designed as in Stifani et al. (1992), and 100 µg/ml stock) or affinity-purified rabbit α-Αnk (1:2500; 1 mg/ml used in a PCR reaction with the above cDNA pools using the stock) pAbs. The blots were processed and developed as described following conditions: 95¡C, 60 seconds; 40¡C, 60 seconds; 72¡C, 2 (McCartney and Fehon, 1996). Pre-immune sera used as controls for minutes for 45 cycles. An amplified 429 bp band was cloned into these antibodies gave light, nonspecific background banding patterns. Bluescript SK- vector (Stratagene) and sequenced (Sequenase 2.0 kit; Amersham Life Science). Initial clones of LvNotch obtained from a Immunolocalization and image analysis lambda-zap phage mid-gastrula cDNA library (Stratagene), using the Embryos were fixed in ice-cold methanol for 20 minutes and rehy- cloned PCR product as a probe, led to the identification of the 3′ end drated and washed 2× with ice-cold artificial sea water (ASW). of the coding sequence. However, multiple screens and the isolation Embryos were then washed with PBS, blocked for 10 minutes in of 28 clones were necessary to locate the 5′ end of the coding region. PBS/2% normal goat serum (GibcoBRL), incubated overnight at 4¡C Three clones, which together covered the entire open reading frame, in primary antibody, washed, blocked as above, then incubated at were subcloned and subjected to a transposon-based sequencing room temperature in secondary antibody (Cy5- or Cy3-conjugated; strategy (Strathmann et al., 1991) to obtain the full sequence for both Jackson Immunoresearch Laboratories) for 1.5 hours. Embryos were strands of DNA encoding LvNotch. DNA sequences were analyzed washed and mounted in 9:1 (v/v) glycerol:1 M Tris, pH 8.0. For all using AssemblyLign (International Biotechnologies, Inc.). Alignment images shown, pAbs generated to the fusion protein Bam1 were used; of the 5′ and 3′ ends of all clones identified to the final sequence of either from mice whole sera at 1:1000 or affinity-purifiedfrom guinea Localization and regulation of sea urchin Notch 3365 pig at 1:100 (25 µg/ml stock). However, pAbs generated from a Drosophila Notch and vertebrate Notch1 and 2. An alignment minimum of two non-overlapping fusion proteins were analyzed for of Notch family members, including at least one of the four all stages shown to confirm the specificity of the staining pattern. The vertebrate Notch proteins and Drosophila Notch (GCG PileUp PMC-specific monoclonal antibody (mAb) 1G8 (McClay et al., 1983), program, gap weights: 3, 10 and 15), revealed a deletion of α α β and guinea pig -LvCadherin and -Lv -catenin pAbs (Miller, 1995), EGF-repeat 14 in LvNotch (alignment not shown; Fig. 1A). were used with the above fixation and incubation conditions. A Zeiss Overall amino acid sequence comparisons between vertebrate confocal laser scanning microscope was used to aquire all images. Mesenchyme blastula-stage embryos were fixed for cell-count Notch1, 2, 3, 4 and Drosophila Notch to LvNotch showed iden- analysis of LvNotch localization in vegetal plate cells 30-50 minutes tities of 43%, 41%, 41%, 38% and 44%, respectively. after the start of PMC migration (10-45 minutes before vegetal plate To address whether a duplication event or events in the invagination was observed). Lvβ-catenin/LvNotch and 1G8/LvNotch- Notch gene may have occurred before the divergence of sea stained vegetal plates were obtained by sequential confocal section- urchins and vertebrates from a common ancestor, a parsimony ing of double-labeled vegetal plates, rendering 2-D projections of the analysis of Notch protein sequences was undertaken sections, and then overlaying these projections using Adobe (Swofford, 1993; MACCLADE 3.04, Maddison and Photoshop 3.0.5. Maddison, 1992). The resulting phylogenetic tree (Fig. 1B) LiCl and NiCl treatment and embryo manipulation suggests that the duplication events that produced multiple 2 Notch genes in vertebrates occurred in the vertebrate lineage Batches of fertilized eggs were divided into control and treated cultures. Embryos were treated with 30 mM LiCl from the 2-4 cell after the divergence of vertebrates and sea urchins from a stage until the mid-mesenchyme blastula stage; embryos treated with common ancestor. NiCl2 were placed in 1 mM NiCl2 immediately after fertilization until the early gastrula stage (Hardin et al., 1992). After treatment, embryos LvNotch expression and localization were concentrated, rinsed 3× with ASW and cultured in ASW. Treated LvNotch mRNA was present during all stages of sea urchin and untreated embryos were immunostained in parallel. Single embryogenesis (Fig. 2). Low levels of maternal mRNA were embryo manipulations and antibody staining was performed as found in the egg. After fertilization the amount of LvNotch described (McClay and Logan, 1996). mRNA appeared to increase, peaking in abundance at the gastrula stage, and then decreasing to lower levels in the Phylogenetic analysis pluteus larva. Full-length Notch family member protein products were obtained in To determine the temporal and spatial pattern of LvNotch a BLAST search (Version 8, 1994, Genetics Computer Group (GCG), Madison, WI) and sequence alignment was performed using the protein distribution, eight fusion proteins were made, encom- PileUp program of GCG (gap weight: 3.0; length weight: 0.100). passing different regions of LvNotch (Fig. 3A). Specific anti- Ambiguously aligned positions were excluded from phylogenetic bodies were generated to five LvNotch fusion proteins (Bam1, analysis, which produced the same tree using both weighted 3, 4, Pet1, Ank), and were used to examine the expression of parsimony (MACCLADE 3.04, Maddison and Maddison, 1992) and LvNotch (see Materials and Methods). unordered parsimony (PAUP 3.1; Swofford, 1993). Bootstrap values Examination of western blots of gastrula protein extracts were obtained for the unordered parsimony tree using PAUP 3.1 using intracellular (α-Ank), and extracellular (α-Bam1) anti- (Swofford, 1993). bodies revealed prominent immunoreactive bands at 116×103 3 Mr and 260×10 Mr, respectively, as well as additional lower abundance fragments (Fig. 3B). This banding pattern RESULTS suggested that the majority of endogenous LvNotch product is present as two fragments, corresponding roughly in size to the Isolation of sea urchin LvNotch cDNA clones intracellular and extracellular domains. Rapid lysis with the Using degenerate oligonucleotide primers (Stifani et al., 1992), addition of an extensive protease cocktail during protein a single band at the predicted size of 400 bp was amplifed by extraction did not reduce the amount of fragmented LvNotch RT-PCR using staged pools of cDNA from embryos of the sea product (data not shown). Similar complex fragmentation of urchin Lytechinus variegatus. This amplification produced a the Notch receptor has been reported in Drosophila, for C. single PCR fragment homologous to previously isolated Notch elegans GLP-1, and in cell lines expressing vertebrate Notch genes, and was used to screen a mid-gastrula stage cDNA homologs (Fehon et al., 1990; Crittenden et al., 1994; Zagouras library. This led to the identification and the sequencing of et al., 1995). The functional significance of this processing or three cDNA clones, which together make up a 7,646 bp cDNA degradation of Notch is unknown. A developmental western sequence with a predicted open reading frame of 2,531 amino blot showed that LvNotch was fragmented at all stages where acids (Fig. 1A). Based on homology to other Notch genes we it was expressed, and that abundance peaked in the gastrula have named this gene LvNotch. embryo, consistent with the mRNA expression profile (Fig. 3C). While western analysis suggested that LvNotch may exist Deduced amino acid sequence and phylogenetic predominantly as an extracellular and intracellular fragment, analysis of LvNotch whole-mount immunofluorescence using these extracellular- Alignment of the predicted LvNotch protein sequence with and intracellular-specific antibodies revealed no differences in Drosophila Notch demonstrated that LvNotch contains all localization pattern (data not shown). conserved domains shared by Notch proteins (Fig. 1A). Whole-mount immunofluorescent analysis showed that LvNotch has multiple EGF-like repeats, three Notch/Lin-12 LvNotch protein was dynamically expressed at the cellular and repeats, a transmembrane domain, six Ankyrin repeats and a subcellular level after cleavage stages. During cleavage stages, putative PEST domain. However, LvNotch contains only 35 expression was uniform on the surfaces of all blastomeres (Fig. EGF-like repeats, in contrast to the 36 repeats found in 4A). In the early blastula embryo (6 hours in development), 3366 D. R. Sherwood and D. R. McClay A

