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