QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sherwood, D. R. Articles by McClay, D. R. Search for Related Content PubMed PubMed Citation Articles by Sherwood, D. R. Articles by McClay, D. R. Social Bookmarking                         What's this?

LvNotch signaling plays a dual role in regulating the position of the ectoderm-endoderm boundary in the sea urchin embryo

David R. Sherwood^* and David R. McClay

Developmental, Cell and Molecular Biology Group, Box 91000, Duke University, Durham, NC 27708, USA
^* Present address: California Institute of Technology, Division of Biology 156-29, Pasadena, CA 91125, USA

View larger version (66K): [in a new window]   Fig. 1. Activation and inhibition of LvNotch signaling throughout the embryo shifts the ectoderm-endoderm boundary. (A) Zygotes were injected with LvNotch mRNA constructs and allowed to develop. At the 16-cell stage, individual mesomeres were labeled with DiI, and the distribution of the descendants of these cells determined in 36-42 hour pluteus larvae. (B,C) Nearly all labeled mesomeres from embryos injected with LvNact contributed progeny to the gut (Table 1). A lateral view of a larva from a LvNact injected embryo (B) shows that the DiI labeled mesomere (C) contributed descendants to the aboral ectoderm (arrow), and all three endoderm-derived gut compartments (h,m,f in B; arrowhead). (D,E) Almost no labeled mesomeres from embryos injected with LvNneg contributed descendants to the gut (Table 1). A larva from a LvNneg injected embryo (D) shows that the DiI labeled mesomere (E) contributed progeny to the ectoderm along the left arm (arrow), but not to the gut (arrowhead). The characteristic smaller size of larvae from LvNact injected embryos (B) and larger appearance of larvae from LvNneg injected embryos (D) was probably the result of a respective decrease and increase in the amount of ectoderm present after the shift in the ectoderm-endoderm boundary, as the amount of ectoderm in the embryo is thought to regulate the size of sea urchin larvae (Ettensohn and Malinda, 1993). a, animal pole; f, foregut; h, hindgut; m, midgut; v, vegetal pole. Scale bar: 100 µm.

View larger version (65K): [in a new window]   Fig. 2. Alteration of vegetal LvNotch signaling shifts the ectoderm-endoderm boundary cell non-autonomously in overlying animal cells. (A) LvNotch mRNAs were co-injected with the lineage tracer fluorescein dextran into individual blastomeres at the eight-cell stage. Embryos containing injected macromere/micromere pairs were isolated at the 16-cell stage, and single mesomeres overlying injected macromeres were labeled with DiI. The fates of DiI labeled mesomeres were then determined in pluteus larvae. (B,C) Activation of LvNotch signaling vegetally within macromere/micromere pairs increased the percentage of overlying uninjected mesomeres that contributed progeny to gut tissue (Table 2). A larva that had a DiI-labeled mesomere (C, red) labeled over a LvNact injected macromere/micromere pair (C, green) is shown. The overlying mesomere contributed cells both to the gut (large arrowhead), and oral ectoderm (arrow). The injected micromere gave rise to primary mesenchyme cells (PMCs), which are not visible because of decreased staining after fusion with uninjected PMCs (see Hodor and Ettensohn, 1998). The injected macromere gave rise predominantly to SMCs (scattered green cells within the larvae) in response to activated LvNotch, as well as a small portion of foregut tissue (small arrowhead). (D,E) In contrast, inhibition of endogenous vegetal LvNotch decreased the percentage of overlying mesomeres that contributed progeny to the gut (Table 2). Shown is an example of a larva (D) that had a DiI labeled mesomere (E, red) labeled over a LvNneg injected macromere/micromere pair (E, green). The overlying mesomere contributed progeny only to ectoderm, along the right arm (arrow). The macromere has given rise to tissue in all three gut compartments (arrowhead) and ectoderm, but not SMCs. Scale bar: 100 µm.

View larger version (63K): [in a new window]   Fig. 3. Perturbation of LvNotch signaling within the animal region of the embryo alters ectoderm-endoderm boundary position. (A) LvNotch mRNAs were co-injected with the lineage tracer fluorescein dextran into blastomeres at the eight-cell stage. Embryos in which injected blastomeres gave rise to pairs of mesomeres at the 16-cell stage were isolated, and the fate of the injected mesomere pairs determined in pluteus larvae. (B,C) Activation of LvNotch signaling within mesomeres increased the percentage of these cells that contributed progeny to the gut compared with controls (Table 2). An example of a pluteus larva (B) in which the LvNact injected mesomere pair (C) contributed descendants to ectoderm along the ciliated band (arrow) and right arm, as well as gut tissue up to the midgut/foregut boundary (arrowhead). (D,E) Conversely, inhibition of endogenous LvNotch signaling within mesomeres reduced the number of mesomeres that contributed descendants to the gut compared with controls (Table 2). A representative pluteus larva is shown (D); the mesomere pair injected with LvNneg (E) contributed progeny only to aboral ectoderm (arrow) and not the gut. Scale bar: 100 µm.