LvNotch ------MRRTTHSGISIFCLPKVGLFLVVVISTL------TSYGASGTSTCSPNPCNGGTCIPPDTQDGEVTCSCERTHVGDRCQFENPCYHAEICQNDGECEVRFNSDGSNPYFHCNCPLGYELSLCEGEVDNVCKL 126 Drosophila Notch MQSQRSRRRSRAPNTWICFWINKMHAVASLPASLPLLLLTLAFANLPNIVRGTDTALVAASCTSVGCQNGGTCVTQ--LNGKTYCACDSHYVGDYCEHRNPC-NSMRCQNGGTCQVTFR--NGRPGISCKCPLGFDESLCEIAVPNACD- 144 12 3

EEPCQNGGTCRLTTSLWDYECFCTPANTGENCTDDNHCVSNPCLNGAVCT--SSSDGYSCECAQGFQGDTCNNDINECTRPNGPTVCYNGGTCFNQYGGFQCECPLGFTGDQCELVYEPCSPDPCRNGGQCASTGPYTFTCTCQNGFTGE 274 HVTCLNGGTCQLKT-LEEYTCACANGYTGERCETKNLCASSPCRNGATCTALAGSSSFTCSCPPGFTGDTCSYDIEEC----QSNPCKYGGICVNTHGSYQCMCPTGYTGKDCDTKYKPCSPSPCQNAGICRSNG-LSYECKCPKGFEGK 288 45 6

TCELNLNDCTQHQCLNGGTCIDGVNDYTCSCLKEFTGTYCEMDFDECDTAVDP-CFNGGTCSNTYGNFSCICVRGWEGQTCEINKDDCTPNPCQFEGECEDRVASFKCTCPPGRTGLLCHLEDACMSNPCHHTAQCSTSVVDGSFICDCA 423 NCEQNYDDCLGHLCQNGGTCIDGISDYTCRCPPNFTGRFCQDDVDECAQRDHPVCQNGATCTNTHGSYSCICVNGWAGLDCSNNTDDCKQAACFYGATCIDGVGSFYCQCTKGKTGLLCHLDDACTSNPCHADAICDTSPINGSYACSCA 438 78 910

TGYQGFNCSEDIDECSLSMDSICQSGGTCQNFDGGWSCLCSSGFTGSRCETDIDECDDDPCYNGGTCLNKRGGYACICLTGFTGTLCETDINECSSNPCLNGASCFDITGRFECACLAGYTGTTCQVNIDDCQSSPCEN------562 TGYKGVDCSEDIDEC--DQGSPCEHNGICVNTPGSYRCNCSQGFTGPRCETNINECESHPCQNEGSCLDDPGTFRCVCMPGFTGTQCEIDIDECQSNPCLNDGTCHDKINGFKCSCALGFTGARCQINIDDCQSQPCRNRGICHDSIAGY 586 11 12 13 14

------GGTCIDGVNQFTCLCETGYEGHRCEMDSDECASRPCMNGGVCEDLIGFYQCNCPVGTSGDNCEYNHYDCSSNPCVNDGTCVDGINEYTCMCHEGYRGLNCEEDIDDCESRPCHNGGTCVDEVNG 686 SCECPPGYTGTSCEININDCDSNPCHRGKCIDDVNSFKCLCDPGYTGYICQKQINECESNPCQFDGHCQDRVGSYYCQCQAGTSGKNCEVNVNECHSNPCNNGATCIDGINSYKCQCVPGFTGQHCEKNVDECISSPCANNGVCIDQVNG 736 15 16 17 18 EGF-like YHCLCPIGYHDPFCMSNINECSSNPCVNGGSCHDGVNEYSCECMAGYTGTRCTDDFDECSSNPCQHGGTCDNRHAFYNCTCQAGYTGLNCEVNIDDCVDEPCLNGGICIDEVNSFQCVCPQTFVGLLCETERSPCEDNQCQNGATCVYSE 836 repeats YKCECPRGFYDAHCLSDVDECASNPCVNEGRCEDGINEFICHCPPGYTGKRCELDIDECSSNPCQHGGTCYDKLNAFSCQCMPGYTGQKCETNIDDCVTNPCGNGGTCIDKVNGYKCVCKVPFTGRDCESKMDPCARNRCKNEAKCTPSS 886 19 20 21 22

DYAGYSCRCTSGFQGNFCDDDRNEC-LFSPCRNGGSCTNLEGSFECSCLPGYDGPICEINIDECASGPCTNGGICTDLIDDYFCSCQRGFTGKNCQNDTDECLSSPCRNGATCHEYVDSYTCSCLVGFSGMHCEINDQDCTTSSCLYGGT 985 NFLDFSCTCKLGYTGRYCDEDIDECSLSSPCRNGASCLNVPGSYRCLCTKGYEGRDCAINTDDCASFPCQNGRTCLDGIGDYSCLCVDGFDGKHCETDINECLSQPCQNGATCSQYVNSYTCTCPLGFSGINCQTNDEDCTESSCLNGGS 1036 23 24 25 26

CIDGVNSYTCECVTGYTGSNCQIEINECDSDPCENGATCQDRFGSYSCHCDVGFTGLNCEHVVQWCSPQNNPCYNGATCVAMGHLYECHCASNWIGKLCDVPKVSCDIAASDKNVTRSELCLNGGTCIDATSSHSCLCQDGYTGSYCEVN 1135 CIDGINGYNCSCLAGYSGANCQYKLNKCDSNPCLNGATCHEQNNEYTCHCPSGFTGKQCSEYVDWCG--QSPCENGATCSQMKHQFSCKCSAGWTGKLCDVQTISCQDAADRKGLSLRQLC-NNGTCKDYGNSHVCYCSQGYAGSYCQKE 1183 27 28 29

IDECASAPCHNGGTCTDGVYSYTCSCLPGFEGPRCQQNINECASSPCHNGGQCHDMVNGYTCSCPAGTQGTDCSINLDDCYEGACYHGGVCIDQVGTYTCDCPLGFVGQHCEGDVNECLSNPCDPVGSQDCVQLINNYQCVCKPGYTGQD 1285 IDECQSQPCQNGGTCRDLIGAYECQCRQGFQGQNCELNIDDCAPNPCQNGGTCHDRVMNFSCSCPPGTMGIICEINKDDCKPGACHNNGSCIDRVGGFECVCQPGFVGARCEGDINECLSNPCSNAGTLDCVQLVNNYHCNCRPGHMGRH 1333 30 31 32 33

CEQEIPNCQNDPCQNNGLCLPSDEGYYCDCLRGFTGVHCETKLTPCGTHPCQNEGTCMEYGDDFDDYTCMCPSGVSGDNCEID-YNECASSPCINGGTCLDEYGQYRCDCPATWNGRNCHLFD---PTFAGGIGMDPEIPTEPPVTIPIT 1431 CEHKVDFCAQSPCQNGGNCNIRQSGHHCICNNGFYGKNCELSGQDCDSNPCR-VGNCVVADEGF-GYRCECPRGTLGEHCEIDTLDECSPNPCAQGAACEDLLGDYECLCPSKWKGKRCDIYDANYPGWNGGSGSGND------R 1470 34 35 36

