Reis AH and Sokol SY (2020), Rspo2 antagonizes FGF signaling during vertebra...

XB-ART-56959

Development 2020 May 27;14710:. doi: 10.1242/dev.189324.

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Rspo2 antagonizes FGF signaling during vertebrate mesoderm formation and patterning.

Reis AH, Sokol SY.

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R-spondins are a family of secreted proteins that play important roles in embryonic development and cancer. R-spondins have been shown to modulate the Wnt pathway; however, their involvement in other developmental signaling processes have remained largely unstudied. Here, we describe a novel function of Rspo2 in FGF pathway regulation in vivo Overexpressed Rspo2 inhibited elongation of Xenopus ectoderm explants and Erk1 activation in response to FGF. By contrast, the constitutively active form of Mek1 stimulated Erk1 even in the presence of Rspo2, suggesting that Rspo2 functions upstream of Mek1. The observed inhibition of FGF signaling was accompanied by the downregulation of the FGF target genes tbxt/brachyury and cdx4, which mediate anterioposterior axis specification. Importantly, these target genes were upregulated in Rspo2-depleted explants. The FGF inhibitory activity was mapped to the thrombospondin type 1 region, contrasting the known function of the Furin-like domains in Wnt signaling. Further domain analysis revealed an unexpected intramolecular interaction that might control Rspo2 signaling output. We conclude that, in addition to its role in Wnt signaling, Rspo2 acts as an FGF antagonist during mesoderm formation and patterning.

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Species referenced: Xenopus laevis
Genes referenced: cdx4 eef1a1 fgf2 fgfr1 map2k1 mapk1 msgn1 rspo2 smad2 tbxt
GO keywords: formation of primary germ layer [+]
???displayArticle.antibodies??? FLAG Ab1 GFP Ab7 Mapk1/3 Ab23 Mapk1 Ab7
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???displayArticle.disOnts??? cancer [+]
???displayArticle.omims??? COLORECTAL CANCER; CRC