View larger version (32K): [in a new window]   Fig. 4. Ectopic gut tissue from constitutive activation of LvNotch signaling in mesomeres consists solely of LvNact injected cells. (A-C) Lateral and enlarged anal (inset) views of a pluteus larva in which the mesomere pair injected with LvNact gave rise to aboral ectoderm and additional gut tissue (arrow) attached to the normal hindgut (arrowhead). Note that the ectopic gut tissue in (A) is composed exclusively of fluorescent LvNact injected cells (B, images overlaid in C). Scale bars: 100 µm (25 µm in the insets).

View larger version (45K): [in a new window]   Fig. 5. The position of the ectoderm-endoderm boundary is not altered in cells neighboring LvNact-injected mesomere pairs. (A) Uninjected mesomeres neighboring LvNact/fluorescein dextran-injected mesomere pairs were lineage labeled with DiI. In contrast to LvNact-injected mesomeres, neighboring DiI-labeled cells did not contribute progeny to the gut at an increased frequency (Table 2). (B,C) Lateral view of a pluteus larva (B) shows that the LvNact-injected mesomere pair (C, green) contributed progeny to the gut tissue (arrowhead) and oral ectoderm, while the DiI labeled mesomere (C, red) gave rise only to aboral ectoderm (arrow). (D) Uninjected macromeres underlying pairs of LvNact or LvNactANK5/fluorescein dextran-injected mesomeres were labeled with DiI. No difference in ectoderm contribution was found for macromeres underlying LvNact- versus LvNactANK5-injected mesomeres in pluteus larvae. (E-G) Anal view of a larva showing the lineage of a DiI labeled macromere underlying a pair of mesomeres injected with LvNact /fluorescein-dextran. (E) The DiI-labeled macromere has given rise primarily to gut (overexposed and out of focus fluorescence in center and left); however, the macromere has also contributed to a number of anal ectoderm cells bordering the ciliated band (arrows; individual cells clearly distinguished by perinuclear membrane staining). (F) The pair of LvNact- and fluorescein dextran-injected mesomeres has given rise to ectoderm (small arrowhead; mostly along oral surface and not in view – similar to mesomere in C), as well as gut tissue (large arrowhead). (G) Overlay of DiI-labeled macromere descendants (red), LvNact- and fluorescein dextran-injected mesomere descendants (green), and DIC image (gray). Scale bar: 100 µm in B,C; 25 µm in E-G.

View larger version (78K): [in a new window]   Fig. 6. Isolated animal caps containing constitutively activated LvNotch form gut tissue. (A) Zygotes were injected with LvNact and cultured to the eight-cell stage, when animal and vegetal halves were separated. (B) A 24 hour untreated animal cap has developed into a ciliated ectodermal vesicle, containing no endoderm. (C) In contrast, an archenteron has begun to invaginate in a 24 hour animal cap containing LvNact, which (by 48 hours) has given rise to gut tissue (D, arrow) that expresses the hindgut/midgut marker Endo1 (E, arrow). Scale bar: 100 µm.

View larger version (42K): [in a new window]   Fig. 7. Distribution and number of LvNact- and LvNactANK5-injected macromere and mesomere pair descendants in the late mesenchyme blastula embryo. Embryos are shown viewed along the A-V axis and co-immunostained for the LvNact protein product (green), which localizes to the nucleus, and ß-catenin (red), which localizes to all epithelial adherens junctions, thus outlining the shape of the embryo. The number and distribution of injected macromere or mesomere pair descendants containing LvNact or LvNactANK5 was determined by examining the number and distribution of nuclei containing the LvNact or LvNactANK5 proteins. (A) An example of LvNact distribution in an embryo in which a macromere/micromere pair was injected with LvNact. (B) A typical example of LvNact distribution in an embryo in which a mesomere pair was injected with LvNact. Embryos injected with LvNact showed similar nuclear distributions and numbers of cells expressing the LvNact protein, as compared with those injected with LvN actANK5 (Table 3). Scale bar: 25 µm.