VATTTEPPRC---SQECLRKKGDYFCDEECNTHLCEWDGKDCSLNLDPWENCTAANVPCWNRFGDGSCDRECNNHGCLFDGFDCVDQKPEECGE--DAFCLERYGNGFCDEECNNIGCLYDGLDCE---ENPEFAHGVLIVDVMIPYAEM 1573 Notch/Lin-12 YAADLEQQRAMCDKRGCTEKQGNGICDSDCNTYACNFDGNDCSLGINPWANCTAN--ECWNKFKNGKCNEECNNAACHYDGHDC-ERKLKSCDTLFDAYCQKHYGDGFCDYGCNNAECSWDGLDCENKTQSPVLAEGAMSV-VMLMNVEA 1616 repeats 123

LTLNRSQMFLQDMSVVLNTVVLFAPTADNQNKIEPWENDPRAVRAPVRRDVLAVSREGKLTVKKRQAELTPDDGTKCFLLLDNYKCHQDRPGNCFETAADAAAYLAALVTSQLLPTSLPITGVGSETTPAQ----TVSPSVLIAVAIIGA 1719 Transmembrane FREIQAQ-FLRNMSHMLRTTVRLKKDALGHDIIINWKDN---VRVPEIEDT-DFARKNKILY----TQQVHQTGIQIYLEIDNRKC-----TECFTHAVEAAEFLAATAAKHQLRNDFQIHSVRGIKNPGDEDNGEPPANVKYVITGIIL 1752 domain

FVIAAAC---VIARKRFRTNG-TWFPSNFVRTSSTKSENPKVPRECPIGNESPPSTICSSIDAGEDAM---NALECRAPKRQRISRLSPVDYGQ------GHTTL----DTDTPWMTMTSNGPLHPSMALTPPRDQEPPF--ITNKGPDG 1850 VIIALAFFGMVLSTQRKRAHGVTWFPEGFRAPAAVMSRRRRDPHGQEMRNLNKQVAMQSQGVGQPGAHWSDDESDMPLPKRQRSDPVSGVGLGNNGGYASDHTMVSEYEEADQRVWSQAHLDVVDVRAIMTPPAHQDGGKHDVDARGPCG 1902 1Ankyrin repeats LTPLMLAVMRSKASITDQE----EETHAHFIEDLITRGGDINHRTETRGETALHLAARYNLPVAARKLLEYSVNTNAEDNMGETPLHAAVRADAIEVFRLLIQNRSTQIDAKTKAGYTPMIIAVRLMVENMVEELELAGADTAATDNDGR 1996 LTPLMIAAVRGGGLDTGEDIENNEDSTAQVISDLLAQGAELNATMDKTGETSLHLAARFARADAAKRLFHAGADANCQDNTGRTPLHAAVAADAMGVFQILLRNRATNLNARMHDGTTPLILAARLAIEGMVEDLITADADINAADNSGK 2052 2345

SALMWAAALNHLPALEFLLKKGSNKDLQDNQEQTPLFLAAKEGHVGAVKMLLDHGANREITDHLDMSPREIANRMSLQDVVRLLDTYSMMSP---ALTPNSHVTSP------MDMHNGELGYSNGAKPKKKRGTNKNGDVRERGKEKR 2135 TALHWAAAVNNTEAVNILLMHHANRDAQDDKDETPLFLAAREGSYEACKALLDNFANREITDHMDRLPRDVASERLHHDIVRLLDEHVPRSPQMLSMTPQAMIGSPPPGQQQPQLITQPTVISAGNGGNNGNGNASGKQSNQTAKQKAAK 2202 6

KGKKRPSSDE------SLPPLVPSPPESVESP-----QGYNRTPPPGGIYSLPVTTQNVMMPQD------HYQLKEVARHGQGPHVVDMHPNT-IMNGRLYVPGNNPSPAKGSMGGENMDI---- 2238 KAKLIEGSPDNGLDATGSLRRKASSKKTSAASKKAANLNGLNPGQLTGGVSGVPGVPPTNSAVQAAAAAAAAVAAMSHELEGSPVGVGMGGNLPSPYDTSSMYSNAMAAPLANGNPNTGAKQPPSYEDCIKNAQSMQSLQGNGLDMIKLD 2352

----PGASDLVSQTIWSPDHALLQN-QRSMHDPNVMVPGTTSQAMAMIRQAPSTQSMQQTLSPEARDSSPKPCTMVPSPTISNQYIMASSPNLPSTTSQAT------HYARANNPLYAGSPHRGKVPQQIH 2358 NYAYSMGSPFQQELLNGQGLGMNGNGQRNGVGPGVLPGGLC-GMGGLSGAGNGNSREQGLSPPYSNQSP-PHSVQSSLALSPHAYLGSPSPAKSLPSLPTSPTHIQAMRHATQQKQFGGSNLNSLLGGANGGGVVGGGGGGGGGVGQGPQ 2500

SMYQSQNRMNPRACISELPNGHPNSPENC--MQEYMIREPELLNPAKQSMYVVNGKVPNGQHRDILSEKTSFS------NPHNNNGSTVDPSCTYTMV------AHMPDH------2454 NSPVSLGIISPTGSDMGIMLAPPQSSKNSAIMQTISPQQQQQQQQQQQQQHQQQQQQQQQQQQQQQQQLGGLEFGSAGLDLNGFCGSPDSFHSGQMNPPSIQSSMSGSSPSTNMLSPSSQHNQQAFYQYLTPSSQHSGGHTPQHLVQTLD 2650

SFPTPSPESPSKWSNPSPTSASDWSGSEAISSPPVPPNQQQVSGQELMNGHTGRLLTGHDMPLGISLQGSCTEVAYI 2531 PEST SYPTPSPESPGHWSSSSPRSNSDW--SEGVQS--PAANNLYISGGHQANKGSEAIYI...... 2703 domain

B Mouse1 100 10% 100 Rat1

Notch1 Fig. 1. (A) An alignment of the deduced amino acid sequence of LvNotch 100 Human (Tan1)

(GenBank # AF00634) and Drosophila Notch (for example, see Wharton 100 et al., 1985) proteins using the BestFit program (GCG; gap weight, 3.0, Xenopus1 length weight, 0.001). Identical amino acids are shaded. Positions of the 83 ﬁrst amino acid residues in EGF-repeats, Notch/Lin-12 repeats and Zebrafish

