Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA
*Author for correspondence at present address: Department of Biological Sciences, College of Science, Rochester Institute of Technology, 85 Lomb Memorial Drive, Rochester, NY 14623, USA (e-mail: hxssbi{at}rit.edu)
Accepted 15 January 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Delta, Notch, Sea urchin, Micromere, Induction, Mesoderm, Endoderm
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Notch signaling pathway also plays an important role in mesoderm specification during sea urchin development (Sherwood and McClay, 1997; Sherwood and McClay, 1999
; Sherwood and McClay, 2001
). This conserved pathway controls many cell fate decisions in diverse animal embryos (reviewed by Artavanis-Tsakonis et al., 1999
). In the sea urchin embryo, activation of the Notch signaling pathway causes excess non-skeletogenic mesoderm development, whereas blocking the pathway causes severe deficiencies in the development of all non-skeletogenic mesodermal cell types (Sherwood and McClay, 1999
).
The Notch signaling pathway is normally activated during the blastula stages (Sherwood and McClay, 1999) and the presence of the micromeres is necessary for this activation, suggesting that a signal from the micromeres might directly activate the Notch receptor (Sweet et al., 1999
). Providing further evidence for this idea, a signal from blastula stage micromere descendants (eight through tenth cleavage) is sufficient to activate the Notch protein (McClay et al., 2000
). Eighth cleavage stage micromere descendants have signaling abilities, as micromere derivatives from this stage have a strong potential to induce animal cells to generate an archenteron (Minokawa and Amemiya, 1999
). These studies support the hypothesis that descendants of the micromeres activate the Notch signaling pathway during the blastula stage by providing a signal in the form of a Notch ligand.
The purpose of this study is to examine further the molecular mechanisms of non-skeletogenic mesoderm specification. We cloned and characterized the sea urchin homologue of Delta and found it to be expressed by micromere derivatives during the blastula stage, and by macromere derivatives during later stages. Experiments with chimeric embryos demonstrate that micromere-derived LvDelta is necessary and sufficient for induction of pigment and blastocoelar cells, whereas macromere-derived LvDelta is involved in the development of blastocoelar and muscle cells. In addition, we find that LvDelta expression is sufficient to endow blastomeres with the powerful inductive properties first recognized by Hörstadius the ability to act as an organizing center to coordinate the development of animal cells into a pluteus larva.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell isolation and transplantation
Micromere removal at the 16-cell stage, animal cap isolation at the 8-cell stage, and cell transplantations were performed by hand using a glass needle as described (Sweet et al., 1999).
Antibody staining
Antibody staining was performed as described (Sweet et al., 1999). Primary antibodies included 6a9 (Ettensohn and McClay, 1988
) and 6e10 (Ingersoll, 1993
) (both are markers for skeletogenic mesoderm), SMC2 and SMC1 (a blastocoelar cell marker and a marker specific for prospective secondary mesenchyme cells (SMCs) in the vegetal plate, respectively) (Hodor, 1998
; Sweet et al., 1999
), Endo1 (an endodermal marker) (Wessel and McClay, 1985
), anti-myosin (a muscle marker) (Wessel et al., 1990
), and CAD-1 (an antibody against LvG-cadherin) (Miller and McClay, 1997
). Secondary antibodies included fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM for SMC1, SMC2 and 6a9, Texas Red-conjugated goat anti-mouse IgG for 6e10, and Cy3-conjugated goat anti-guinea pig IgG for CAD-1 (Jackson ImmunoResearch Laboratories, Inc.).
