Kim YJ et al. (2015), Xenopus laevis FGF receptor substrate 3 (XFrs3)...

XB-ART-49972

Dev Biol 2015 Jan 01;3971:129-39. doi: 10.1016/j.ydbio.2014.10.019.

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Xenopus laevis FGF receptor substrate 3 (XFrs3) is important for eye development and mediates Pax6 expression in lens placode through its Shp2-binding sites.

Kim YJ , Bahn M , Kim YH , Shin JY , Cheong SW , Ju BG , Kim WS , Yeo CY .

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Members of the fibroblast growth factor (FGF) family play important roles during various developmental processes including eye development. FRS (FGF receptor substrate) proteins bind to FGFR and serve as adapters for coordinated assembly of multi-protein complexes involved in Ras/MAPK and PI3 kinase/Akt pathways. Here, we identified Xenopus laevis Frs3 (XFrs3), a homolog of vertebrate Frs3, and investigated its roles during embryogenesis. XFrs3 is expressed maternally and zygotically with specific expression patterns throughout the early development. Knockdown of XFrs3 using a specific antisense morpholino oligonucleotide (MO) caused reduction of Pax6 expression in the lens placode, and defects in the eye ranging from microphthalmia to anophthalmia. XFrs3 MO-induced defects were alleviated by wild type XFrs3 or a mutant XFrs3 (XFrs3-4YF), in which the putative tyrosine phosphorylation sites served as Grb2-binding sites are mutated. However, another XFrs3 mutant (XFrs3-2YF), in which the putative Shp2-binding sites are mutated, could not rescue the defects of XFrs3 morphants. In addition, we found that XFrs3 is important for FGF or IGF-induced ERK activation in ectodermal tissue. Taken together, our results suggest that signaling through Shp2-binding sites of XFrs3 is necessary for the eye development in Xenopus laevis.