Ankyrin repeats of Drosophila Notch are indicated by number (Coffman 71 Rat2 Notch2 et al., 1990). Conserved structural domains are outlined. (B) Parsimony analysis of LvNotch and the four vertebrate Notch family members 81 Mouse3 Notch3 indicates that the vertebrate Notch proteins are more closely related to each other than to LvNotch. Drosophila Notch was used to root the tree. Mouse4 Notch4 Bootstrap values from 550 pseudoreplicates are indicated above the nodes (PAUP 3.1; Swofford, 1993). Scale bar indicates the number of amino LvUrchin Notch acid substitutions along a given branch length (calculated by MACCLADE; Maddison and Maddison, 1992). Drosophila Notch Localization and regulation of sea urchin Notch 3367

micromere descendants at the center of the vegetal plate (data not shown). At this same time, LvNotch, which previously was localized predominantly basolaterally in cells of the animal half of the embryo, was now distributed uniformly in cell membranes. Examination of cell surfaces in the early mesenchyme blastula vegetal plate revealed a slight upregulation Fig. 2. Developmental northern blot of LvNotch expression. The of LvNotch along the apical surface of cells bordering the + amount of poly(A) RNA was 3 µg/lane, calculated by OD260 (left) central vegetal plate (Fig. 4D). Midway through the mes- or estimated by isolation from the same number of embryos (right). enchyme blastula stage (approx. 11.5 hours) a subset of cells E, egg; 7th, 7th cleavage; TVB, thickened vegetal plate blastula; EG, began to express high levels of apical LvNotch in a striking early gastrula; LG, late gastrula; Pr, prism; Pl, pluteus larva. cell-by-cell manner (Fig. 4D,E). By the end of this stage (12- 12.5 hours), cells with high levels of apical LvNotch formed an asymmetric ring, with one side of the ring having both loss of LvNotch expression was seen in a sector of the embryo increased levels of apical expression and more cells express- that later becomes the center of the vegetal plate (Fig. 4B). An ing apical LvNotch (Fig. 4F). In the mid-gastrula (14 hours), increase in basolateral membrane staining was observed at this high levels of apical LvNotch expression were maintained in time in cells that continued to express LvNotch. This pattern cells now identiﬁable as presumptive endoderm cells, while the of expression persisted through the thickened vegetal plate SMCs at the tip of the archenteron lacked detectable LvNotch stage (10 hours), when the area lacking detectable LvNotch expression (Fig. 4H). By the late gastrula (16 hours), when was identiﬁed as the central thickened vegetal plate (Fig. 4C). most of the SMCs have migrated away from the tip of the Only 1 to 2 hours later (mid to late-mesenchyme blastula archenteron, apical LvNotch extended to the tip of this stage), LvNotch was strongly upregulated on the apical surface structure (Fig. 4I). Notably, throughout most of gastrulation the of cells surrounding the central region of the vegetal plate highest levels of apical LvNotch were distributed along one (referred to as apical LvNotch; Fig. 4G). Low levels were also side of the archenteron (Fig. 4H, inset; Fig. 4I), except in the observed inconsistently in the cytoplasm of the eight small last presumptive endoderm cells to invaginate, where LvNotch

100 aa

*Pet 1 2s *Bam 4 *Ank Pet 2 *Bam 3 *Bam 1 Clone B

hydrophobic sequences Ankyrin repeats

Pest sequence EGF-like repeats

Notch/Lin-12 repeats

Fig. 3. (A) Schematic diagram of LvNotch. Regions to which eight fusion proteins were made are indicated. * marks fusion proteins that produced specific pAbs. (B,C) Western analysis of protein extracts using intracellular-directed rabbit α-Ank and extracellular-directed guinea pig α-Bam1 pAbs. (B) Long exposure of gastrula protein extracts (200 embryos/lane); arrow indicates what is presumably full length 3 LvNotch at 350×10 Mr, and arrowheads the predominant intracellular and 3 3 extracellular fragments of LvNotch at 116×10 Mr and 260×10 Mr, respectively. (C) Developmental western analysis of protein extracts (200 embryos/lane) shows that fragmentation of LvNotch occurs at all stages where LvNotch is expressed (arrowheads). Affinity-purified antibodies from two separate animals for both fusion proteins yielded identical banding patterns, while pre-immune serum gave nonspecific, weaker banding. * denotes yolk protein, which cross-reacts with α-Bam1 pAb. 16, 16- cell stage; other abbreviations as in Fig. 2. 3368 D. R. Sherwood and D. R. McClay

Fig. 4. LvNotch displays dynamic cellular and subcellular distribution during sea urchin development. (A) LvNotch is expressed uniformly along all cell surfaces in the 60-cell- stage embryo. (B) In the early blastula, LvNotch is downregulated in a sector of the embryo (between arrowheads) and becomes concentrated along basolateral membranes of cells. (C) In the thickened vegetal plate stage, LvNotch maintains a concentration along basolateral membranes of cells, and the region of LvNotch downregulation is identifiable as the thickened vegetal plate (vp). (D-F) Surface views of mesenchyme blastula-stage embryos. (D) The vegetal plate of a mid-mesenchyme blastula embryo shows a narrow ring of cells expressing slightly increased levels of apically localized LvNotch and a single cell expressing higher levels. (E) In a slightly older embryo (side view), more cells express apical LvNotch. (F) In the late mesenchyme blastula embryo, vegetal plate cells express apical LvNotch in an asymmetric ring. (G) A cross-section of a late mesenchyme blastula embryo shows that apical LvNotch (arrow) extends into cells in the center of the vegetal plate, beyond the limits of cells in which LvNotch is expressed basolaterally (arrowhead). In contrast to the vegetal plate, the cells of the animal half of the embyro express LvNotch at lower levels with a nonpolar distribution in membranes. (H) In the mid-gastrula stage, high levels of apical LvNotch expression are found in the presumptive endoderm while expression is absent in SMCs at the tip of the archenteron (arrow). A cross-section of the archenteron at the level of the presumptive endoderm (inset) shows that the asymmetic distribution of apical LvNotch continues into the archenteron. (I) LvNotch expression extends to the end of the archenteron in the late gastrula, and continues to be asymmetrically distributed along the sides. Bars: 25 µm figures; 10 µm inset. was expressed at high levels in a symmetric manner (data not thus speculated that the expression of apical LvNotch might shown). correlate with the formation of the ZA. Previous electron micrograph studies have indicated that the ZA forms during the Relationship of apical LvNotch expression to the early blastula stage in the sea urchin embryo (Spiegel and zonula adherens junction and to developmental Howard, 1983). To directly examine the localization of compartments LvNotch in relation to the ZA, embryos were double stained The striking appearance and specific localization of apical for LvNotch and sea urchin Lv-cadherin and Lvβ-catenin LvNotch in vegetal plate cells of the mesenchyme blastula proteins (conserved components of the ZA; Kemler, 1993). The embryo suggested that this specific distribution may have a ZA was present between all cells in the mid-blastula stage functional significance. As a first step towards understanding embryo (9 hours in development), a time when LvNotch was possible functions for this localization pattern, we undertook a localized primarily in basolateral membranes of cells (Fig. detailed analysis of the expression of apical LvNotch in 5A,B). In the early gastrula (13 hours), cells that expressed relation to cellular and developmental compartments. apical LvNotch had LvNotch colocalized with the ZA, as well as over the apical surface (Fig. 5C,D). These results demon- The appearance of apical LvNotch does not correlate strate that the apical shift in LvNotch localization that occurs with the formation of the zonula adherens junction in the mesenchyme blastula embryo is neither correlated with In Drosophila, Notch also undergoes changes in subcellular the formation of the ZA, nor restricted to this junction. distribution during development, from a lateral membrane distribution in early embryonic epidermal cells to a close associ- Apical LvNotch is coincident with the presumptive SMC- ation with the zonula adherens (ZA) junctions in embryonic endoderm boundary hindgut cells and imaginal epithelia (Fehon et al., 1991). We Apical LvNotch in the mid-gastrula embryo appeared to be Localization and regulation of sea urchin Notch 3369

Table 1. Numbers and asymmetry of cells expressing apical LvNotch in the mesenchyme blastula stage vegetal plate Number of cells Number of cells with apical lacking LvNotch Asymmetry of Treatment* n LvNotch† in center of VP† LvNotch ring‡ Untreated Late MB 20 128.5±4.3 75.5±1.6 1.8 Vegetalized (LiCl treated)¤ Untreated Mid-Late MB 12 71.8±1.2a 81.5±4.6a 2.1 LiCl treated Mid-Late MB 12 129.7±7.0b 97.75±2.7b 1.5 Ventralized (NiCl2 treated) Untreated Late MB 9 112.8±3.8a 76.6±3.2a 1.8 b a NiCl2 treated Late MB 8 80±5.5 74.4±1.8 1.4