The SMC2 antibody is specific for blastocoelar cells in the pluteus larva; however, antibody staining with SMC2 is often prone to high background staining (Hodor, 1998; Sweet et al., 1999
). To distinguish between SMC2-positive blastocoelar cells and background staining, confocal microscopy was used to image whole larvae stained with SMC2. In each confocal z-section, blastocoelar cells were identified by SMC2 staining, cell morphology and location in the larva, and were counted. The total number of blastocoelar cells for a single larva is the sum of the blastocoelar cells counted from each z-section. Images of SMC2 staining in Figs 5 and 8 correspond to one or two confocal z-sections.
|
|
Northern blotting
Total RNA was isolated from embryos using Trizol. 10 µg of RNA from each developmental stage was loaded onto a 1% agarose formaldehyde gel, separated by electrophoresis and blotted onto Nylon membrane (Roche) using Turboblot (Schleicher and Schuell). A DIG-labeled antisense RNA probe was synthesized from the LvDelta clone (base pairs 1-1927) using a T7 Megascript Kit (Ambion) and was used to probe the RNA blot using DIG Easy Hyb (Roche). The RNA blot was developed using chemiluminescence (Roche).
In situ hybridization
Whole-mount in situ hybridizations were carried out as described previously (Zhu et al., 2001). The same probe used in the northern analysis of LvDelta was used for in situ hybridization.
mRNA injection
The full-length LvDelta clone was linearized with Xho1 and used as a template to generate 5' capped mRNA using the T3 mMessage mMachine kit (Ambion). In addition, mRNA encoding a truncated form of LvDelta was used as a control. Using the Quik Change Site-Directed Mutagenesis kit (Stratagene) a G-to-T point mutation was introduced at nucleotide position 981, which generated an internal stop codon following amino acid 135. This mutation results in the translation of a short, N-terminal fragment of the protein lacking any signaling activity (Henderson et al., 1997). Truncated LvDelta mRNA was generated as described above. mRNA injection solution consisted of 20% glycerol, 0.5% RITC-dextran (Sigma) and 2 µg/µl mRNA. Fertilized eggs were placed in a row onto protamine sulfate-treated Petri dish covers and injected with 45-65 pg mRNA solution using Picospritzer II (General Valve Corporation). Embryos were used for further analysis only if they had four micromeres at the 16-cell stage.
Morpholino injection
Morpholino antisense oligonucleotides were designed against LvDelta sequence (5'-CAAGAAGGCAGTGCAGCCGATTCGT-3'; from 32 to 8 relative to the translational start codon) and produced by GeneTools, LLC. To account for possible nonspecific toxic effects of morpholino solution, a morpholino complementary to the 5'-UTR of the SpAristaless transcript (Zhu et al., 2001) was used as a control. It is presumed that the sequence of SpAristaless is sufficiently divergent from the sequence of LvAristaless mRNA because SpAristaless morpholino causes a phenotypic effect when injected into the eggs of S. purpuratus (C. A. E., unpublished observations) but not when injected into the eggs of L. variegatus. Morpholino injection solution consisted of 3.5 mM morpholino, 20% glycerol and 0.5% RITC-dextran. Fertilized eggs were injected with 22-34 pl of morpholino solution.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The finding that micromere derivatives express LvDelta at the blastula stage is consistent with the hypothesis that LvDelta is the micromere-derived signal that activates the Notch signaling pathway and induces mesoderm. In addition, the expression of LvDelta by macromere derivatives at later stages raises the possibility that the protein has other developmental roles.
Perturbation of LvDelta function affects mesoderm development
To determine whether LvDelta is necessary for mesoderm development, an antisense morpholino oligonucleotide designed to interfere with endogenous LvDelta translation was injected into fertilized eggs. Effects on mesoderm development were assessed by morphology and by staining embryos with a collection of mesoderm-specific antibodies. Mesenchyme blastula-stage embryos were stained with SMC1, a marker for prospective SMCs (Sweet et al., 1999) and pluteus larvae were examined for the differentiation of pigment cells, blastocoelar cells (SMC2 staining) and muscle fibers (anti-myosin staining). To control for the possibility of non-specific effects of the LvDelta morpholino, embryos were injected with the same concentration of an unrelated morpholino designed to block SpAristaless function.