View larger version (36K): [in a new window]   Fig. 8 LvNotch signaling alters nuclear ß-catenin localization at the ectoderm-endoderm boundary. (A-C) Confocal sections along the A-V axis of untreated and mRNA-injected late mesenchyme blastula embryos stained with a ß-catenin-specific polyclonal antibody. (A) An untreated embryo shows nuclear ß-catenin localized in presumptive endoderm cells that border the presumptive ectoderm (arrows). Angle indicates the territory vegetal to nuclear ß-catenin (the presumptive endoderm and SMCs). ß-catenin is also present at high levels within the small micromeres at the vegetal pole (arrowhead; Miller and McClay 1997). (B) An embryo injected with LvNact shows a clear shift in nuclear ß-catenin localization toward the animal pole (arrows). (C) Conversely, nuclear ß-catenin distribution was found more vegetally (arrows) in LvNneg-injected embryos. (D) The volume of the embryo vegetal to nuclear ß-catenin (±s.e.m.) was approximately 50% greater in LvNact-injected embryos and 30% smaller in LvNneg-injected embryos compared with untreated controls. Volume was calculated using the angle and the equation Volume=0.5(1-cos)/2 (see Reynolds et al., 1992). Asterisk denotes significant difference from untreated embryos (P<0.01; two-sample t-test). Scale bar: 25 µm.

View larger version (25K): [in a new window]   Fig. 9. Vegetal LvNotch signaling shifts the localization of nuclear ß-catenin at the ectoderm-endoderm boundary cell non-autonomously. (A-C) LvNact was injected into pairs of mesomeres placing activated LvNotch at or slightly above the ectoderm-endoderm boundary. Co-immunostaining of late mesenchyme blastula embryos with an intracellular directed LvNotch antibody and with a ß-catenin antibody revealed the LvNact protein in nuclei of injected cells (A, bracket), and nuclear ß-catenin at the ectoderm-endoderm boundary (B, arrowheads) as well as in the small micromeres (B, arrow). An overlay (C) of LvNact (green) and ß-catenin (red) staining shows that LvNact did not shift the localization of nuclear ß-catenin animally, even when expressed in cells directly neighboring nuclear ß-catenin (arrowhead; compare left and right sides of embryo). (D-E) In contrast, an example of an embryo with LvNact injected into a macromere/micromere pair (bracket, D) shows that the localization of nuclear ß-catenin (E; arrowheads) was shifted animally on the side of the embryo containing LvNact. An overlay (F) of LvNact (green) and ß-catenin (red) staining reveals the shift of nuclear ß-catenin (arrowhead) into cells that do not contain activated LvNotch. Scale bar: 25 µm.

View larger version (32K): [in a new window]   Fig. 10. Schematic summarizing the effects of perturbing LvNotch signaling along the A-V axis and the relationship of LvNotch to other signaling pathways that regulate ectoderm-endoderm boundary position in the sea urchin embryo. (A) A surface fate map of an untreated mesenchyme blastula stage embryo showing (from vegetal to animal pole) the SMC, endoderm and ectoderm territories. Activation of LvNotch signaling throughout the embryo expands the SMC territory at the expense of neighboring presumptive endoderm cells (Sherwood and McClay, 1999), and shifts the endoderm territory animally at the expense of presumptive ectoderm (this study). Inhibition of LvNotch signaling eliminates the specification of SMCs in the mesenchyme blastula embryo (Sherwood and McClay, 1999) and shifts the endoderm territory vegetally, thus expanding the amount of ectoderm in the embryo (this study). Activation or inhibition of LvNotch has no effect on primary mesenchyme cell specification, which are not shown as they have ingressed inside the blastocoel at this time (Sherwood and McClay, 1999). (B) Summary of signaling pathways that affect the position of the ectoderm-endoderm boundary at the late mesenchyme blastula stage when the ectoderm-endoderm boundary is thought to be established (Logan and McClay, 1998). BMP 2/4 signaling in the animal region of the embryo has been shown in the sea urchin Strongylocentrotus purpuratus to promote ectoderm formation and inhibit endoderm specification (Angerer et al., 2000). In the sea urchin Lytechinus variegatus LvNotch signaling and nuclear ß-catenin signaling promote endoderm formation at the boundary (this study and M. Ferkowicz and D. M., unpublished). In the vegetal region of the embryo LvNotch regulates the expression of a signal (possibly a Wnt homolog) that promotes endoderm formation in overlying cells (this study). dnLvNotch, dominant negative LvNotch; SMC, secondary mesenchyme cell.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter