Wright KD et al. (2015), Cooperative and independent functions of FGF an...

XB-ART-51426

BMC Dev Biol 2015 Oct 06;15:33. doi: 10.1186/s12861-015-0083-8.

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Cooperative and independent functions of FGF and Wnt signaling during early inner ear development.

Wright KD , Mahoney Rogers AA , Zhang J , Shim K .

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BACKGROUND: In multiple vertebrate organisms, including chick, Xenopus, and zebrafish, Fibroblast Growth Factor (FGF) and Wnt signaling cooperate during formation of the otic placode. However, in the mouse, although FGF signaling induces Wnt8a expression during induction of the otic placode, it is unclear whether these two signaling pathways functionally cooperate. Sprouty (Spry) genes encode intracellular antagonists of receptor tyrosine kinase signaling, including FGF signaling. We previously demonstrated that the Sprouty1 (Spry1) and Sprouty2 (Spry2) genes antagonize FGF signaling during induction of the otic placode. Here, we investigate cross talk between FGF/SPRY and Wnt signaling during otic placode induction and assess whether these two signaling pathways functionally cooperate during early inner ear development in the mouse. METHODS: Embryos were generated carrying combinations of a Spry1 null allele, Spry2 null allele, β-catenin null allele, or a Wnt reporter transgene. Otic phenotypes were assessed by in situ hybridization, semi-quantitative reverse transcriptase PCR, immunohistochemistry, and morphometric analysis of sectioned tissue. RESULTS: Comparison of Spry1, Spry2, and Wnt reporter expression in pre-otic and otic placode cells indicates that FGF signaling precedes and is active in more cells than Wnt signaling. We provide in vivo evidence that FGF signaling activates the Wnt signaling pathway upstream of TCF/Lef transcriptional activation. FGF regulation of Wnt signaling is functional, since early inner ear defects in Spry1 and Spry2 compound mutant embryos can be genetically rescued by reducing the activity of the Wnt signaling pathway. Interestingly, we find that although the entire otic placode increases in size in Spry1 and Spry2 compound mutant embryos, the size of the Wnt-reporter-positive domain does not increase to the same extent as the Wnt-reporter-negative domain. CONCLUSIONS: This study provides genetic evidence that FGF and Wnt signaling cooperate during early inner ear development in the mouse. Furthermore, our data suggest that although specification of the otic placode may be globally regulated by FGF signaling, otic specification of cells in which both FGF and Wnt signaling are active may be more tightly regulated.

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Species referenced: Xenopus
Genes referenced: ctnnb1 foxi2 fzd3 fzd7 pax2 pax8 spry1 spry2 wnt1 wnt5a wnt5b wnt6 wnt8a