During gastrulation, control embryos produce SMCs at the tip of the archenteron (Fig. 4A). Following the injection of LvDelta morpholino, however, archenteron formation was delayed and gastrula-stage embryos exhibited a very smooth archenteron with few SMCs apparent at the tip (Fig. 4B). In normal late blastula-stage embryos, SMC1 stains prospective mesoderm cells in the vegetal plate (Fig. 4D) (Sweet et al., 1999). SMC1-positive cells were rarely seen in embryos containing LvDelta morpholino (Fig. 4E), however, indicating that mesodermal specification had been affected prior to the start of gastrulation. Fig. 5 (A,D,G) shows examples of the numbers of pigment cells, blastocoelar cells and muscle fibers typically observed in control embryos (quantitative data are presented in Table 1). In embryos injected with LvDelta morpholino, few pigment cells, blastocoelar cells or muscle cells developed (Fig. 5B,E,H) (Table 1). Embryos injected with a control morpholino exhibited nearly normal levels of these mesodermal cell types (Table 1). Overall, embryos injected with LvDelta morpholino closely resembled those overexpressing a dominant negative form of the LvNotch receptor, based both on morphology and the expression of mesodermal markers (Sherwood and McClay, 1999
). This suggests that, like LvNotch, LvDelta plays a critical role in the development of non-skeletogenic mesoderm.
|
|
LvDelta functions differently in micromere and macromere descendants
As LvDelta transcripts are expressed by both micromere and macromere descendants at different times during development, we examined the function of LvDelta in these different cells. Chimeric embryos were generated such that LvDelta function was blocked only in micromere descendants, or only in mesomere and macromere descendants. To block LvDelta function specifically in micromeres, the quartet of micromeres was removed from a normal embryo and replaced with micromeres containing LvDelta morpholino and a lineage tracer (Fig. 6A). The resulting embryos developed few pigment cells and blastocoelar cells compared to sham controls (Fig. 6B; Table 2). The same phenotype is observed following micromere removal (Sweet et al., 1999), supporting the hypothesis that LvDelta is the micromere-derived signal involved in the development of these cell types. Normal levels of muscle fibers developed in these chimeric embryos (Fig. 6C), however, consistent with other evidence that micromere signaling is not required for muscle development (Sweet et al., 1999
).
|
|
LvDelta expression is sufficient for induction of mesoderm and endoderm
The experiments described above demonstrate that micromere-derived LvDelta is necessary for the induction of certain types of mesoderm. To test whether LvDelta is sufficient to induce mesoderm, fertilized eggs were injected with full-length LvDelta mRNA and a lineage tracer, and at the 16-cell stage, single mesomeres expressing LvDelta were transplanted to uninjected hosts from which the micromeres had been removed (Fig. 7A). To serve as a control, parallel transplants were performed using mesomeres expressing the truncated form of LvDelta mRNA. We scored the formation of pigment cells in these embryos because this mesodermal cell type is the most dramatically affected by micromere signaling (Sweet et al., 1999).
|
In the presence of a mesomere expressing the full-length form of LvDelta, embryos developed many pigment cells (Fig. 7D; average 80.0, n=13). The transplanted mesomere gave rise to endoderm (9/13) and coelomic pouches, as well as other mesoderm (11/13; Fig. 7E). As the descendants of the transplanted cell generated mesoderm, it is likely that the Notch signaling pathway was activated within the clone of the original transplanted cell. It is clear, however, that the LvDelta-expressing cells also induced host cells to generate mesoderm, including pigment cells, because many mesodermal cells were not labeled with the RITC-dextran lineage tracer (Fig. 7E). These results indicate that LvDelta is sufficient to induce neighboring macromere derivatives to develop mesoderm.
