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LvDelta is a mesoderm-inducing signal in the sea urchin embryo and can endow blastomeres with organizer-like properties

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA

View larger version (66K): [in a new window]   Fig. 1. Structure of sea urchin Delta homologues. (A) Schematic of LvDelta protein. SP, signal peptide; DSL, Delta/Serrate/LAG-2 domain; EGF, EGF repeats; TM, transmembrane domain; ICD, intracellular domain. (B) Nucleotide sequence of the LvDelta and SpDelta clones. Translational start and stop codons are boxed. Motifs common to Delta homologues are indicated. The site of the G-to-T mutation used to generate a premature stop codon and a truncated form of LvDelta is labeled with an arrowhead. The LvDelta clone continues to the poly(A) tail although the 3' untranslated region was not completely sequenced. The SpDelta clone terminates in the region encoding the intracellular domain. GenBank accession numbers for LvDelta and SpDelta are AY074791 and AY074792, respectively.

View larger version (91K): [in a new window]   Fig. 2. Alignment of the predicted amino acid sequences of several Delta proteins. Amino acid sequences of Delta proteins from S. purpuratus, L. variegatus, X. laevis, (Chitnis et al., 1995), M. musculus (Bettenhausen et al., 1995) and D. melanogaster (Kopczynski et al., 1988) were aligned using Pileup and Pretty in the GCG Wisconsin Package. Conserved domains are labeled. The mutation site used to generate truncated LvDelta is indicated by an arrowhead.

View larger version (111K): [in a new window]   Fig. 3. LvDelta is activated zygotically and is expressed transiently during the blastula and early gastrula stages. (A) Developmental northern blot. Total RNA (10 µg) from each of the stages indicated was hybridized with DIG-labelled LvDelta RNA probe. Ethidium bromide staining of the 18S rRNA bands served as a loading control. Molecular mass markers (kb) are indicated. A large transcript (5.3 kb) begins to accumulate by 6 hours after fertilization (seventh cleavage). A second, smaller transcript (4.8 kb) accumulates by 8 hours after fertilization. Both transcripts decrease in abundance after 8 hours until they are barely detectable by 16 hours (gastrula stage). UFE, unfertilized embryo. (B,C) In situ hybridization. (B) At the late blastula stage (8 hours), LvDelta is expressed in a ring of cells at the vegetal pole. (C) At the late mesenchyme blastula/early gastrula stage (12 hours), LvDelta is expressed by macromere derivatives in the vegetal plate, but not by PMCs (large micromere derivatives; arrowhead).

View larger version (72K): [in a new window]   Fig. 4. Mesoderm development following manipulation of LvDelta levels. (A-C) Gastrulation morphology in living embryos; (D-F) Immunostaining of blastula stage embryos with SMC1 (green), a marker for non-skeletogenic mesoderm, and CAD-1 (red), which labels cell boundaries. (A,D) In control embryos, SMCs ingress from the archenteron during gastrulation. SMC1-positive cells are evident in the central region of the vegetal plate at the mesenchyme blastula stage. (B,E) Following injection of LvDelta morpholino, gastrulation is delayed and few SMCs ingress from the archenteron. SMC1 immunostaining shows that there are few prospective mesodermal cells in the vegetal plate (one SMC1-positive cell is visible in this embryo). (C,F) Following injection of LvDelta mRNA, the archenteron produces excessive numbers of SMCs and the vegetal plate contains greatly increased numbers of SMC1-positive cells.

View larger version (125K): [in a new window]   Fig. 5. Later differentiation of mesodermal cells following manipulation of LvDelta levels. (A-C) Pigment cells; (D-F) SMC2 staining (blastocoelar cells are indicated by arrowheads; there is background staining in the ectoderm and endoderm, showing the outline of the larva and the midgut, respectively); (G-I) anti-myosin staining showing muscle fibers. (A,D,G) Control pluteus larvae showing normal numbers of pigment cells, blastocoelar cells and muscle fibers. (B,E,H) Following injection of LvDelta morpholino, the resulting larvae have few pigment cells, blastocoelar cells and muscle fibers. (C,F,I) Following injection of LvDelta mRNA, increased numbers of pigment cells, clusters of blastocoelar cells and increased muscle fibers are apparent.

