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The coordinated movement of germ layer progenitor cells reaches its peak at the dorsal side, where the Bmp signaling gradient is low, and minimum at the ventral side, where the Bmp gradient is high. This dynamic cell movement is regulated by the interplay of various signaling pathways. The non-canonical Wnt signaling cascade serves as a pivotal regulator of convergent and extension cellular movement, facilitated by the activation of small GTPases such as Rho, Rab, and Rac. However, the underlying cause of limited cell movement at the ventral side remains elusive. To explore the functional role of a key regulator in constraining gastrulation cell movement at the ventral side, we investigated the Bmp4-direct target gene, sizzled, to assess its potential role in inhibiting non-canonical Wnt signaling. In our current study, we demonstrated that ectopic expression of sizzled led to gastrulation defects in a dose-dependent manner, without altering cell fate specification. Overexpression of sizzled resulted in decreased elongation of Activin-treated animal cap and Keller explants. Furthermore, our immunoprecipitation assay unveiled the physical interaction of Sizzled with non-canonical Wnt ligand proteins (Wnt5 and Wnt11). Additionally, the activation of small GTPases involved in Wnt signaling mediation (RhoA and Rac1) was diminished upon sizzled overexpression. In summary, our findings suggest that Bmp4 signaling negatively modulates cell movement from the ventral side of the embryo by inducing sizzled expression during early Xenopus gastrulation.
Fig. 1. Bmp4 inhibits activin-induced CE movement in Xenopus embryos. (A) Embryos were injected with bmp4 and dnbr mRNAs (1 ng/5 nL each) alone and in combination treated with or without activin protein (50 ng/mL). Animal caps were dissected at early stage 8 and incubated in L-15 media until stage 11 (mid gastrula). Ectodermal explants elongation was observed in each treated animal caps sample sets. (B) Dnbr mRNA (1 ng/5 nL) were injected to embryos in the ventral marginal zone (VMZ) at 4-cell stage and grown until late neurula stage. The lateral and dorsal areas were shown to observe the effect of Bmp signaling inhibition on embryos. (C) Dorsal marginal zone (DMZ) and VMZ were dissected from embryos injected with dnbr mRNA into the VMZ at 4-cell stage. Cell elongation assay were performed to analyze the effect of Bmp inhibition. mRNA, messgenger RNA.
Fig. 2. Bmp regulative response elements reside within the sizzled promoter. (A) List of dorso-ventral marker genes selected from affymetrix gene chip data including sizzled (frzb3). (B) List of dorso-ventral marker genes selected from transcriptome data including sizzled. (C-E) qRT-PCR analysis of the gene expression levels in animal cap explants from embryos injected with BMP4, Smad2, and DNBR. qRT-PCR values were determined from the ΔΔCt for the target genes relative to ODC. Significantly different results for treatments used 1-way ANOVA with Graph Pad Prism; P < .05. (F) Coverage plot of 3F. Smad1 within the sizzled promoter proximal region. (G) Coverage plot of 3flag-Ventx1.1 within the sizzled promoter proximal region. The asterisks represent the statistical significance of the multiple comparisons. Each number of stars (1, 2, 3, or 4) corresponds with a p-value. The more stars there are, the smaller the p-value and the greater the significance. ANOVA, analysis of variance; DNBR, dominant negatine Bmp receptor; ODC, ornithine decarboxylase.
Fig. 3. Sizzled and Frzb3 regulate cell movement during gastrulation. (A) Frzb3 mRNA in a dose-dependent manner (0.5, 1, and 2 ng) were injected into 1-cell and observed morphological changes in embryos. (B) HA-Frzb3 protein expression was confirmed through western blot and the statistical evaluation was done as shown in graph. (C) Both szl/frzb3 wild types and mutants were injected and into 1-cell stage and the dorsal lip maturation was observed. (D) Szl/frzb3 wild and mutant types injected Keller explants were treated with and without Activin protein at early blastula stage. Animal cap elongation assay was performed.
Fig. 4. Frzb3 or sizzled induces gastrulation defects without altering the expression of cell fate marker genes. (A) Frzb3 both variants, S and L mRNAs were injected at 1-cell stage and the animal caps were dissected at stage 8 and incubated in the presence and absence of activin (50 ng/mL) protein. RT-PCR was performed to check the lineage specific marker genes. (B) Frzb3 and ventx1.1 mRNAs were injected to compare their effect on relative expression of cell fate specification marker genes. (C and D) RT-PCR was performed from frzb3 and ventx1.1 mRNAs injected animal cap explants along with activin treated and untreated. RT-PCR, reverse transcription-polymerase chain reaction.
Fig. 5. Frizzled physically interacts with noncanonical Wnt signaling molecules and reduces small GTPases Rac1 and RhoA activation. (A) Flag-tagged Frzb3 were injected alone and in combination with different Myc-tagged Wnt ligands (both canonical and noncanonical Wnts) into 1-cell stage embryos and immunoprecipitation assay were performed to check the physical interaction between Frzb3 and Wnts. (B) and (C) Ha-Rac1 were injected with and without frzb3 mRNA and HA-RhoA were injected with, without, and in combination of frzb3 and szl mRNAs into 1-cell stage. Small GTPase Pulldown assay was performed to observe the small GTPases activation. (D) Proposed model of noncanonical Wnt signaling-mediated cell movement inhibition via Bmp synexpression gene, sizzled.
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