To explore further whether LvDelta-mediated signaling is sufficient for mesoderm induction, single mesomeres containing full-length or truncated LvDelta mRNA were transplanted to animal caps isolated at the 8-cell stage (Fig. 8A). Animal caps combined with a mesomere expressing truncated LvDelta rarely gastrulated (1/12) and usually formed hollow balls of ectoderm (Fig. 8B) in which the transplanted mesomere contributed to the epithelium (Fig. 8C). In contrast, animal caps recombined with a mesomere expressing full-length LvDelta, usually gastrulated (10/15) and frequently developed into miniature plutei (6/15; Fig. 8D). These plutei contained blastocoelar cells (Fig. 8F), skeletogenic cells (Fig. 8G) and pigment cells (average 4.2 cells; n=15; not shown). In embryos that gastrulated, the descendants of the transplanted mesomere formed endoderm (9/10) and mesoderm (8/10; Fig. 8E). The finding that some derivatives of the LvDelta-expressing mesomere generate mesoderm suggests that Notch signaling is activated within the clone of the transplanted cell. Endoderm and mesoderm also developed from unlabeled host cells (Fig. 8E), indicating that the descendants of the transplanted LvDelta-expressing mesomere induced host cells to generate these cell types.
These blastomere transplantation experiments clearly show that LvDelta-mediated signaling is sufficient to induce macromeres and mesomeres to develop mesoderm and endoderm. Expression of LvDelta is sufficient to endow blastomeres with properties of an organizer; i.e. the ability to function as a signaling center that can coordinate the development of an animal cap into a pluteus larva.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The two sea urchin homologues of Delta described here show 100% amino acid identity in the intracellular domain (ICD). Broader phylogenetic comparisons suggest, however, that the ICD is generally one of the least conserved domains of Delta (Lissemore and Starmer, 1999). The functions of the ICD are unclear and may vary among Delta/Serrate/Lag-2 (DSL) proteins (see Henderson et al., 1997
). There is considerable evidence that soluble, monomeric Delta protein cannot signal and that clustering of the proteins is required for signaling (Varnum-Finney et al., 2000
). In Xenopus and Drosophila, the ICD is essential for Delta function (Chitnis et al., 1995
; Sun and Artavanis-Tsakonas et al., 1996
) and it has been proposed that this domain might function to cluster Delta in the cell membrane. It is also possible that the ICD in Drosophila Delta is important for the endocytosis of Delta, which appears to be a critical part of the signaling process in this organism (Parks et al., 2000
). In C. elegans, although membrane association is required for signaling, the ICD is dispensable (Henderson et al., 1997
). The unusual degree of conservation seen between the SpDelta and LvDelta ICDs suggests that this domain has an important functional role in the sea urchin.
LvDelta is the micromere-derived signal that activates Notch and induces non-skeletogenic mesoderm
Previous studies have shown that a signal from the micromere descendants activates Notch in the central region of the overlying macromere territory and induces mesoderm (Sweet et al., 1999; McClay et al., 2000
). Several studies suggest that the key signaling interaction occurs at the blastula stage, between the eighth and tenth cleavages (Minokawa and Amemiya, 1999
; McClay et al., 2000
) (H. C. Sweet and C. A. Ettensohn, unpublished observations). We find that micromere derivatives express LvDelta during the blastula stage, at the time when these cells are known to provide a mesoderm-inducing signal. Moreover, our morpholino and mRNA overexpression studies show that LvDelta function is both necessary and sufficient for mesoderm induction. The phenotypes of embryos described in this study are strikingly similar to those observed following expression of dominant-negative and activated LvNotch constructs (Sherwood and McClay, 1999
). The effect of blocking LvDelta function in the micromeres closely resembles the effect of removing the micromeres, in that there is a reduction in the number of pigment cells and blastocoelar cells but little or no effect on the development of muscle cells. Taken together, our findings strongly support the hypothesis that LvDelta is the micromere-derived signal that activates the Notch signaling pathway and results in the development of non-skeletogenic mesoderm.
