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First published online October 26, 2007
doi: 10.1242/10.1242/dev.010272

Development 134, 4095-4106 (2007)
Published by The Company of Biologists 2007
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Lrp6 is required for convergent extension during Xenopus gastrulation

Emilios Tahinci1, Curtis A. Thorne1, Jeffrey L. Franklin1,2, Adrian Salic3, Kelly M. Christian1, Laura A. Lee1, Robert J. Coffey1,2 and Ethan Lee1,*

1 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
2 Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
3 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.


Figure 1
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  		Fig. 1. Loss or gain of Lrp6 function inhibits convergent extension without affecting tissue patterning. (A) Dorsal injections of Lrp6 morpholino (LRP6MO, 40 ng) or mRNA (1 ng) cause defects in neural and mesodermal convergent extension. In contrast to uninjected or ß-catenin (50 pg) injected siblings, embryos with altered Lrp6 levels are not fully elongated (less than 70% of the length of controls) and have split or thicker notochords resembling that of Xdd-(2 ng) or Dkk1-(400 pg) injected embryos. Lrp6 or ß-catenin mRNA levels were chosen so as to cause similar percentages of anterior duplication when injected ventrally (bar graph). For each injection, Tor70 antibody staining for notochord is shown in the lower panels. Scale bars: 0.5 mm. (B) The anteriorizing effect of Lrp6MO injection (30 ng) is rescued by co-injecting full-length lrp6 mRNA (LRP6FL, 500 pg) or ß-catenin DNA (100 pg). Convergent-extension defects due to Lrp6MO injections are rescued by co-injecting lrp6FL mRNA, but not ß-catenin DNA. Anteriorized embryos had an average dorsoanterior index (DAI) of 8 (Kao and Elinson, 1988Go). ß-catenin DNA injection resulted in posteriorized embryos (60% affected, 72 embryos injected) having an average DAI of 2. Error bars indicate standard deviation. Numbers of embryos scored are indicated in parentheses. (C) Mesendodermal [Bra (Xbra), Chordin, Goosecoid (Gsc), Sizzled, Endodermin (Endd)] and ectodermal (Otx2, Xag1, epidermal Keratin) markers are expressed in developing embryos injected with lrp6 mRNA (1 ng), dkk1 mRNA (200 pg) or Lrp6MO (40 ng), indicating that the effect of Lrp6 on convergent extension is not due to altered tissue patterning. Loading control: ODC (ornithine decarboxylase).

 

Figure 2
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  		Fig. 2. Loss and gain of Lrp6 function in explants affects convergent-extension movements. (A) lrp6 mRNA (LRP6; 1 ng) or Lrp6MO (LRP6MO; 40 ng) injections block activin-mediated animal cap elongation, in a similar manner to injection of Wnt11 (400 pg) control. By contrast, injection of ß-catenin at levels that cause complete axis duplication (50 pg) has no effect. (B) RT-PCR analysis shows that mesendodermal markers (Xbra, Chordin, Goosecoid, Sizzled, Endodermin) are induced by activin in animal caps and their expression is unaffected by injections of Lrp6MO or lrp6 mRNA. Note induction of Xbra and Chordin after injections of similar amounts of dsh mRNA (1 ng). Loading control: ODC (ornithine decarboxylase). (C) Keller sandwiches elongate when DMZ explants are cultured in apposition. Keller sandwiches injected with Lrp6MO (40 ng) or lrp6 mRNA (1 ng) show impaired elongation (compared to control uninjected or ß-catenin expressing explants) and resemble those expressing Xdd (2 ng). ANE, anterior neural ectoderm; PNE, posterior neural ectoderm; IM, involuting mesoderm. Student's t-test was used for statistical analysis. Error bars indicate standard deviation. Asterisks mark differences that are statistically significant from control (P<0.01). Numbers of explants scored are indicated in parentheses.

