December 1, 2009;
The involvement of lethal giant larvae and Wnt signaling in bottle cell formation in Xenopus embryos.
Lethal giant larvae
) plays a critical role in establishment of cell polarity in epithelial cells. While Frizzled/Dsh
signaling has been implicated in the regulation of the localization and activity of Lgl
, it remains unclear whether specific Wnt ligands are involved. Here we show that Wnt5a
triggers the release of Lgl
from the cell cortex into the cytoplasm
with the concomitant decrease in Lgl
stability. The observed changes in Lgl
localization were independent of atypical PKC (aPKC
), which is known to influence Lgl
distribution. In ectodermal cells, both Wnt5a
triggered morphological and molecular changes characteristic of apical constriction, whereas depletion of their functions prevented endogenous and ectopic bottle cell
formation. Furthermore, Lgl
RNA partially rescued bottle cell
formation in embryos injected with a dominant negative Wnt5a
construct. These results suggest a molecular link between Wnt5a
that is essential for apical constriction during vertebrate gastrulation.
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References [+] :
Fig. 4. Wnt5a and Lgl are required for activin-dependent induction of ectopic bottle cells. (A) DN-Wnt5a RNA (1.5 ng), Lgl MO1 (40 ng) or Lgl MO2 (4 ng) interfere with activin βB RNA (4 pg)-induced pigment accumulation, which marks ectopic bottle cells. (B) Morphological changes induced by activin βB in ectodermal cells are inhibited by DN-Wnt5a, Lgl MO1 or Lgl MO2, but not by the control MO. Four-cell embryos were injected with RNAs and MOs as indicated, using mCherry RNA as a lineage tracer. mCherry (red) was visualized in cryosections of superficial ectoderm at early gastrula stages directly using epifluorescence. (C) Definition of the apical index (A.I.), which is the cell length (L) divided by the apical width (A.W). (D) Apical index in ectodermal cells from embryo cryosections depicted in (B). Error bars denote standard deviation. n, the number of cells measured for each group. (E) DN-Wnt5a RNA and Lgl MOs do not influence target gene activation by activin βB in animal cap explants as analyzed by RT-PCR. FGFR, a loading control. AC, uninjected animal cap cells. −RT, no reverse transcriptase control. WE, whole embryo.
Fig. 6. Roles for Wnt5a and Lgl in apical F-actin and phospho-MLC accumulation in endogenous bottle cells. (A) DN-Wnt5a, but not Lgl MO, inhibits apical accumulation of F-actin in bottle cells. Phenotype frequencies: Co MO, 79% (n = 34); DN-Wnt5a, 88% (n = 52); Lgl MO1, 66% (n = 44); Lgl MO2, 71% (n = 42). (B) DN-Wnt5a and Lgl MO interfere with apical localization of phospho-MLC in bottle cells. Phenotype frequencies: Co MO, 75% (n = 40); DN-Wnt5a, 85% (n = 40); Lgl MO1, 85% (n = 42); Lgl MO2, 80% (n = 40). (A, B) Arrowheads mark presumptive dorsal blastopore. Arrows indicate the orientation of bisected embryos. Vg, vegetal pole; An, animal pole. Embryos coinjected dorsally with DN-Wnt5a (1.5 ng), Lgl MO1 (40 ng) or Lgl MO2 (5 ng) with mCherry RNA (400 pg) were fixed at stage 10.5, bisected midsagittally and then stained with phallodin-Alexa-488 (A) or an anti-phospho-MLC antibody (B).
Fig. 7. Lgl acts downstream of Wnt5a in regulating bottle cell formation. (A) The inhibitory effect of DN-Wnt5a on blastopore formation is rescued by Lgl. (B) Quantification of the ability of Lgl to rescue the effect of DN-Wnt5a on blastopore formation. (A, B) Embryos were injected dorsally with LacZ RNA (100 pg) as a lineage tracer with or without combination of DN-Wnt5a (1.5 ng) and Lgl (600 pg) as indicated. Arrowheads and arrows indicate the absence and reappearance of endogenous bottle cells in injected areas, respectively. (C) Coinjection of Lgl reverses the disruption of apical F-actin accumulation by DN-Wnt5a. Frequencies of embryos with F-actin accumulation: mCherry control, 100% (n = 30); DN-Wnt5a, 12% (n = 52); DN-Wnt5a + Lgl, 72% (n = 62). Embryos were injected with mCherry RNA alone or with the same combination of RNAs as in (A), midsagittally bisected and stained with phalloidin-Alexa488. Arrow indicates reappearance of the blastopore groove and apical accumulation of F-actin.
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