LvDelta and LvNotch play an additional role in micromere-independent mesoderm development
Following the removal of the micromeres, some mesoderm (mostly blastocoelar cells and muscle cells) develops. This suggests that there is a micromere-independent pathway leading to mesoderm development. The micromere-independent mesoderm pathway probably involves Notch signaling because the elimination of Notch function essentially eliminates all mesoderm development (Sherwood and McClay, 1999). We propose that the micromere-independent pathway also involves LvDelta. LvDelta transcripts are found in macromere derivatives within the vegetal plate domain of the mesenchyme blastula-stage embryo. More significantly, elimination of LvDelta function in macromeres results in a severe effect on blastocoelar cell and muscle cell development, a result similar to that observed following the elimination of Notch function throughout the embryo (Sherwood and McClay, 1999
). This evidence supports the idea that Delta/Notch signaling occurs among macromere descendants in the vegetal plate, mediating the specification of blastocoelar and muscle cells.
Delta/Notch signaling and endoderm development
Although Delta/Notch signaling clearly plays a critical role in mesoderm induction, the role of this pathway in endoderm development is less clear. Activation of the Notch pathway in animal blastomeres is sufficient to induce ectopic endoderm and Notch signaling normally plays a role in positioning the ectoderm-endoderm boundary (Sherwood and McClay, 2001) (see below). Notch signaling does not appear to be necessary for endoderm specification, however (Sherwood and McClay, 1999
). We report similar findings; expression of LvDelta by mesomeres is sufficient to induce animal cells to form endoderm, but suppression of LvDelta function throughout the embryo does not block endoderm development. It is not known whether endoderm specification in such embryos occurs by a normal mechanism or by an alternative pathway. Further analysis of the timing and pattern of expression of various endodermal gene markers should elucidate that point. The present results suggest, however, that the normal role of Delta/Notch signaling in the vegetal blastomeres may be limited to establishing the mesodermal domain within the vegetal plate and, possibly as a consequence, shifting the position of the prospective endoderm toward the animal pole.
It remains possible that the experimental methods used to block Delta/Notch function are not completely effective and that low levels of Delta/Notch function are sufficient to mediate endoderm specification. The normal expression patterns of LvDelta and LvNotch are consistent with the possibility that Delta/Notch signaling might be involved in normal endoderm development. At the mesenchyme blastula stage, vegetal plate cells express LvDelta and cells surrounding the vegetal plate express apical LvNotch (Sherwood and McClay, 1997). It is unclear whether the Delta morpholino and Notch dominant negative construct are effective at this relatively late stage, although our chimeric embryo experiments argue that Delta function in the macromeres at later stages is suppressed by the morpholino, as assayed by effects on muscle cell development.
Delta/Notch signaling in animal blastomeres
In a series of classic experiments in experimental embryology, Hörstadius demonstrated the organizing ability of the micromeres by transplanting the micromeres to the animal pole of the embryo and demonstrating the induction of a secondary archenteron and skeletal organizing centers (reviewed by Hörstadius, 1973). Hörstadius also transplanted micromeres to animal caps and found that micromeres can induce these cells, which would normally make only ectoderm, to form a complete pluteus larva. These results have been repeated and extended by many others (e.g. Khaner and Wilt, 1991
; Ransick and Davidson, 1993
; Amemiya, 1996
; Sweet et al., 1999
). Here we report for the first time that the expression of one signaling molecule can endow sea urchin blastomeres with powerful, organizer-like properties. We cannot exclude the possibility that there are multiple signaling molecules produced by micromeres, but signaling by LvDelta alone is clearly sufficient to entrain the repatterning of animal blastomeres to give rise to an organized pluteus.
The mechanism by which LvDelta-expressing cells mediate their organizing influence remains to be determined. One possibility is that the LvDelta-expressing cells act like micromere derivatives, inducing neighboring host cells (as well as some cells derived from the transplanted mesomere) to form pigment and blastocoelar cells, and entraining a normal cascade of signaling interactions that patterns the animal tissue. There is evidence that activation of Notch signaling shifts the endoderm/ectoderm boundary toward the animal pole through the production of a secondary signal (Sherwood and McClay, 2001). Sherwood and McClay found no evidence, however, of the activation of a secondary, non-autonomous signal when Notch signaling was activated specifically in animal cells. These two observations suggest that Notch signaling might act differently in animal and vegetal cells. It will be important to compare the effects of manipulating of Delta/Notch signaling on the expression of downstream regulatory molecules (Brachyury, Wnt-8, etc.) in vegetal cells and animal cells.