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Species referenced: Xenopus laevis
Genes referenced: cdx4 eef1a1 egr2 en2 evx1 fgf2 fgf4 fgf8 fgfr1 foxg1 frs2 frs3 grb2 gsc igf1 mapk1 myc odc1 otx2 pax6 pitx3 ptbp1 ptpn11 rax rax2 sox3 sox7 tbxt vegt ventx1 ventx1.2
???displayArticle.antibodies??? Actb Ab2 Akt1 Ab1 Akt1 Ab6 FLAG Ab1 MapK1 Ab5 Mapk1 Ab3
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	Fig. 1. Temporal expression patterns of XFrs3. (A) Schematic representation of the domain structure of XFrs3. Six putative tyrosine phosphorylation sites (Y191, 305, 350, 398, 445, and 480) are marked in red. Myr, myristoylation sequence (MGSCCS, amino acids 1-6); PTB, phosphotyrosine binding domain (aa 7-117); Grb2, putative Grb2-binding sites; Shp2, putative Shp2-binding sites. (B) Levels of XFrs3 transcript are analyzed by RT-PCR using RNA isolated from whole embryos at indicated stages. ODC is used as a loading control. (C, D) Spatial expression patterns of XFrs3 in mid-blastula (C, stage 9) and early gastrula (D, stage 10.5) are analyzed by RT-PCR. Embryos were dissected to animal, marginal and vegetal regions at stage 9, or bisected to dorsal and ventral regions at stage 10.5. WE indicates RNA from whole embryos, and WE (cut) indicates RNA isolated with all dissected regions combined. Levels of marker gene mRNA for each region are examined to access the accuracy of dissection: Sox3, animal region marker; Sox7 and VegT, vegetal region markers; Gsc, dorsal marker; Xvent1, ventral marker. For the negative control of RT-PCR (–RT), RNA from whole embryos was processed as others without reverse transcriptase.
	Fig. 2. Spatial expression patterns of XFrs3. (A) Whole-mount in situ hybridization of XFrs3. Embryos at indicated stages (13, 15, 20, 25, 28 and 35) were subjected to in situ hybridization with XFrs3 antisense probe. For stages 25, 28 and 35, right panels show magnified images of embryos at left panels. At early neurula stage (stage 13), XFrs3 is expressed broadly in the presumptive anterior neuroectoderm, and its expression is gradually localized to the eye field, cement gland and neural tube at mid-neurula and late neurula stages (stages 15 and 20). At early tailbud stages (stages 25 and 28) and tadpole stage (stage 35), XFrs3 transcript is localized in anterior structures of neuroectodermal origin [branchial arches (ba), cement gland (cg), eyes (e) and otic vesicles (ov)], brain (b), spinal cord (sc), somites (s) and pronephros (pn). (B) XFrs3 expression pattern in the eye at stage 35. Embryos at stages 35 were subjected to whole-mount in situ hybridization with XFrs3 antisense probe and then cross-sectioned. XFrs3 is expressed in the lens, the retina, and the brain.
	Fig. 3. Overexpression of wild type or mutant XFrs3 disrupts mesoderm patterning. Indicated amounts of mRNA (per embryo) were injected into equatorial region of 2-cell embryos, and the embryos are scored for phenotypes at stage 33/34. XFrs3 WT, wild type XFrs3; XFrs3-6YF, mutant XFrs3 with Tyr to Phe substitution for all 6 putative tyrosine phosphorylation sites; XFD, dominant negative mutant form of XFGFR1. Anterior is to the left. In the tables, numbers and numbers in parentheses respectively indicate the number of embryos and the percentage of embryos showing particular defect. (A) Indexes I-IV represent the degrees of anterior defects: normal (I) to most severe anterior defects (IV). (B) Indexes I-IV represent the degrees of posterior defects: normal (I) to most severe posterior defects (IV).
	Fig. 4. Knockdown of XFrs3 causes defects in the eye and anterior structures. (A) Indicated amounts (per embryo) of XFrs3 MO or standard negative control MO were injected into equatorial region of 2-cell embryos, and the embryos are scored for phenotypes at stage 33/34. Representative embryos are also photographed at higher magnification to show defects in the eye. (B) Indicated combinations of XFrs3 MO (MO, 7.5 pmole/embryo) and XFrs3 mRNA (5–50 pg/embryo) were injected into equatorial region of 2-cell embryos, and the embryos are scored for phenotypes at stage 38.
	Fig. 5. Knockdown of XFrs3 reduces the expression of eye-specific marker Pax6 but not Rx at late neurula stage. XFrs3 MO or random negative control (Ran.) MO (7.5 pmole each/embryo) was injected along with nuclear β-galactosidase mRNA (250 pg/embryo) into one dorsal blastomere of 4-cell embryos. Embryos were fixed at stage 20, stained for β-galactosidase (red), and then subjected to whole-mount in situ hybridization with indicated antisense RNA probe. Embryos are viewed from anterior with dorsal sides towards the top. The right most panels for Pax6, Pitx3, and Rx are magnified images of embryos on the left. Injections were carried out with 4 separate batches of embryos, and 2 batched of embryos were pooled and randomly chosen for in situ hybridization analyses.
	Fig. 6. Knockdown of XFrs3 reduces the expression of Rx at tailbud stage. (A) XFrs3 MO or random negative control (Ran.) MO (7.5 pmole each/embryo) was injected into one dorsal blastomere of 4-cell embryos. Stage 30 embryos are analyzed by in situ hybridization for Rx. Embryos are viewed from the injected side. (B) XFrs3 MO or random MO (7.5 pmole each/embryo) was injected with or without XFrs3 mRNA (50 pg/embryo) into one dorsal blastomere of 4-cell embryos. Stage 20 embryos are analyzed by in situ hybridization for Pax6. Red and black arrows respectively indicate Pax6 expression in the eye on the MO alone and MO/mRNA-injected sides. For panel B, injections were carried out with 2 separate batches of embryos, and embryos were pooled and processed for in situ hybridization analyses.