*Each treatment was performed on a different batch of embryos. Similar results were observed, but not quantiﬁed, on a least two additional batches of embryos for each treatment. †Means ± SEM followed by the same letter within a treatment in this column are not signiﬁcantly different (P>0.05; by ANOVA). ‡Asymmetry of apical LvNotch was determined by calculating the average number of cells expressing apical LvNotch in a 6-cell wide group of cells on the side of the ring with the lowest expression, and dividing this into the average number of cells in a similar swath on the directly opposing side. ¤This batch of embryos was sampled 20-30 minutes earlier in the mesenchyme blastula stage than the other treatment groups, and as a result the apical LvNotch ring was examined earlier in its formation. This timing difference accounts for the fewer number of cells expressing apical LvNotch in the untreated condition. Abbreviations: MB, mesenchyme blastula; VP, vegetal plate.

restricted to the presumptive endoderm and excluded from All of these embyros (19/19) had higher levels of apical most, if not all, SMCs (Fig. 4H). In addition, the absence of LvNotch along the dorsal side of the presumptive endoderm expression in the center of the vegetal plate of the mesenchyme (Fig. 6C). Apical LvNotch thus both delineates a boundary blastula embryo suggested that apical LvNotch may be specifically excluded from the presumptive SMCs even before invagination begins (Fig. 4G). We therefore asked if the apical expression was indeed coincident with the SMC-endoderm boundary in the mesenchyme blastula vegetal plate by comparing the number of cells either expressing or lacking apical LvNotch to a fate-map of cells of this stage (Fig. 6A,C; Ruffins and Ettensohn, 1996). The number of cells lacking apical LvNotch expression in the center of the vegetal plate correlated almost exactly with the number of presumptive mesoderm cells fate-mapped to this position, 75.5 versus 74 (66 SMC precursors and 8 small micromere descendants), respectively (Table 1). The cells (roughly 130) that express apical LvNotch were therefore coincident with the fate map’s 155 endodermal precursor cells that surround the presumptive mesoderm cells. Levels of apical LvNotch expression are differentially localized along the dorsoventral axis of the presumptive endoderm The expression of high levels of apical LvNotch along one side of the presumptive endoderm led us to examine whether this polarity correlated with the dorsoventral axis of the embryo. This was achieved by comparing the position of the asymmetric ring of apical LvNotch in the early gastrula embryo to the orientation of the ventrolateral clusters of primary mes- Fig. 5. Apical shifts in LvNotch subcellular localization do not enchyme cells (PMCs), one of the ﬁrst morphological features correlate with the formation of the ZA. (A-D) Embryos were double- distinguishing the ventral from the dorsal side of the embryo. labeled with guinea pig α-Lv-cadherin pAb and mouse α-LvNotch Double staining with a PMC-speciﬁc monoclonal antibody, pAb. (A, B) In the post-hatch blastula embryo, LvNotch (A) 1G8, and LvNotch showed that 86% (12/14 embryos) of the predominates in the basolateral membranes of cells. LvG-cadherin (B) is concentrated in the ZA, which in cross-section appears as LvNotch apical rings examined had a clear dorsal polarity bright, apical points between cells. (C,D) In the early gastrula while the remaining embryos appeared to have either embryo, cells that express high levels of apical LvNotch (C) have symmetric (1/14) or lateral concentrations of apical LvNotch LvNotch distributed over the apical surface (inset), as well as co- (1/14; Fig. 6B). To determine if this asymmetry was main- localized to the ZA (arrowheads). Double staining for Lvβ-catenin tained in the presumptive endoderm during vegetal plate and LvNotch produced identical results (data not shown). Bars, 25 invagination, late-gastrula embryos were similarly evaluated. µm (10 µm in insets). 3370 D. R. Sherwood and D. R. McClay

conversion (Fig. 7A). Immediately after removing the presumptive SMCs, apical LvNotch extended to the tip of the archenteron (12/14 embryos; Fig. 7B,C). In embryos allowed to recover for only 1.5 hours, however, apical LvNotch was no longer present at the tip of the archenteron in 88% of embryos examined (Fig. 7D; 14/16). Apical LvNotch thus appears to be rapidly downregulated in response to the lineage conversion of presumptive endoderm to SMCs. Perturbations of the animal/vegetal and dorsoventral axes cause specific alterations in apical LvNotch expression The expression of apical LvNotch along the presumptive SMC- endoderm boundary and increased expression on the dorsal side of the vegetal plate suggested that this specific pattern might be tied to both the animal/vegetal and dorsoventral axes of the embryo. To test this possibility, embryos were first treated with lithium chloride (LiCl), which increases the amount of vegetally derived tissues (Horstadius, 1973). LiCl treatment caused an expansion in the number of presumptive SMCs that lacked LvNotch and in the number of presumptive endoderm cells that express apical LvNotch in mesenchyme blastula embryos (Fig. 8A,B): the number of cells that lacked Fig. 6. Apical LvNotch expression in the mesenchyme blastula detectable LvNotch expression in the center of the vegetal plate vegetal plate delineates the presumptive SMC-endoderm boundary increased 36% (P<0.05), and the number of surrounding cells and has a dorsal bias. (A) Cells expressing or lacking apical LvNotch that expressed apical LvNotch increased 60% (P<0.05; Table in the vegetal plate were quantified (Table 1) by double-labeling α β 1). LiCl also reduced the asymmetry of apical LvNotch, con- embryos with guinea pig -Lv -catenin pAb (red), which outlines sistent with its less well-characterized, but known radializing the apical sides of epithelial cells, and mouse α-LvNotch pAb (blue). (B,C) Embryos double-stained with 1G8 mAb (green) specific for effect (Horstadius, 1973; Table 1). The expansion of cells PMCs and guinea pig α-LvNotch pAb (blue). The bias of apical lacking and expressing apical LvNotch in LiCl-treated LvNotch expression is towards the dorsal side of the embryo, both in embryos provides further evidence that apical LvNotch is the vegetal plate of the early gastrula (B) and in the presumptive specifically expressed in the presumptive endoderm and is endoderm of the archenteron in the late gastrula (C). (D) A schematic excluded from the presumptive SMCs. map of apical LvNotch expression (blue) on the mesenchyme To determine if the dorsal bias in LvNotch expression blastula vegetal plate was inferred by combining the above results, changes with experimental perturbation of the dorsoventral (A-C; Table1) and the mesenchyme blastula fate map (Ruffins and axis, this axis was disrupted by treatment with NiCl2, which Ettensohn, 1996). The number of cells lacking LvNotch expression increases ventral ectoderm at the expense of dorsal ectoderm, in the center of the vegetal plate is shown in parenthesis next to the and alters the patterning of mesodermal structures associated estimated number of presumptive mesoderm cells, and the number of cells expressing apical LvNotch is similarly shown next to the with the endoderm and ectoderm (Hardin et al., 1992). NiCl2 estimated number of presumptive endoderm cells. Note: the fate map treatment did not affect the number of cells lacking LvNotch of Ruffins and Ettensohn (1996) did not determine the outer expression in the center of the mesenchyme blastula vegetal boundary of the presumptive endoderm in relation to the dorsoventral plate (P>0.05; Table 1). However, disruption of the dorsoven- axis. Therefore cell counts cannot place it at this boundary. Based on tral axis did cause a marked reduction in the overall intensity, the restriction of high levels of apical LvNotch expression to the asymmetry and number of cells expressing apical LvNotch in presumptive endoderm in the late gastrula, we draw it at the edge of the vegetal plate (28% fewer cells; P<0.05; Table 1; Fig. 9A,B). the presumptive endoderm boundary in (D). This decrease in apical LvNotch continued through most of the gastrula stage (Fig. 9C,D); protein extractions revealed that mid-gastrula embryos treated with NiCl2 had an approximately between the presumptive endoderm and mesoderm of the 30% decrease in LvNotch protein (Fig. 9E). Thus, the presence vegetal plate, and has a consistent dorsal polarity (Fig. 6D). of a dorsoventral axis appears to be required for the asymmetric expression of apical LvNotch. A rapid downregulation of apical LvNotch accompanies conversion of endoderm cells to SMCs Previous studies have shown that, after microsurgical removal DISCUSSION of the presumptive SMCs early in gastrulation, a new SMC population is established via conversion of presumptive Identification of a sea urchin Notch endoderm cells to SMCs at the tip of the recovering archen- We report the identification of the first echinoderm Notch gene teron (McClay and Logan, 1996). To confirm the association LvNotch, isolated from the sea urchin Lytechinus variegatus. of apical LvNotch with the presumptive SMC-endoderm The deduced amino acid sequence of LvNotch shares signifi- boundary, we repeated this experiment and asked whether cant homology to Drosophila and vertebrate Notch proteins apical LvNotch expression was altered during this cell lineage (see Artavanis-Tsakonas, 1995; Uyttendaele et al., 1996). A Localization and regulation of sea urchin Notch 3371