Patterning via Delta/Notch signaling: an overview
Delta/Notch signaling appears to be involved in at least three signaling events in the sea urchin embryo (Fig. 9). The first signaling event takes place during the blastula stage (eighth through tenth cleavage divisions), as LvDelta is expressed in the large micromere territory and activates the LvNotch receptor in neighboring macromere descendants (Fig. 9A). This results in the activation of a mesodermal developmental pathway specifically promoting the development of pigment cells and blastocoelar cells. In the second signaling event, LvDelta is expressed by macromere derivatives in the vegetal plate at the mesenchyme blastula and early gastrula stages (Fig. 9B; double arrow). We speculate that this second phase of Delta expression is independent of micromere signaling, although we have not yet tested this directly. Expression of LvDelta again activates the Notch signaling pathway within the macromere territory but now, perhaps as a result of changes in cell competence, stimulates a developmental pathway that specifies muscle cells as well as blastocoelar cells. As LvNotch is downregulated in the vegetal plate at this stage (Sherwood and McClay, 1997), it is possible that the second inductive event occurs only in cells at the periphery of the vegetal plate that still express LvNotch protein. During the third event, which may occur at the same time as the second, cells in the vegetal plate express LvDelta and activate LvNotch in adjacent cells (Fig. 9B; single arrows). This recruits cells at the periphery of the vegetal plate into the endoderm and shifts the position of the endoderm/ectoderm boundary toward the animal pole, possibly via the production of a secondary signaling molecule such as Wnt-8 (see Sherwood and McClay, 2001
). To gain additional evidence either for or against this framework of a model it will be necessary to develop means of detecting and manipulating Delta/Notch signaling within the vegetal plate with even greater spatial and temporal resolution.
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amemiya, S. (1996). Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres. Dev. Growth Differ. 38, 465-476.
Angerer, L. M. and Angerer, R. C. (2000). Animal-vegetal axis patterning mechanisms in the early sea urchin embryo. Dev. Biol. 218, 1-12.[Medline]
Artavanis-Tsakonas, S., Rand, R. D., Lake, R. J. (1999). Notch signaling: Cell fate control and signal integration in development. Science 284, 770-776.
Bettenhausen, B., de Angelis, M. H., Simon, D., Guénet, J.-L. and Gossler, A. (1995). Transient and restricted expression during mouse embryogenesis of Dll1, a murine gene closely related to Drosophila Delta. Development 121, 2401-2418.
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C. (1995). Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375, 761-766.[Medline]
Ettensohn, C. A. and McClay, D. R. (1988). Cell lineage conversion in the sea urchin embryo. Dev. Biol. 125, 396-409.[Medline]
Ettensohn, C. A. and Sweet, H. C. (2000). Patterning the early sea urchin embryo. Curr. Top. Dev. Biol. 50, 1-44.[Medline]
Guss, K. A. and Ettensohn, C. A. (1997). Skeletal morphogenesis in the sea urchin embryo: Regulation of primary mesenchyme gene expression and skeletal rod growth by ectoderm-derived cues. Development 124, 1899-1908.
Henderson, S. T., Gao, D., Christensen, S. and Kimble, J. (1997). Functional domains of LAG-2, a putative signaling ligand for LIN-12 and GLP-1 receptors in Caenorhabditis elegans. Mol. Biol. Cell 8, 1751-1762.[Abstract]
Hodor, P. G. (1998). Cell-cell and cell-matrix interactions responsible for morphogenesis of the sea urchin primary mesenchyme. PhD Thesis, Carnegie Mellon University.
Hörstadius, S. (1973). Experimental Embryology of Echinoderms. Oxford: Clarendon Press.
Ingersoll, E. P. (1993). Identification of an extracellular matrix determinant that plays a key role in sea urchin gastrulation. PhD Thesis, Carnegie Mellon University.
Khaner, O. and Wilt, F. H. (1991). Interactions of different vegetal cells with mesomeres during early stages of sea urchin development. Development 112, 881-890.[Abstract]
Kopczynski, C. C., Alton, A. K., Fechtel, K., Kooh, P. J. and Muskavitch, M. A. (1988). Delta, a Drosophila neurogenic gene, is transcriptionally complex and encodes a protein related to blood coagulation factors and epidermal growth factor of vertebrates. Genes Dev. 12B, 1723-1735.
Lissemore, J. L. and Starmer, W. T. (1999). Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG-2 (DSL) proteins. Mol. Phylogenet. Evol. 11, 308-319.[Medline]
Logan, C. Y. and McClay, D. R. (1997). The allocation of early blastomeres to the ectoderm and endoderm is variable in the sea urchin embryo. Development 124, 2213-2223.
McClay, D. R., Peterson, R. E., Range, R. C., Winter-Vann, A. M. and Ferkowicz, M. J. (2000). A micromere induction signal is activated by ß-catenin and acts through Notch to initiate specification of secondary mesenchyme cells in the sea urchin embryo. Development 127, 5113-5122.
Miller, J. R. and McClay, D. R. (1997). Characterization of the role of cadherin in regulating cell adhesion during sea urchin development. Dev. Biol. 192, 323-339.[Medline]
Minokawa, T. and Amemiya, M. (1999). Timing of the potential of micromere-descendants in echinoid embryos to induce endoderm differentiation of mesomere-descendants. Dev. Growth Differ. 41, 535-547.[Medline]
Parks, A. L., Klueg, K. M., Stout, J. R. and Muskavitch, M. A. (2000). Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127, 1373-1385.
Ransick, A. and Davidson, E. H. (1993). A complete second gut induced by transplanted micromeres in the sea urchin embryo. Science 259, 1134-1138.[Medline]
Ransick, A. and Davidson, E. H. (1995). Micromeres are required for normal vegetal plate specification in sea urchin embryos. Development 121, 3215-3222.
Ruffins, S. W. and Ettensohn, C. A. (1996). A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) mesenchyme blastula. Development 122, 253-263.
Sherwood, D. R. and McClay, D. R. (1997). Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation. Development 124, 3363-3374.
Sherwood, D. R. and McClay, D. R. (1999). LvNotch signaling mediates secondary mesenchyme specification in the sea urchin embryo. Development 126, 1703-1713.
Sherwood, D. R. and McClay, D. R. (2001). LvNotch signaling plays a dual role in regulating the position of the ectoderm-endoderm boundary in the sea urchin embryo. Development 128, 2221-2232.
Sun, X. and Artavanis-Tsakonas, S. (1996). The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands. Development 122, 2465-2474.
Sweet, H. C., Hodor, P. G. and Ettensohn, C. A. (1999). The role of micromere signaling in Notch activation and mesoderm specification during sea urchin embryogenesis. Development 126, 5255-5265.
Varnum-Finney, B., Wu, L., Yu, M., Brashem-Stein, C., Staats, S., Flowers, D., Griffin, J. D. and Bernstein, I. D. (2000). Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J. Cell Sci. 113, 4313-4318.
Wessel, G. M. and McClay, D. R. (1985). Sequential expression of germ-layer specific molecules in the sea urchin embryo. Dev. Biol. 111, 451-463.[Medline]
Wessel, G. M., Zhang, W. and Klein, W. H. (1990). Myosin heavy chain accumulates in dissimilar cell types of the macromere lineage in the sea urchin embryo. Dev. Biol. 140, 447-454.[Medline]
Zhu, X., Mahairas, G., Illies, M., Cameron, R. A., Davidson, E. H. and Ettensohn, C. A. (2001). A large-scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo. Development 128, 2615-2627.