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First published online 1 August 2007
doi: 10.1242/dev.009092

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Gene regulatory networks and developmental plasticity in the early sea urchin embryo: alternative deployment of the skeletogenic gene regulatory network

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

View larger version (138K): [in this window] [in a new window]   Fig. 1. WMISH analysis of skeletogenic genes during NSM transfating. (A) Lvp16 (ventral view). (B) Lvp58 (lateral view). (C) Lvsm30 (ventral view). (D) Lvsm50 (scheitel region). Embryos were fixed 15 hours (A-C) or 36 hours (D) after PMC removal. Arrows point to the syncytial network of transfated cells. Arrowhead in C indicates transfated cells in the ventral region that do not express Lvsm30.

View larger version (70K): [in this window] [in a new window]   Fig. 2. PMC removal triggers ectopic expression of Lvalx1 in cells near the tip of the archenteron. WMISH analysis of Lvalx1 (A-G) and Lvtbr (H) expression. (A) Control mesenchyme blastula. Lvalx1 is expressed specifically by PMCs (arrow). (B) A PMC-deficient embryo immediately after surgery. (C,D) PMC-deficient embryos 4 hours after surgery, viewed laterally (C) and along the AV axis (D). Cells near the tip of the archenteron express Lvalx1 (C, arrow). In many embryos, Lvalx1-expressing cells are located predominantly on one side of the archenteron (D, arrow). (E,F) PMC-deficient embryos 5-6 hours after surgery, viewed laterally (E) and along the AV axis (F). Lvalx1 is expressed by cells at the tip of the archenteron (arrows), some of which have begun to migrate out of the epithelium. (G) PMC-deficient embryo, 9-10 hours after surgery. Lvalx1-expressing cells are migrating within the blastocoel (e.g. arrow) and some remain associated with the archenteron tip. (H) Lvtbr expression in a PMC-deficient embryo, 10-11 hours after surgery. Transfating NSM cells are organizing in a subequatorial ring pattern and express Lvtbr (arrow).

View larger version (40K): [in this window] [in a new window]   Fig. 3. LvAlx1 is required in macromere descendants for transfating. (A) Experimental protocol. (B-I) Embryos were immunostained with mAb 6a9 and the same embryos were photographed using both brightfield (left column) and epifluorescence (right column) optics. (B,C) Control late gastrula stage embryo, 20 hours postfertilization. (D,E) LvAlx1 MO-injected embryo, 48 hours postfertilization. Most morphogenetic processes are unaffected by LvAlx1 knockdown but the embryo lacks 6a9-positive cells and skeletal elements. (F,G) Micromere-deficient [micromere(-)] embryo, 24 hours postfertilization. Transfating has occurred, leading to the formation of many 6a9-positive cells (arrow) and two skeletal primordia. (H,I) LvAlx1 MO-injected, micromere-deficient embryo, 48 postfertilization. The embryo has undergone extensive morphogenetic changes but lacks 6a9-positive cells and a skeleton.

View larger version (50K): [in this window] [in a new window]   Fig. 4. LvAlx1 is required in mesomere descendants for transfating. (A) Experimental protocol. (B-G) Embryos were immunostained with mAb 6a9 and the same embryos were photographed using brightfield (B-D) and epifluorescence (E-G) optics. (B,E) Animal cap isolated at the 16-cell stage and cultured for 24 hours in the absence of LiCl. The embryo lacks mesoderm and endoderm and no 6a9-positive cells are present. (C,F) Animal cap treated with 50 mM LiCl for 3 hours after the operation and cultured for 24 hours. Endoderm and mesoderm have formed, including many 6a9-positive cells. (D,G) Animal cap isolated from a LvAlx1 MO-injected embryo, treated with 50 mM LiCl, and cultured for 48 hours. Although a gut (arrow) has formed, no 6a9-positive cells are present. (H) RT-PCR experiments showing that animal caps treated with 50 mM LiCl express Lvalx1 and one of its downstream targets (Lvp16). Each experiment was repeated three times (R1-R3).

View larger version (62K): [in this window] [in a new window]   Fig. 5. Lvalx1 is upstream of the signal that suppresses NSM transfating. (A) Experimental protocol. (B-G) Recombinant embryos were photographed using brightfield (upper row) and epifluorescence (lower row) optics. (B,E) Animal cap + micromere recombinant, 36 hours after fertilization. Micromere progeny (labeled with Rhodamine dextran) have formed the embryonic skeleton (arrow) and have induced the formation of a complete embryonic axis. (C,F) Animal cap + LvAlx1 MO-micromere recombinant, 48 hours after fertilization. LvAlx1 MO-injected micromeres have induced a complete embryonic axis. In contrast to B,E, however, the progeny of the micromeres (labeled with fluorescein dextran) are not arranged along the skeletal rods (C, arrowhead). Instead, they are scattered in the blastocoel or remain associated with the tip of the archenteron (arrow). (D,G) Animal cap + LvAlx1 MO-micromere recombinant, 48 hours after fertilization. Mesomere descendants have transfated, as shown by 6a9 immunostaining, and are associated with skeletal elements (G, arrow).

View larger version (125K): [in this window] [in a new window]   Fig. 6. MAPK signaling is required for transfating. Right panels show embryo whole mounts immunostained with mAb 6a9 and examined by confocal microscopy. The remaining panels show living embryos viewed with differential interference optics, except H and K, which are in situ hybridizations. (A-C) Control embryos at the late gastrula stage. 65-70 PMCs are arranged in a subequatorial ring pattern (arrow). (D-F) Embryos treated with 6 µM U0126 from the 2-cell stage. Arrows in E indicate locations adjacent to ectodermal thickenings where ventrolateral clusters of PMCs normally form. Arrow in F indicates one of two 6a9-positive cells that formed in this embryo, which was treated with U0126 until sibling controls reached the prism stage. (G-I) Control PMC-deficient embryos (not treated with U0126). Transfating is indicated by skeleton formation (G; 24 hours after surgery), Lvalx1 mRNA expression (H; 8 hours after surgery), and 6a9 immunostaining (I; 12 hours after surgery). (J-L) PMC-deficient embryos transferred to 6 µM U0126 immediately after surgery (J,K and L show embryos 24, 8 and 24 hours after surgery, respectively).

View larger version (79K): [in this window] [in a new window]   Fig. 7. Overexpression of LvAlx1 is sufficient to increase numbers of skeletogenic cells. (A) Projection of a z-stack of confocal images of a 6 hour (early blastula) embryo expressing LvAlx1-GFP. LvAlx1 protein accumulates in the nuclei of all blastomeres. (B) Projection of a z-stack of the vegetal plate of a mesenchyme blastula-stage embryo expressing LvAlx1-GFP. Cells throughout the vegetal plate have tagged protein in their nuclei. (C-E) Embryos expressing wild-type or mutant forms of LvAlx1, fixed at the late gastrula stage and immunostained with mAb 6a9. (C) Embryo injected with Lvalx1.STOP mRNA (0.75 mg/ml). Numbers of 6a9-positive cells are similar to those observed in uninjected sibling embryos (Fig. 6A and Fig. 8). (D,E) Lateral and vegetal views of two different embryos injected with Lvalx1.WT mRNA (0.38 mg/ml). These embryos have large numbers of 6a9-positive cells (100-120 cells) arranged in a radially symmetrical band. (F) Lvp16 expression (arrow) in PMCs of a control embryo. (G) A sibling embryo injected with Lvalx1.WT mRNA (0.38 mg/ml). Overexpression of LvAlx1 leads to increased numbers of Lvp16-expressing cells, which are arranged in a circumferential belt (arrow).

View larger version (30K): [in this window] [in a new window]   Fig. 8. The effect of Alx1 overexpression on numbers of 6a9-positive cells. The results of six independent trials are shown. Each vertical bar indicates a mean calculated from 10-30 embryos. Vertical lines represent one standard deviation. 20% glycerol, the mRNA carrier solution, was used as a control; LvAlx1.STOP is a mutant that cannot bind to DNA.