Fig. 7. Sequence of SMC removal, recovery and LvNotch apical expression in early gastrula embryos. (A) Diagram illustrating SMC removal via micropipet. (B) Prior to surgery, apical LvNotch is absent in the tip of the archenteron. (C) Immediately after surgery, apical LvNotch extends to the tip of the archenteron (arrowhead), but is rapidly downregulated by 1.5 hours later (D) and a new boundary of apically expressed LvNotch is established (arrow). Bar, 25 µM. phylogenetic analysis of the relationship of LvNotch to ver- common ancestor. Interestingly, EGF-repeat 14 is the same tebrate Notch genes suggests that the duplication of Notch repeat in which the split mutation in Drosophila Notch is genes in vertebrates occurred after the divergence of sea found; a mutation that results in an eye-speciﬁc phenotype urchins and vertebrates from a common ancestor, which is consistent with the proposed scenario of two main phases of gene duplication early in the vertebrate lineage (Holland et al., 1994). This analysis also implies that a single Notch gene may be present in the sea urchin genome, as appears to be the case in Drosophila. To date we have neither isolated additional sea urchin Notch genes with the degenerate primers and PCR search nor detected other cross-reacting Notch proteins with the extensive polyclonal antibodies we have generated. These data are consistent with the presence of a single Notch gene in sea urchins, but do not rule out the possiblity that additional Notch genes have been created by independent duplication events within the sea urchin lineage. The most distinctive difference of LvNotch to other family members is the absence of EGF-repeat 14. Since two of the four known vertebrate Notch proteins contain the full comple- ment of 36 EGF-repeats found in Drosophila Notch, the loss of EGF-repeat 14 most likely occurred in the sea urchin lineage after the divergence of sea urchins and vertebrates from a

Fig. 9. NiCl2 decreases the asymmetry and the amount of apical LvNotch expression in the vegetal plate and archenteron. Compare surface views of untreated (A) and NiCl2-treated (B) vegetal plates of mesenchyme blastula embryos stained for LvNotch, as well as sections of untreated (C) and NiCl2-treated (D) late gastula archenterons. (E) Quantitative western analysis of protein extracts (200 embryos/lane) reveals a 28±5.3% s.e.m. decrease in LvNotch protein abundance in NiCl2-treated mid-gastrula embryos. The × 3 Fig. 8. The apical LvNotch ring and cells within this ring are abundance of the intracellular fragment (116 10 Mr) recognized by expanded in mesenchyme blastula embryos treated with LiCl. the rabbit α-Ank pAb was measured, and three different extracts Untreated (A) or LiCl-treated (B) were double-labeled for guinea from two separate trials within the linear range of exposure were pig α-Lvβ-catenin pAb (red) and mouse α-LvNotch pAb (blue). analyzed (Image QuaNT; Molecular Dynamics). Bars, 25 µm (A,B); Bar, 25 µm. 10 µm (C,D). 3372 D. R. Sherwood and D. R. McClay

(Hartley et al., 1987; Kelley et al., 1987). While it is tempting expression of LvNotch could retain much of the presumptive to speculate that a specific function(s) of EGF-repeat 14 endoderm in an uncommitted state. Consistent with this notion necessary for eye development in Drosophila has been lost and is the substantial regulative capacity of the developing is not required in the sea urchin, it has been shown that two endoderm throughout gastrulation, indicated by experiments in Notch-like proteins in C. elegans with different numbers of which removal of the presumptive SMCs in the early gastrula EGF-like repeats are redundant in embryonic development and results in a recovery of this mesodermal population by a group functionally interchangeable (Lambie and Kimble, 1991; of presumptive endoderm cells switching to an SMC fate Fitzgerald et al., 1993). Therefore, loss of EGF-repeat 14 in (McClay and Logan, 1996). The rapid loss of LvNotch sea urchin may not necessarily affect the range of interactions expression at the tip of the recovering archenteron from which that LvNotch participates in during development. the SMCs have been removed suggests that presumptive endoderm cells that switch to an SMC fate rapidly downregu- LvNotch expression has an early polarity along the late apical LvNotch expression. It is thus possible that Notch animal-vegetal axis and reveals regionalization of signaling, by maintaining much of the presumptive endoderm the mesenchyme blastula vegetal plate in an uncommitted state, normally prevents these cells from The earliest differential pattern of LvNotch protein expression adopting a mesodermal fate. that we detected was the absence of LvNotch protein in the While the ventral bend of the pluteus larval endoderm and early blastula vegetal pole, and localization to primarily baso- bilateral symmetry of associated mesodermal structures is con- lateral membranes in cells throughout the animal half of the sistent with the sea urchin endoderm containing dorsoventral embryo. This cellular pattern of expression is similar to the polarity, the bias in LvNotch expression is the first reported mRNA localization of three genes (VEB genes; Reynolds et evidence that a specific dorsoventral molecular asymmetry al., 1992). Perturbation experiments with one of the VEB gene exists in the endoderm of the sea urchin. The Notch pathway products, BP10/SpAN, which is a secreted metalloprotease and provides a potential mechanism for establishing dorsoventral homolog of the human BMP-1 and Drosophila tolloid protein, polarity in this structure. Consistent with this notion is that has demonstrated a role for this protein in the patterning of the NiCl2, which causes the loss of the apparent morphological sea urchin embryo along the animal-vegetal axis (Lepage et al., dorsoventral polarity in the endoderm (Hardin et al., 1992), 1992). It is therefore possible that LvNotch may similarly also results in a reduction or loss of the LvNotch molecular mediate cell-cell interactions important in the specification of asymmetry. Involvement of the Notch pathway in axial organ- cell-types along the early animal-vegetal axis. ization would not be unprecedented, as it is essential for Lineage analysis has demonstrated that the differential spec- dorsoventral polarity of the Drosophila wing and in the early ification of SMCs and endoderm could occur at the mes- C. elegans embryo (for examples see de Celis et al., 1995; enchyme blastula stage (Ruffins and Ettensohn, 1996). Mello et al., 1994). However, previous studies have not revealed the expression of molecular markers distinguishing these two germ-layers until LvNotch expression and implications for Notch invagination of the vegetal plate has begun during the gastrula signaling stage (Ransick et al., 1993; Harada et al., 1995, 1996; reviewed Several experiments using overexpressed fragments of Notch by Davidson, 1993). Our analysis of the expression of apically in cell lines and embryos have suggested that Notch may be localized LvNotch demonstrates that the SMCs and endoderm activated by translocation of the intracellular domain into the are in fact differentially specified at least by the mid- to late nucleus (Jarriault et al., 1995; Kopan et al., 1996; reviewed in mesenchyme blastula stage, earlier than previous molecular Artavanis-Tsakonas et al., 1995). Similar to observations in studies have indicated. In addition, the dorsal bias in apical Drosophila and C. elegans (Fehon et al., 1991; Crittenden et LvNotch localization and specific change in expression of al., 1994), however, we have not yet detected this domain of apical LvNotch in vegetalized and ventralized embryos estab- LvNotch in the nucleus. While these results do not exclude the lishes that the late mesenchyme blastula vegetal plate is possibility that the intracellular domain acts in the nucleus organized with reference to both the animal/vegetal and during development, they indicate that this domain is either dorsoventral axes of the embryo before invagination of the difficult to detect or rapidly degraded upon entering the vegetal plate begins. The continued polarity of apical LvNotch nucleus. along the dorsal side of the presumptive endoderm during The specific and dynamic distributions of Drosophila Notch invagination, and reduction of this expression by NiCl2 and C. elegans GLP-1 during development have led to the treatment, further shows that: (1) the presumptive endoderm proposal that the activity of the Notch signaling pathway may has a dorsoventral polarity in register with the ectodermal in part depend upon the precise localization of the receptor dorsoventral axis, (2) cells of the presumptive endoderm (Fehon et al., 1991; Crittenden et al., 1994). Our results with maintain a dorsal bias in apical LvNotch expression during the LvNotch are consistent with and extend these findings. We convergent-extension movements that extend the archenteron, show that dynamic cellular distributions of LvNotch to specific and (3) the endoderm may be ventralized by NiCl2 treatment, developmental compartments of the embryo are often accom- much like the ectoderm is. panied by distinct subcellular localizations of LvNotch in apical or basolateral membrane domains. For example, in the Potential roles for the Notch pathway in vegetal early blastula embryo, LvNotch is absent from the vegetal pole plate regionalization but concentrated in basolateral cell membranes throughout the It has been proposed that Notch signaling can maintain cells in animal half of the embryo (Fig. 4C). Later in development, an uncommitted developmental state (reviewed in Artavanis- high levels of LvNotch are specifically localized on the apical Tsakonas et al., 1995). If this is true, the specific apical surface and colocalized with the ZA of cells that are restricted Localization and regulation of sea urchin Notch 3373 to the presumptive endoderm, and the highest levels of apical REFERENCES expression are on the dorsal side of this structure (Fig. 6). We also demonstrate, using known components of the ZA (i.e. Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M. E. (1995). Notch cadherin and β-catenin), that the ZA are present in these epi- signaling. Science 268, 225-32. Bachman, E. S. and McClay, D. R. (1995). Characterization of moesin in the thelial cells prior to these membrane domain changes in sea urchin Lytechinus variegatus: redistribution to the plasma membrane LvNotch localization. Therefore, changes in LvNotch following fertilization is inhibited by cytochalasin B. J. Cell Sci. 108, 161-71. membrane domain localization do not correlate with the Cameron, R. A., Smith, L. C., Britten, R. J. and Davidson, E. H. (1994). formation of the ZA; rather, they appear to be specific to Ligand-dependent stimulation of introduced mammalian brain receptors alters spicule symmetry and other morphogenetic events in sea urchin distinct cellular distributions of LvNotch in developmental embryos. Mech. Dev. 45, 31-47. compartments of the embryo. Coffman, C., Harris, W. and Kintner, C. (1990). Xotch, the Xenopus homolog While the C. elegans Notch-like GLP-1 protein has not been of Drosophila Notch. Science 249, 1438-41. shown to undergo similar subcellular shifts in membrane local- Conlon, R. A., Reaume, A. G. and Rossant, J. (1995). Notch1 is required for ization (Crittenden et al., 1994), Drosophila Notch shifts from the coordinate segmentation of somites. Development 121, 1533-45. Crittenden, S. L., Troemel, E. R., Evans, T. C. and Kimble, J. (1994). GLP- lateral membranes in embryonic epithelia to colocalize with the 1 is localized to the mitotic region of the C. elegans germ line. Development ZA in embryonic hindgut and imaginal epithelia (Fehon et al., 120, 2901-11. 1991). In addition, in the Drosophila ovary, high levels of Davidson, E. H. (1993). Later embryogenesis: regulatory circuitry in Notch are expressed over the apical surface of follicle cells morphogenetic fields. Development 118, 665-90. within specific developmental stage egg-chambers (Xu et al., de Celis, J. F., Garcia-Bellido, A. and Bray, S. J. (1996). Activation and function of Notch at the dorsal-ventral boundary of the wing imaginal disc. 1992). A direct comparison of Drosophila Notch with ZA com- Development 122, 359-69. ponents has not been performed; however, Notch is not con- Fehon, R. G., Kooh, P. J., Rebay, I.,Regan, C. L., Xu, T., Muskavitch, M. A. centrated on the apical surface or colocalized to the ZA in the T., Artavani-Tsakonas, S. (1990). Molecular interactions between the late gastrula embryonic epidermis (Fehon et al., 1991), a time protein products of the neurogenic loci Notch and Delta, two EGF- homologous genes in Drosophila. Cell 61, 523-534. when the ZA is present in these cells (Tepass and Hartenstein, Fehon, R. G., Johansen, K., Rebay, I. and Artavanis-Tsakonas, S. (1991). 1994). Therefore, apical shifts of Drosophila Notch also appear Complex cellular and subcellular regulation of Notch expression during to be independent of ZA formation and are likely to be specific embryonic and imaginal development of Drosophila: implications for Notch to developmental compartments of the embryo as well. function. J. Cell Biol. 113, 657-69. Together, these results offer compelling evidence that the Fitzgerald, K., Wilkinson, H. A. and Greenwald, I. (1993). glp-1 can substitute for lin-12 in specifying cell fate decisions in Caenorhabditis specific subcellular localization of Notch proteins in apical and elegans. Development 119, 1019-27. basolateral membrane domains is a conserved and important Goode, S., Melnick, M., Chou, T.-B. and Perrimon, N. (1996). The element in Notch receptor signaling. neurogenic genes egghead and brainiac define a novel signaling pathway Interestingly, a recent study in Drosophila oogensis has essential for epithelial morphogenesis during Drosophila oogenesis. Development 122, 3863-3879. suggested that Notch may mediate distinct functions on apical Harada, Y., Akasaka, K., Shimada, H., Peterson, K. J., Davidson, E. H. and and lateral membrane domains (Goode et al., 1996). Molecular Satoh, N. (1996). Spatial expression of a forkhead homologue in the sea analysis of the Notch receptor has raised the possibility that urchin embryo. Mech. Dev. 60, 163-173. Notch could be acting as a multifunctional receptor capable of Harada, Y., Yasuo, H. and Satoh, N. (1995). A sea urchin homologue of the interacting with numerous ligands (Rebay et al., 1991). Shifts chordate Brachyury (T) gene is expressed in the secondary mesenchyme founder cells. Development 121, 2747-54. in localization of Notch may therefore represent interactions Hardin, J. (1996). The cellular basis of sea urchin gastrulation. In Current with ligands differentially localized at the subcellular level. Topics in Developmental Biology (ed. R. A. Peterson and G. P. Schatten), pp. Alternatively, laterally and apically localized LvNotch could 159-246. Academic Press, Inc, San Diego. be interacting with the same ligands, but generating distinct Hardin, J., Coffman, J. A., Black, S. D. and McClay, D. R. (1992). responses in different membrane domains, as occurs in some Commitment along the dorsoventral axis of the sea urchin embryo is altered in response to NiCl2. Development 116, 671-85. cellular contexts with the vertebrate EGF-receptor (Lund et al., Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold 1996). Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Knowledge of the cellular and subcellular expression of Hartley, D. A., Xu, T. A. and Artavanis-Tsakonas, S. (1987). The embryonic proteins involved in Notch signaling, combined with experi- expression of the Notch locus of Drosophila melanogaster and the implications of point mutations in the extracellular EGF-like domain of the mental manipulation of these components and Notch, will be predicted protein. EMBO J. 6, 3407-17. needed to understand the the full significance and functions Holland, P. W., Garcia-Fernandez, J., Williams, N. A. and Sidow, A. (1994). that Notch may mediate on apical versus basolateral membrane Gene duplications and the origins of vertebrate development. Dev. Suppl. domains. Recent advances in techniques to overexpress 125-33. specific proteins in the sea urchin (Cameron et al., 1994; Mao Horstadius, S. (1973). Experimental Embryology of Echinoderms. Clarendon Press, Oxford. et al., 1996) should allow us to pursue these studies, and enable Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R. and Israel, A. a more direct understanding of the function of this pathway in (1995). Signalling downstream of activated mammalian Notch. Nature 377, the development of the sea urchin embryo. 355-8. Kelley, M. R., Kidd, S., Deutsch, W. A. and Young, M. W. (1987). Mutations We are indebted to Rick Fehon for advice and encouragement altering the structure of epidermal growth factor-like coding sequences at the throughout this work, and thank Cliff Cunningham for his expertise Drosophila Notch locus. Cell 51, 539-48. Kemler, R. (1993). From cadherins to catenins: cytoplasmic protein with the phylogenetic analysis. We thank John Matese for help with interactions and regulation of cell adhesion. Trends Genet. 9, 317-321. figures and Nina Tang Sherwood, Gabrielle Kardon, Phil Hertzler and Kopan, R., Schroeter, E. H., Weintraub, H. and Nye, J. S. (1996). Signal Rebecca Lamb for comments on the manuscript. This work was transduction by activated mNotch: importance of proteolytic processing and supported by NIH grant HD14483 to D. R. M., NSF equipment grant its regulation by the extracellular domain. Proc. Nat. Acad. Sci. USA 93, BIR-9318118, and NIH training grant 5T32GM07184 to D. R. S. 1683-8. 3374 D. R. Sherwood and D. R. McClay