 

Figure 3
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  		Fig. 3. Lrp6 controls the morphology and motility of mesodermal cells. (A) Frames from time-lapse movies of DMZ and VMZ cells in shaved Keller explants from embryos injected with Lrp6MO (LRP6MO; 40 ng) and Stbm (Xstbm; 200 pg) show alterations in motility, shape, orientation, protrusion distribution along the cell periphery and protrusion stability as indicated in the bar charts. Outlines of randomly chosen cells were traced throughout each movie, and traces from the first (red) and last (blue) frame were superimposed to assess cell translocation. For measuring protrusion distributions, each cell was divided into four quadrants emanating from the center (black lines), and each quadrant was subdivided into two sectors by marking the midpoint along the cell membrane (green lines). Each protrusion was assigned to the sector to which it most closely localized (long axis in blue, short axis in red). The angle ({theta}) between the long axis of the cell and the mediolateral axis (purple line) of the explant was used to calculate its orientation in the explant. Protrusion stability was measured by counting the number of stable (present throughout the 15 minute movie) and transient protrusions (appearing after the first frame and/or disappearing before the last). (B) Similar cellular changes were observed in isolated DMZ cells of embryos injected with Lrp6MO. DMZ and VMZ cells from stage 10.5 embryos were dissociated, plated on fibronectin, immediately fixed and stained with Rhodamine-phalloidin. DMZ cells from wnt11 mRNA (Xwnt; 160 pg)-, stbm mRNA (Xstbm; 200 pg)-, or Lrp6MO (LRP6MO; 40 ng)-injected embryos have decreased length-to-width ratios, increased number of total cytoplasmic protrusions, and increased numbers of cytoplasmic protrusions along the short axes versus the long axes compared to GFP-injected (200 pg) control. VMZ cells from wnt11-, stbm-, or Lrp6MO-injected embryos show no obvious length-to-width ratio changes, but show a greater number of protrusions along their long axes and fewer protrusions along their short axes compared to controls. Statistical analyses in A and B were done using Student's t-test. Error bars indicate standard deviation. Asterisks mark differences that are statistically significant from control (P<0.01). Numbers of cells analyzed are indicated in parentheses. DMZ and VMZ cells used to determine length-to-width ratios were also used to determine number of protrusions per cell. Cells and explants were co-injected with myristoylated GFP (200 pg) to highlight plasma membranes and trace injected cells. Scale bars: 25 µm in A; 20 µm in B.

 

Figure 4
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  		Fig. 4. Lrp6 localizes in a polarized distribution in cells that undergo convergent extension and is adjacent to actin-rich regions in cellular processes. (A) Animal cap cells show uniform distribution of injected VSVG-lrp6 (VSVG-LRP6; 2 ng). In Activin-treated caps the staining of VSVG-Lrp6 is polarized. (B) DMZ explants from VSVG-Lrp6-injected embryos show preferential localization of Lrp6 at the posterior cell edge. High magnification views of cells in the DMZ are shown in the bottom panels. Injection of myristoylated, plasma membrane localized GFP (mGFP; 500 pg) or anti-actin staining was performed as controls for animal cap and DMZ experiments, respectively. Explants are outlined with solid lines to make their borders readily identifiable. (C) Lrp6 is adjacent to actin-rich regions of cellular extensions. DMZ cells injected with VSVG-Lrp6 were dissociated, plated on fibronectin, immediately fixed, and stained with anti-VSVG (green) and phalloidin (red). Scale bars: animal caps, 30 µm; DMZ (40x), 200 µm; high-magnification DMZ (75x), 50 µm; dissociated cells (200x), 20 µm; high-magnification dissociated cells (350x), 5 µm.

 

Figure 5
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  		Fig. 5. Lrp6 antagonizes Wnt/PCP signaling upstream of Dishevelled and JNK. (A) Injection of suboptimal amounts of wnt11 mRNA (Xwnt; 160 pg), an activator of Wnt/PCP signaling, or suboptimal amounts of lrp6 mRNA (LRP6; 1 ng) causes incomplete block of activin-induced animal cap elongation. Injection of suboptimal lrp6 mRNA (1 ng) reverses the partial block by Wnt11 (160 pg) of activin-mediated animal cap elongation, suggesting that Lrp6 opposes Wnt11-mediated activation of the Wnt/PCP pathway. Injection of suboptimal Lrp6MO (30 ng) reinforces the Wnt11-mediated partial block of activin-treated animal caps, further demonstrating that Lrp6 antagonizes Wnt/PCP signaling. Double asterisks for Wnt11+Lrp6 (Xwnt11/LRP6) indicate statistically significant differences from Wnt11 and Lrp6 (P<0.01). Triple asterisks for Wnt11+Lrp6MO mark differences statistically significant from Wnt11 and Lrp6MO (P<0.01). (B) Dsh-GFP translocates to the cell cortex upon Wnt/PCP activation. Injection of Wnt11 (400 pg) or Lrp6MO (40 ng) promotes cortical translocation of Dsh-GFP. By contrast, injection of the Wnt/ß-catenin ligand, Wnt8 (Xwnt; 40 pg), has no significant (bar chart) effect on XDsh-GFP localization. (C) Jun-N-terminal kinase (JNK) is phosphorylated and localized to the nucleus in animal caps injected with dsh (XDsh; 1 ng) (160 pg) or Lrp6MO (40 ng). Animal caps injected with ß-catenin mRNA (50 pg) demonstrate minimal phospho-JNK staining or nuclear localization similar to uninjected control. Student's t-test was used for statistical analysis. Error bars indicate standard deviation. Asterisks mark differences that are statistically significant from control (P<0.01). Numbers of explants/cells scored are indicated in parentheses. Scale bars: 500 µm in A; 50 µm in B; 100 µm in C.

 

Figure 6
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  		Fig. 6. A small intracellular domain of Lrp6 is sufficient to mediate its convergent-extension activity. (A) Serial deletions of the intracellular domain of Lrp6 demonstrate that a 36 amino acid fragment, Lrp6-B, can mediate its convergent-extension activity. Dark red boxes indicate PPP(S/T)P motifs. All constructs have an N-terminal myristoylation sequence. Percentages of normal (>70% ACEL; average control embryo length), mildly affected (30-70% ACEL), or severely affected (<30% ACEL) embryos are shown (bar graph). Each construct (500 pg mRNA) was used in at least three independent experiments. Numbers of embryos scored are shown in parentheses. (B) Sequence comparison of the B domain of human Lrp6 (hLRP6-B), mouse Lrp6 (mLRP6-B) and Xenopus Lrp6 (XLRP6-B). Numbers in parentheses indicate the amino acid position of the start of the B domain. Identical amino acids are in blue. (C) Embryos and explants from embryos injected with lrp6-B mRNA (2 ng) show severe convergent-extension defects; Lrp6-B (1.6 ng) synergizes with Lrp6 (1 ng) in blocking animal cap elongation. Lateral views of stage 26 embryos are shown. In the bar graph, single asterisks indicate statistically significant differences from control (P<0.01), and double asterisks indicate statistically significant differences from Lrp6 and Lrp6-B values (P<0.01). Numbers of caps scored are indicated in parentheses. (D) Lrp6-B does not induce expression of Wnt/ß-catenin target genes, siamois and nr3 (Xnr3), or act in a dominant-negative manner to inhibit Wnt8-induced expression of siamois and nr3 in animal caps. Loading control: ODC (ornithine decarboxylase). Whole embryos (WE) were used as positive control. RT, reverse transcriptase. (E) In contrast to dominant negative Dishevelled (Xdd), transient transfection of Lrp6-B in HEK293 cells does not upregulate TOPFLASH or inhibit Wnt3a-induced TOPFLASH activity. Student's t-test was used for statistical analysis. Error bars indicate standard deviation. Scale bar: 500 µm in C.

 

Figure 7
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  		Fig. 7. Evidence for coupling of Lrp6 functions: inhibition of Wnt/PCP signaling and activation of Wnt/ß-catenin signaling. (A) In contrast to Lrp6, injection of a mutant Lrp6 that cannot activate the Wnt/ß-catenin pathway (LRP6-m5) does not inhibit activin-mediated elongation of animal caps. Equivalent amounts of lrp6 or lrp6-m5 mRNA (2 ng) were injected. (B) lrp6-B mRNA (3 ng) alone does not induce secondary axis formation in Xenopus embryos, but potentiates the activity of wnt8 mRNA (6 pg) in secondary axis assays. Numbers of embryos injected are shown in parentheses. Asterisk indicates statistically significant differences (P<0.01). (C) Model in which Wnt ligands promote assembly of active Lrp6/Frizzled (FZ) receptor complexes that recruit Dishevelled and actively inhibit Wnt/PCP signaling (via as yet unknown mechanisms) through the B domain of Lrp6.

 

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© The Company of Biologists Ltd 2007