View larger version (55K): [in a new window]   Fig. 6. LvDelta function is required in both micromere and macromere derivatives. Cell transplantation was used to test the effects of blocking LvDelta function in either the micromere descendants or the macromere and mesomere descendants. (A-C) Micromeres containing LvDelta morpholino and a lineage tracer were combined with normal macromeres and mesomeres. (A) Diagram of the experimental design. The resulting embryos have few pigment cells (B), but anti-myosin staining of muscle fibers appears normal (C). (D-F) Macromeres and mesomeres containing LvDelta morpholino and a lineage tracer were combined with normal micromeres. (D) Diagram of the experimental design. The resulting larvae produce many pigment cells (E) but few muscle fibers (F).

View larger version (85K): [in a new window]   Fig. 7. LvDelta expressed by mesomeres is sufficient to induce pigment cell formation by macromeres. (A) Experimental design. Micromeres were removed from an unlabeled embryo to generate the host. Fertilized eggs were injected with the truncated or full-length form of LvDelta mRNA and one mesomere was isolated at the 16-cell stage and transplanted to the vegetal pole of the host embryo. (B) A micromere(–) embryo recombined with a mesomere expressing truncated LvDelta develops few pigment cells. (C) Epifluorescence image of the larva in B demonstrates that the transplanted mesomere contributes to the foregut and midgut. (D) A micromere(–) embryo recombined with a mesomere expressing full-length LvDelta develops many pigment cells. (E) Epifluorescence of the larva in D shows that the transplanted mesomere contributes to the foregut and mesoderm. Many mesodermal cells do not contain the lineage tracer and were derived from host tissue.

View larger version (55K): [in a new window]   Fig. 8. LvDelta expressed by mesomeres is sufficient to induce animal caps to develop mesoderm and endoderm. (A) Experimental design. To generate the host, an animal cap was isolated at the 8-cell stage. Fertilized eggs were injected with the truncated or full-length form of LvDelta mRNA and one mesomere was isolated at the 16-cell stage and transplanted to the vegetal pole of the animal cap. (B) An animal cap recombined with a mesomere expressing truncated LvDelta develops into a hollow ball of ectoderm. (C) Epifluorescence image of the larva in B demonstrates that the transplanted mesomere contributes to the epithelium. (D) An animal cap recombined with a mesomere expressing full-length LvDelta develops into a pluteus larva. (E) Epifluorescence of the larva in D shows that the transplanted mesomere contributes to the foregut, midgut and mesoderm, though some endoderm and mesoderm is not labeled with the lineage tracer. These larvae develop blastocoelar cells (F) and skeletogenic cells (G).

View larger version (31K): [in a new window]   Fig. 9. Model for Delta/Notch signaling in the sea urchin embryo. There appear to be at least three roles for Delta/Notch signaling during early development. (A) Blastula stage (eighth-tenth cleavage stage). Micromere derivatives (red) express LvDelta (arrows), and activate the Notch protein in neighboring cells to promote the specification of pigment cells and blastocoelar cells in the region that will become the non-skeletogenic mesoderm territory (pink). (B) Mesenchyme blastula stage to early gastrula stage. Prospective nonskeletogenic mesoderm (pink) in the vegetal plate expresses LvDelta (double-ended arrow) to promote the development of prospective muscle cells and blastocoelar cells. LvDelta expression by these cells may also activate the Notch protein in neighboring cells (single arrows from pink to yellow), promoting the development of prospective endoderm (yellow). Delta/Notch signaling also results in the expression of a secondary signal (arrows entirely within the yellow region) that promotes endoderm development in more animal cells (Sherwood and McClay, 2001).

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