Lambie, E. J. and Kimble, J. (1991). Two homologous regulatory genes, lin- Reynolds S. D., Angerer, L. M., Palis, J., Nasir, A. and Angerer, R. C. 12 and glp-1, have overlapping functions. Development 112, 231-40. (1992). Early mRNAs, spatially restricted along the animal-vegetal axis of Lepage, T., Ghiglione, C. and Gache C. (1992). Spatial and temporal sea urchin embryos, include one encoding a protein related to tolloid and expression pattern during sea urchin embryogensis of a gene coding for a BMP-1. Development 114, 769-86. protease homologous to the human protein BMP-1 and to the product of the Ruffins, S. W. and Ettensohn, C. A. (1996). A fate map of the vegetal plate of Drosophila dorsal-ventral patterning gene tolloid. Development 114, 147- the sea urchin (Lytechinus variegatus) mesenchyme blastula. Development 164. 122, 253-63. Lund, K., Schultheiss, K., Hill, V., Clifton, P., Kuwada, S. and Amsler, K. Smith, D. B. and Johnson, K. S. (1988). Single-step purification of (1996). Epidermal growth factor (EGF) receptors generate distinct responses polypeptides expressed in Escherichia coli as fusions with glutathione S- from different cellular compartments. Mol. Biol. Cell 7, 66a. transferase. Gene 67, 31-40. Maddison, W. P. and Maddison, R. (1992). MACCLADE, Analysis of Spiegel, E. and Howard, L. (1983). Development of cell junctions in sea- Phylogeny and Character Evolution (Sinauer, Sunderland, MA), Version 3.0. urchin embryos. J. Cell Sci. 62, 27-48. Mao, C. A., Wikramanayake, A. H., Gan, L., Chuang, C. K., Summers, R. Stifani, S., Blaumueller, C. M., Redhead, N. J., Hill, R. E. and Artavanis- G. and Klein, W. H. (1996). Altering cell fates in sea urchin embryos by Tsakonas, S. (1992). Human homologs of a Drosophila Enhancer of split overexpressing SpOtx, an orthodenticle-related protein. Development 122, gene product define a novel family of nuclear proteins. Nature Genet. 2, 343. 1489-98. Strathmann, M., Hamilton, B. A., Mayeda, C. A., Simon, M. I., McCartney, B. M. and Fehon, R. G. (1996). Distinct cellular and subcellular Meyerowitz, E. M. and Palazzolo, M. J. (1991). Transposon-facilitated patterns of expression imply distinct functions for the Drosophila DNA sequencing. Proc. Nat. Acad. Sci. USA 88, 1247-1250. homologues of moesin and the neurofibromatosis 2 tumor suppressor, Swofford, D. L. (1993). PAUP, Phylogenetic Analysis Using Parsimony merlin. J. Cell Biol. 133, 843-52. (Illinois Nat. Hist. Survey, Champaign, IL) Version 3.11. McClay, D. R., Cannon, G. W., Wessel, G. M., Fink, R. D. and Marchase, R. Tepass, U. and Hartenstein, V. (1994). The development of cellular junctions B. (1983). Patterns of antigenic expression in early sea urchin development. in the Drosophila embryo. Dev. Biol. 161, 563-96. In Time, Space and Pattern in Embryonic Development (ed. W.R. Jeffries and Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D. and Kitajewski, R. A. Raff), pp. 157-169. Alan R. Liss, Inc, New York. J. (1996). Notch4/int-3, a mammary proto-oncogene, is an endothelial cell- McClay, D. R. and Logan, C. Y. (1996). Regulative capacity of the specific mammalian Notch gene. Development 122, 2251-9. archenteron during gastrulation in the sea urchin. Development 122, 607-16. Wharton, K. A., Johansen, K. M., Xu, T. and Artavanis-Tsakonas, S. Mello, C. C., Draper, B. W. and Priess, J. R. (1994). The maternal genes apx- (1985). Nucleotide sequence from the neurogenic locus Notch implies a gene 1 adn glp-1 and establishment of dorsal-ventral polarity in the early C. product that shares homology with proteins containing EGF-like repeats. elegans embryo. Cell 77, 95-106. Cell 43, 567-81. Miller, J. (1995). Cellular and molecular analysis of morphogenesis in the sea Xu, T., Caron, L. A., Fehon, R. G. and Artavanis-Tsakonas, S. (1992). The urchin. PhD Thesis. Duke University, Durham, NC. involvement of the Notch locus in Drosophila oogenesis. Development 115, Ransick, A., Ernst, S., Britten, R. J. and Davidson, E. H. (1993). Whole 913-22. mount in situ hybridization shows Endo 16 to be a marker for the vegetal Zagouras, P., Stifani, S., Blaumueller, C. M., Carcangiu, M. L. and plate territory in sea urchin embryos. Mech. Dev. 42, 117-24. Artavanis-Tsakonas, S. (1995). Alterations in Notch signaling in neoplastic Rebay, I., Fleming, R. J., Fehon, R. G., Cherbas, L., Cherbas, P. and lesions of the human cervix. Proc. Nat. Acad. Sci. USA 92, 6414-8. Artavanis-Tsakonas, S. (1991). Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67, 687-99. (Accepted 26 June 1997)