	Fig. 7. The putative Shp2-binding sites, but not the putative Grb2-binding sites, are important for XFrs3 function during eye development. Indicated combinations of XFrs3 MO or random (Ran.) MO (7.5 pmole each/embryo) along with wild type (WT) or mutant (4YF, 2YF, or 6YF) XFrs3 mRNA (50 pg each/embryo) were injected into equatorial region of 2-cell embryos. (A) Embryos are scored for phenotypes at stage 38. The percentages of embryos with microphthalmia or anophthalmia are tabulated in the bottom panel. Injections were carried out with 3 separate batches of embryos. (B) Embryos, scored for particular phenotypes in panel A, were sectioned at the level of eyes and stained with hematoxylin/eosin.
	Fig. 8. XFrs3 is important for FGF or IGF-induced phosphorylation of ERK. Indicated combinations of XFrs3 or random MO (7.5 pmole each/embryo) along with wild type (WT) or mutant (4YF, 2YF, or 6YF) XFrs3 mRNA (50 pg each/embryo) were injected into animal region of 2-cell embryos. Animal caps were explanted at stage 8.5, treated with recombinant basic FGF (bFGF) (50 ng/mL) or IGFII (40 ng/mL) protein, and then harvested when sibling embryos reached stage 10. Activation of ERK (p-ERK) and Akt (p-Akt) are analyzed by immunoblotting. Actin is used as a loading control. For panel A, the total protein levels of ERK and Akt are also compared. For panel B, levels of XFrs3 proteins are compared by anti-Flag immunoblotting [Flag (XFrs3)]. (A) The numbers below p-ERK immunoblot (p-ERK/ERK) indicate relative intensities of p-ERK bands after normalization with corresponding ERK bands. The intensity of p-ERK band in MO non-injected and bFGF-treated sample is considered as 1. The intensities of p-ERK and ERK bands were measured by densitometry. (B) The numbers below p-ERK immunoblot (p-ERK/Actin) indicate relative intensities of p-ERK bands after normalization with corresponding Actin bands. The intensity of p-ERK band in MO/mRNA non-injected and bFGF-treated sample is considered as 1.
	Supplement Fig. 1. Amino acid sequence comparison of Xenopus laevis (X) and human (h) Frs proteins. Identical residues are boxed in black. The conserved PTB domains are underlined in red. Six conserved putative tyrosine phosphorylation sites are highlighted in green. Putative SH3 binding sites (PXXP) are underlined in blue. Dashes denote gaps for maximum homologous alignment.
	Supplement Fig. 2. Phylogenetic tree of vertebrate Frs homologs based on amino acid sequences: Xenopus laevis (X), Xenopus tropicalis (Xt), human (h), mouse (m), chick (c), and zebrafish (z). Numbers in parentheses are percentage identity with XFrs3.
	Supplement Fig. 3. XFrs3 induces and enhances FGF-induced expression of mesoderm markers. eFGF mRNA (20 pg/embryo) was injected along with indicated combinations of wild type or mutant XFrs3 mRNA (1 ng each/embryos) to animal region of 2-cell embryos. Animal cap cells were explanted at stage 8 and harvested at stage 10. The expression of mesoderm markers XBra, XCad3 and XHox3 is examined by RT-PCR. EF1alpha is used as a loading control.
	Supplement Fig. 4. XFrs3 interacts with XFGFR1 and XIGFR1. 293 cells were transfected for indicated combinations of XFrs3-Flag, XIGFR1-Myc, XFGFR1-Myc, XIGF1 and eFGF. The interaction of XFrs3 with XIGFR1 or XFGFR1 is examined by immunoprecipitation for XFrs3 using anti-Flag antibody (IP: Flag) followed by immunoblotting for XIGFR1 or XFGFR1 using anti-Myc antibody (IB: Myc). The levels of XIGFR1 and XFGFR1 proteins in lysates are also compared.
	Supplement Fig. 5. XFrs3 MO does not affect the expression of mesoderm markers. Indicatedcombinations of XFrs3 or standard (Std.) negative control MO (pmole/embryo), XFrs3 mRNA (25 pg/embryo) and XFD mRNA (1 ng/embryo) were injected to equatorial regions of 2-cellembryos. The expression of a mesoderm marker muscle actin (M. actin) is examined from stage 20 embryos by RT-PCR. EF1 is used as a loading control. For the negative control of RTPCR (-RT), RNA from whole embryos was processed as others without reverse transcriptase.
	Supplement Fig. 6. Specificity of XFrs3 MO. 2-cell embryos were injected to equatorial region with indicated combinations of XFrs3-Flag mRNA with (+5' UTR) or without (-5' UTR) XFrs3 5' UTR (50 pg/embryo), and random or XFrs3 MO (7.5 pmole/embryo). Embryos were harvested at stage 10, and the expression levels of XFrs3-Flag are compared by anti-Flag immunoblotting [Flag (XFrs3)]. XFrs3 MO specifically inhibited the translation of XFrs3-Flag from mRNA containing the 5'UTR. Actin is used as a loading control.
	Supplement Fig. 7. XFrs3 MO inhibits eye development. XFrs3 or control random MO (7.5 pmole each/embryo) was injected into one dorsal blastomere at 4-cell stage, and the embryos are photographed at stage 40. The left most row shows uninjected side and the second row shows the injected side. The third row shows the injected side of representative embryo at higher magnification.
	Supplement Fig. 8. XFrs3 Mo does not affect the initial expression of eye markers Pax6 and Rx. XFrs3 or random (Ran.) control MO (7.5 pmole each/embryo) was injected to equatorial region of 2-cell embryos, and embryos are analyzed by in situ hybridization for Pax6 and Rx at indicated stages (St. 13 and 15).
	Supplement Fig. 9. Wild type and mutant XFrs3 mRNAs are translated with similar efficiencies. Indicated combinations of XFrs3 MO (7.5 pmole/embryo) and C-terminal Flagtagged XFrs3 mRNA (XFrs3-Flag, 50 pg each/embryo) were injected to equatorial region of 2-cell embryos. The levels of XFrs3-Flag proteins in stage 10 embryos are compared by anti- Flag immunoblotting [Flag (XFrs3)]. Actin is used as a loading control.
	frs3 (fibroblast growth factor receptor substrate 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, animal view.
	frs3 (fibroblast growth factor receptor substrate 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
	frs3 (fibroblast growth factor receptor substrate 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	frs3 (fibroblast growth factor receptor substrate 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anterior left, dorsal up.