View larger version (35K): [in this window] [in a new window]   Fig. 9. Overexpression of LvAlx1 activates the PMC GRN in macromere descendants. (A) Experimental protocol. Fertilized eggs were injected with Lvalx1.WT mRNA. At the 16-cell stage, one macromere was injected with fluorescein dextran. After 12 hours, the embryos were fixed, immunostained with mAb 6a9 and a Cy5-conjugated secondary antibody, and examined by confocal microscopy. (B) A 2-D projection of a complete z-stack (16 images) of an experimental embryo. Fluorescent dextran is shown in green and 6a9 immunostaining in red. The macromere injected with dextran has contributed to a patch of cells outside the blastopore (arrowhead) as well as cells in the archenteron and a few migrating mesenchyme cells (arrows). This low Mr dextran is concentrated in cell nuclei (Hodor and Ettensohn, 1998). (C) A 2-D projection of a partial z-stack (4 images) from the same embryo. This projection shows more clearly that some 6a9-positive cells are also labeled with fluorescent dextran (arrows).

View larger version (41K): [in this window] [in a new window]   Fig. 10. Lvpmar1 does not activate the skeletogenic GRN network during NSM transfating. (A) The Lvpmar1 locus. (B) Developmental RT-PCR analysis of Lvpmar1 expression. Stages shown are unfertilized egg (UE), 4-, 8-, 16-, 32- and 64-cell stages, late cleavage (LC), early blastula (EB) and mesenchyme blastula (MB). (C) Overexpression of Lvpmar1 activates the PMC GRN in all cells of the embryo. Lvpmar1 mRNA (100 µg/ml) was injected into fertilized eggs and after 24 hours the embryos were fixed and immunostained with mAb 6a9. Lvpmar1 causes a transformation of all cells to a mesenchymal, 6a9-positive phenotype. (D) RT-PCR analysis of Lvpmar1, Lvp16 and Lvactin mRNA expression in PMC-deficient embryos. MB, control mesenchyme blastula stage embryos. Other lanes show PMC-deficient embryos, collected 0, 3, 6 and 9 hours after PMC depletion. Lvp16, a target of Lvpmar1 and Lvalx1, is expressed within 3 hours after PMC depletion and at higher levels at 9 hours. Lvpmar1 mRNA is not detectable at any of the stages examined (the amount of starting material at each stage was equivalent to 1 embryo). Bottom panels show control experiments using identical RT-PCR conditions but with cell lysates prepared from 16-cell stage embryos. Lvpmar1 can be detected when the amount of starting material is equivalent to just 1/100 embryo. R1-R3 are three independent replicates of the experiment, performed using three different batches of embryos.

View larger version (22K): [in this window] [in a new window]   Fig. 11. A comparison of GRN architecture in the large micromere-PMC lineage and NSM cells. In presumptive PMCs (left), maternal ß-catenin activates pmar1, which represses hesC. Components of the MAPK signaling pathway are activated in the PMC lineage by an unknown mechanism and bring about the phosphorylation and activation of Ets1/2. Ets1/2 and alx1 regulate genes that control ingression, migration, and biomineralization, and alx1 has an essential input into PMC signaling. Note that although snail is a transcriptional repressor, it stimulates ingression (Wu and McClay, 2007). In presumptive NSM cells (right), several components of the PMC GRN (e.g. ets1/2, delta and snail) are normally expressed, although little is known concerning their upstream regulators. MAPK signaling is active and presumably causes the phosphorylation of Ets1/2. Alx1 and tbr are normally repressed, directly or indirectly, by the PMC-derived signal (genes not normally expressed in NSM cells are shown in brackets). Key downstream targets of alx1 in the skeletogenic pathway are therefore not expressed. Elimination of the signal induces expression of alx1, which is sufficient to engage all essential, missing elements of the PMC GRN. One consequence of the activation of the PMC GRN in NSM cells is the acquisition of PMC-specific signaling properties (Ettensohn and Ruffins, 1993).

View larger version (43K): [in this window] [in a new window]   Fig. 12. Possible evolutionary changes in the deployment of the skeletogenic GRN. NSM shown as green cells; micromeres and skeletogenic mesenchyme cells are shown in red. See Discussion for details.