Karaulanov E et al. (2009), Unc5B interacts with FLRT3 and Rnd1 to modulate...

XB-ART-39826

PLoS One 2009 May 29;45:e5742. doi: 10.1371/journal.pone.0005742.

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Unc5B interacts with FLRT3 and Rnd1 to modulate cell adhesion in Xenopus embryos.

Karaulanov E , Böttcher RT , Stannek P , Wu W , Rau M , Ogata S , Cho KW , Niehrs C .

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The FLRT family of transmembrane proteins has been implicated in the regulation of FGF signalling, neurite outgrowth, homotypic cell sorting and cadherin-mediated adhesion. In an expression screen we identified the Netrin receptors Unc5B and Unc5D as high-affinity FLRT3 interactors. Upon overexpression, Unc5B phenocopies FLRT3 and both proteins synergize in inducing cell deadhesion in Xenopus embryos. Morpholino knock-downs of Unc5B and FLRT3 synergistically affect Xenopus development and induce morphogenetic defects. The small GTPase Rnd1, which transmits FLRT3 deadhesion activity, physically and functionally interacts with Unc5B, and mediates its effect on cell adhesion. The results suggest that FLRT3, Unc5B and Rnd1 proteins interact to modulate cell adhesion in early Xenopus development.

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Species referenced: Xenopus
Genes referenced: bambi cdh3 chrd dkk1 flrt3 gata2 krt12.4 myf5 myod1 not rnd1 snai1 snai2 sox3 tbxt tubb2b unc5a unc5b unc5d ventx2.2
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	Figure 1. Unc5 proteins are high affinity interactors of FLRT3.(A) FLRT3 ectodomain binds to cells expressing Unc5. HEK293T cells were transfected (T) with the indicated expression constructs, incubated with AP-tagged FLRT3Î´TM conditioned medium (M) and stained for bound alkaline phosphatase (AP) activity. Schematic representation of the FLRT and Unc5 family domain structures is shown on top. (B) FLRT3 and Unc5D ectodomains interact in solution. Equal amounts of V5-tagged FLRT3Î´TM conditioned medium were mixed with the indicated FLAG-tagged condition media and subjected to immunoprecipitation (IP) and immunoblotting (IB). (C,D) Binding curves and Scatchard analyses of AP-FLRT3Î´TM binding to Unc5B or Unc5D transfected cells. The dissociation constants (Kd) are indicated. (E) The LRR domains of FLRT3 mediate binding to Unc5D. Whole cell lysates of HEK293T cells transfected with the indicated Renilla- (RL-) and V5-tagged constructs were immunoprecipitated with anti-V5 antibody. RL activity of individual samples after IP was normalized to the total activity of the respective lysate. (F) Both the IG and the TSP domains of Unc5D are required for FLRT3 binding. HEK293T cells were transfected with the indicated V5-tagged Unc5D expression constructs, incubated with recombinant AP-FLRT3Î´TM and stained for bound AP activity. Similar levels of protein production were confirmed by V5 immunoblotting (data not shown).
	Figure 2. Unc5B and FLRT3 induce cell deadhesion in Xenopus laevis embryos.(A) Embryos were injected with the indicated mRNAs (1.2 ng FLRT3, 4 ng Unc5B variants) and at blastula stage 9 were photographed 10 min after partial excision of the animal caps. (B) V5 and C-cadherin immunoblots for protein production of sibling embryos from panel A. (C) FLRT3 and Unc5B functionally synergize. Embryos were injected with the indicated mRNAs (0.2 ng FLRT3, 1.6 ng Unc5BÎ´D) and cell deadhesion was assessed in animal caps of blastula stage 8.5 embryos (upper row), as well as in whole embryos at neurula stage 13, where ectodermal thinning and blastocoel edema were observed (lower row, indicated is the fraction of affected embryos). PPL, preprolactin (control mRNA). (D) C-cadherin rescue of Unc5B-induced deadhesion. Embryos were injected with the indicated mRNAs (3 ng Unc5BÎ´D, 1.5 ng C-cadherin) and grown until blastula stage 9 in high-salt medium, which promotes ectodermal lesions when adhesion is inhibited (upper row) or in normal medium (lower row, paraffin sections). (E) Quantification of the C-cadherin rescue effect in embryos from panel D (normal medium).
	Figure 3. Unc5B-FLRT3 interaction in Xenopus tropicalis morphogenesis.(A) Developmental quantitative RT-PCR analysis of FLRT3 and Unc5A-D expression. (Bâ€“C) Expression patterns of FLRT3 and Unc5B at blastula stage 9 and gastrula stage 10.5. The dorsal blastopore lip is indicated with an arrow. DM, dorsal mesoderm. (D) Representative phenotypes of embryos injected with FLRT3 and/or Unc5B Morpholinos (MOs). A mixture of two MOs per gene targeting the ATG and the UTR regions was injected (10 ng each). A stunted axis was observed in 0% (nâ€Š=â€Š29), 3% (nâ€Š=â€Š34), 7% (nâ€Š=â€Š28) and 100% (nâ€Š=â€Š31) of the injected embryos, respectively. Lower row: whole mount in situ hybridization of Xnot at late gastrula stage 12.
	Figure 5. Unc5B acts upstream of the small GTPase Rnd1.(A,B) Rnd1 binds to Unc5BÎ´D. Immunoprecipitation (IP) assays followed by immunoblotting (IB) with lysates of HEK293T cells overexpressing the indicated V5- and HA-tagged proteins. (C) Unc5B and Rnd1 synergize in cell deadhesion. X. laevis embryos were injected with the indicated mRNAs (0.8 ng Unc5BÎ´D, 0.16 ng Rnd1). Loss of adhesion was observed as ectodermal thinning and blastocoel edema at neurula stage 13 (upper row), as well as in paraffin sections of blastula stage 9 embryos (lower row). PPL, preprolactin (control mRNA). (D) Quantification of the deadhesion phenotypes from panel C (stage 9 embryos). (E) Rnd1 Morpholino (MO) injection rescues Unc5B-induced deadhesion. X. laevis embryos were injected sequentially with the indicated MO (40 ng) and mRNA (4 ng), and grown until blastula stage 9 in high-salt medium, which promotes ectodermal lesions when adhesion is inhibited (upper row) or in normal medium (lower row, paraffin sections). (F) Quantification of the Rnd1 MO rescue. Two embryo batches (exp.1,2) were injected as in panel E, grown in normal medium and cell deadhesion was assessed at blastula stage 9.
	unc5b (unc-5 homolog B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, lateral view on left, sagital section on right.
	unc5b (unc-5 homolog B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, lateral view on left, sagital section on right.
	Figure 4. Marker gene expression analysis of the FLRT3/Unc5B morphant embryos.Stage 12 (late gastrula) and stage 19 (late neurula) sibling embryos of those analysed in Figure 3D were assessed by quantitative RT-PCR for expression of the tissue markers Xbra (mesoderm), Xnot, Chordin and Dkk1 (dorsal mesoderm), Bambi, Vent2 and Gata2 (ventral mesoderm), MyoD and Myf5 (paraxial mesoderm and somites), Sox3 and N-tubulin (neuroectoderm and neurons), Slug and Snail (neural crest), and Epidermal Keratin.

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Aoki, Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells. 2000, Pubmed