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	Fig. 1. Rspo2 inhibits ectoderm response to FGF but not MEK1. (A-G) Four-cell stage embryos were injected animally with Rspo2 RNA (0.5â€…ng) and Mek1CA RNA (12â€…pg), as indicated. Ectoderm explants were dissected at stage 8 and treated with 100â€…ng/ml FGF2 protein. When control embryos reached stage 13, the explant morphology was imaged (A-D) or lysed for immunoblot or RT-qPCR analysis (E-G). (A) control uninjected ectoderm explants. (B) FGF-treated explants. (C) Rspo2-expressing explants stimulated with FGF. (D) Rspo2-expressing explants. Ten ectoderm explants were used per group in each experiment. The experiments were repeated five times. (E,F) Modulation of Erk1 activation by Rspo2. Immunoblotting was carried out with the antibodies against pErk1 and total Erk1. Data represent three to five independent experiments. (G) Rspo2 inhibits FGF-dependent induction of tbxt. RT-qPCR analysis was performed for tbxt and normalized by eef1a1. The graph shows a representative experiment with triplicate samples from three independent experiments. Data are meanÂ±s.d. Statistical significance was assessed by Student's t-test. **P
	Fig. 2. Rspo2 depletion promotes FGF signaling. (A,B) Two-cell embryos were injected animally with RspoMOATG (10â€…ng) or RspoMOSB (20â€…ng). Ectoderm explants were dissected at stage 8, treated with 25â€…ng/ml of FGF2 and cultured until stage 13. (A) Representative morphology of the embryos. (B) Quantification of the data in A, representative of two independent experiments. (C) RT-qPCR shows enhanced tbxt expression in FGF-stimulated ectodermal explants (stage 13) after Rspo2 depletion. (D,E) Enhanced cdx4 and msgn1 expression in the dorsal marginal zone (DMZ) explants depleted of Rspo2. RT-qPCR was carried out in stage 13 DMZ explants that were isolated at stage 10. Data are meansÂ±s.d. for triplicate samples. Graphs are representative of three independent experiments. (F) In situ hybridization with antisense cdx4 probes was carried out with stage â‰¥10 control embryos and embryos injected marginally four times with 10â€…ng of RspoMOATG. The number of embryos with the displayed phenotype and the total number of injected embryos are shown. (G) Two dorsal animal blastomeres of four-cell embryos were injected with RMOATG or RMOSB (10-20â€…ng each). Representative embryos are shown at stage 39. Arrowheads point to the eye (white) and the cement gland (black). The graph presents frequencies of embryos with head defects (missing eyes, cement gland and reduced facial structures). Numbers of embryos per group are shown at the top of each bar. Data are representative of three to four independent experiments. **PPt-test. Scale bar: 300â€…Âµm.
	Fig. 3. Mapping FGF inhibitory activity to the TSP domain. (A) Schematic of Rspo2 constructs Rspo2, RspoÎ´F and RspoÎ´T. (B-K) Four-cell embryos were injected with 0.5â€…ng Rspo2, RspoÎ´F or RspoÎ´T RNA each, as indicated, and cultured until stage 8. Ectoderm explants were dissected, treated with 100â€…ng/ml of FGF2 with or without SU5402 (100â€…ÂµM, final concentration), and cultured until stage 13. (B-I) Explant morphology is shown for unstimulated explants (B-E) and FGF2-stimulated explants (F-I). (J,K) Effects of Rspo2 constructs on FGF-dependent Erk1 activation. Immunoblot analysis was carried out with the antibodies against pErk1 and total Erk1. Scale bar: 300â€…Âµm.
	Fig. 4. The intramolecular interaction of protein domains in Rspo2. Four-cell stage embryos were injected with RNAs encoding RspoÎ”F-GFP, RspoÎ”T-Flag alone or co-injected. The embryos were cultured until stage 12 and lysed for GFP pulldown. (A) RspoÎ”T-Flag is co-immunoprecipitated (IP) by RspoÎ”F-GFP. (B) RspoÎ”T-Flag is co-immunoprecipitated by Rspo2-GFP. (C) Putative mechanistic model of FGF pathway inhibition by Rspo2. The association of Rspo2 with HSPGs prevents FGF ligand binding and signaling.
	Fig. S1. Phenotypes of embryos injected with Rspo2 RNA. Four-cell stage embryos were injected into two dorsal blastomeres with Rspo2 RNA (0.5 ng) and allowed to develop until neurula (A, B) or tailbud (C) stages. (A) Uninjected control embryo, stage 19. (B-C) Rspo2-expressing embryos. Open blastopore and posterior defects are apparent. Representative embryos are shown, with more than 20 embryos per group from five separate experiments. The number of embryos displaying the phenotype and the total number of embryos are indicated.
	Fig. S2. Validation of Rspo2 knockdown in vivo. A, Embryos were injected with Rspo2-GFP RNA (500 pg) alone or coinjected with increasing amounts of RspoMOATG (10, 20, and 30 ng). Lysates were prepared from injected embryos at stage 11 for immunoblotting with anti- GFP antibody. CoMO (control MO). Co, control uninjected embryo. Erk1 is a control for loading. B, Schematic of RT-PCR to detect changes in Rspo2 RNA splicing. The PCR fragment of 681 bp corresponds to three exons expected in a control embryo. The 412 bp DNA fragment is expected for Rspo2.L RNA with un-spliced exon 2. RT-PCR was carried out with RNA prepared from stage 11 embryos previously injected with RspoMOSB (20 ng). PCR fragments corresponding to a control embryo (Co) and two different embryos injected with RspoMOSB are shown.
	Fig. S3. Expression levels of Rspo2 constructs. RNAs encoding different Flag-tagged Rspo2 constructs (see Fig. 3A) were injected into four cell embryos, ectoderm explants were isolated at midblastula stages and cultured until stage 11 for immunoblotting with anti- Flag antibody.
	Fig. S4. Lack of Rspo2 effects on Activin/Nodal signaling. RNAs encoding different Flag-tagged Rspo2 constructs were injected into four cell embryos, ectoderm explants were isolated at midblastula stages and stimulated with 0.5 ng/ml of Activin A for 30â€™. Cell lysates were separated by PAGE and immunoblotted with anti-phospho-Smad2 and anti-Flag antibody.
	Fig. S5. Lack of Rspo2 association with FGFR1. RNAs encoding different GFP- tagged Rspo2 constructs and/or FGFR1-Flag were injected into four-cell embryos, ectoderm explants were isolated at midblastula stages and cultured until stage 12 for immunoprecipitation (IP) with GFP. FGFR1 is not pulled down with Rspo2-GFP or Rspoâˆ†F-GFP.

References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase