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During Xenopus development, embryonic cells dramatically change their shape and position. Rho family small GTPases, such as RhoA, Rac, and Cdc42, play important roles in this process. These GTPases are generally activated by guanine nucleotide exchange factors (GEFs); however, the roles of RhoGEFs in Xenopus development have not yet been elucidated. We therefore searched for RhoGEF genes in our Xenopus EST database, and we identified several genes expressed during embryogenesis. Among them, we focused on one gene, designated xNET1. It is similar to mammalian NET1, a RhoA-specific GEF. An in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. In addition, transient expression of xNET1 activated endogenous RhoA. These results indicated that xNET1 is a GEF for RhoA. Epitope-tagged xNET1 was localized mainly to the nucleus, and the localization was regulated by nuclear localization signals in the N-terminal region of xNET1. Overexpression of either wild-type or a mutant form of xNET1 severely inhibited gastrulation movements. We demonstrated that xNET1 was co-immunoprecipitated with the Dishevelled protein, which is an essential signaling component in the non-canonical Wnt pathway. This pathway has been shown to activate RhoA and regulate gastrulation movements. We propose that xNET1 or a similar RhoGEF may mediate Dishevelled signaling to RhoA in the Wnt pathway.
Fig. 1 Identification of xNET1 in the Xenopus EST library. (A)
Sequence alignment of xNET1 with human NET1 (hNET1; Chan
et al., 1996) and mouse NET1 (mNET1; Alberts and Treisman,
1998). The nuclear localization signals, NLS1 and 2, are indicated
by brown lines. The DH and PH domains are indicated by blue and
red lines, respectively. The star indicates the mutation site for
mNET1(L321E) and xNET1(L267E). (B) Schematic illustration of
the mutant forms of xNET1 that were constructed for this study.
Deleted regions are shown in gray.
Fig. 2 xNET1 expression in Xenopus embryos. (A) RT-PCR
analysis of xNET1 expression during Xenopus development. H4
(Histone H4) was detected as a control. (B) Whole-mount in situ
hybridization with antisense and sense xNET1 probes. A, anterior;
P, posterior.
Fig. 3 xNET1 binds to and activates RhoA. (A) GST-RhoA, -Rac,
and -Cdc42 were produced in bacteria and then bound to
glutathione Sepharose beads. The cell lysate was prepared from
HEK293T cells transiently expressing xNET1. The lysates and
beads were mixed, and the proteins that bound to the beads were
analyzed by Western blotting. (B) HEK293T cells were transfected
with wild-type and mutant forms of xNET1. The cell lysates were
mixed with the immobilized RhoA-binding domain (RBD), which
specifically binds to GTP-bound RhoA. The bound proteins were
analyzed by Western blotting with an anti-RhoA antibody. WT,
wild-type xNET1; DNLS, xNET1 DNLS112; DDH, xNET1
lacking the DH domain.
Fig. 4 xNET1 is localized to the nucleus. (A) Wild-type xNET1
(WT) and xNET1 lacking both NLS1 and 2 (DNLS112) tagged
with the myc epitope were expressed in HeLa cells. Cells were
stained with the anti-myc antibody and FITC-phalloidin. The scale
bar represents 20 mm. (B) mRNAs encoding wild-type and mutant
forms of xNET1 tagged with the myc epitope were expressed in the
ectodermal cells of Xenopus embryos. Cells were immunostained
with the anti-myc antibody. The scale bar represents 100 mm.
Fig. 5 The phenotype of xNET1 mRNA-injected embryos. mRNAs
encoding wild-type xNET1 (WT), DNLS112 (DNLS) mutant (A),
or xNET1L267E(L267E) (B) were injected into two blastomeres of
four-cell embryos. (C) Water was injected as a control and no
phenotype was observed. (D) To examine mesoderm formation, the
injected embryos were immunostained with the somite-specific
antibody 12/101.
Fig. 6 Flag-tagged xNET1 was co-expressed with myc-tagged
Xenopus Dishevelled (myc-Xdsh) or myc-tagged green fluorescent
protein (myc-GFP) in HEK293 T cells. The myc-tagged proteins
were immunoprecipitated with the anti-myc antibody, and the
precipitated proteins were analyzed by Western blotting.
net1 (neuroepithelial cell transforming 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal up.
net1 (neuroepithelial cell transforming 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view view, anteriorleft.
net1 (neuroepithelial cell transforming 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anteriorleft, dorsal up.