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	Fig. 1. Comparison of Spry1, Spry2, and Wnt reporter expression domains from PPR to otic placode stages. In situ hybridization analysis of Spry1 and Spry2 expression domains compared to Wnt reporter activity in TCF/Lef-lacZ embryos at the stages indicated. Transverse sections are shown, dorsal oriented to the top. a – c Spry1 expression, Spry2 expression, and TCF/Lef-lacZ reporter activity at early somite stages in the posterior PPR. Arrowhead indicates the presumptive otic/epibranchial region. Little or no TCF/Lef-lacZ reporter activity is detected in the posterior PPR at this stage (c). (d – f’) Spry1 expression, Spry2 expression, and TCF/Lef-lacZ reporter activity in anterior (d – f) and posterior (d’ – f’) transverse sections through the OEPD. The entire OEPD is bracketed. (g – i”) Spry1 expression, Spry2 expression, and TCF/Lef-lacZ reporter activity in anterior (g – i), medial (g’ – i’), and posterior (g” – i”) transverse sections through the otic placode (bracketed). Abbreviations: neural ectoderm (ne), endoderm (ed), hindbrain (hb). Scale bar, 50 μm
	Fig. 2. Wnt8a expression in a Spry gene dosage series. In situ hybridization analysis of Wnt8a expression in embryos in which Spry1 and Spry2 genes have been combinatorially inactivated. a – e Wnt8a expression in the hindbrain is indicated (brackets) in dorsal views with anterior to the top. Expansions of gene expression domains are highlighted (asterisk). For each genotype, the percentage of embryos with expanded Wnt8a expression domains is indicated. Scale bar, 100 μm
	Fig. 3. Expression patterns of genes encoding Wnt ligands and receptors are unchanged in Spry1 −/− ; Spry2 −/− embryos. a – j In situ hybridization analyses of genes encoding Wnt ligands and receptors in Spry1 −/− ; Spry2 −/− embryos and controls at OEPD or early otic placode stages. Lateral views of whole-mount embryos are shown; embryos are oriented as indicated. Pre-otic regions are bracketed. k Semi-quantitative reverse-transcriptase PCR analyses of candidate Wnt and Fzd transcript levels in RNA collected from 5 – 7 s hindbrain and OEPD-containing tissue microdissected from the genotypes indicated. Wnt genes (Wnt1, Wnt3a, Wnt6, and Wnt8a) with known expression adjacent to the OEPD are controls. Scale bar, 100 μm
	Fig. 4. Wnt reporter activity in Spry1 −/− ; Spry2 −/− mutant and control embryos at OEPD and otic placode stages. a, b Wnt reporter activity in Spry1 −/+ ; Spry2 −/+ control and Spry1 −/− ; Spry2 −/− mutant embryos. The OEPD region is bracketed; mid-hindbrain region, (mh). Lateral views of whole-mount embryos are shown. c Wnt reporter activity in a Spry1 −/+ ; Spry2 −/+ control embryo. The otic placode is outlined (white dots). The plane of section shown in (d) is indicated with a white line. d Transverse section through the otic placode in a Spry1 −/+ ; Spry2 −/+ control embryo. An example of the location from which LacZ+ placode lengths were measured is shown with a yellow line. An example of the location from which LacZ- placode lengths were measured is shown with a purple line. Total placode lengths represent the sum of LacZ+ and LacZ- length measurements. e Average fold basal area difference between Spry1 −/+ ; Spry2 −/+ control and Spry1 −/− ; Spry2 −/− mutant embryos at 10 – 13 s. Basal area for each placode was calculated as the sum of length measurements multiplied by the thickness of each section. Individual area measurements were normalized by the average area measurement in Spry1 −/+ ; Spry2 −/+ controls. , p < 0.05; * p < 0.001. f Graphical representation of medial-to-lateral total placode, LacZ+, and LacZ- lengths of the same embryos represented in (e). Measurements for each individual otic placode are shown: total placode lengths are shown in grey, LacZ+ lengths in light blue, and LacZ- lengths in light purple. For each genotype, measurements from individual otic placodes were aligned by the maximal total placode length, represented by “0” on the x-axis. The average total placode length, LacZ- length, and LacZ- length are shown with a darker line. Scale bar (a – c), 100 μm
	Fig. 5. Partial rescue of otic phenotypes Spry1 −/− ; Spry2 −/− mutants by reducing the dosage of β-catenin. a – c In situ hybridization analysis to detect Pax8 expression in the otic placode (outlined with white dots). c No rescue of otic placode expansions were observed in Spry1 −/− ; Spry2 −/− ; β-catenin −/+ embryos, as indicated. d – f In situ hybridization analysis to detect Foxi2 expression in epidermal/epibranchial cells surrounding the otic placode. The Foxi2-negative, otic region is outlined with white dots. f A Spry1 −/− ; Spry2 −/− ; β-catenin −/+ embryo in which the Foxi2 expression pattern appeared more similar to normal control embryos (d), rather than Spry-deficient embryos (e). The percentage of Spry1 −/− ; Spry2 −/− ; β-catenin −/+ embryos with partial rescue of the Foxi2 expression pattern is indicated. g – i E-cadherin antibody stain on whole-mount embryos to reveal the extent of closure of the otic cup. i A Spry1 −/− ; Spry2 −/− ; β-catenin −/+ embryo in which the otic cup is more closed than any Spry-deficient control (see H). The percentage of Spry1 −/− ; Spry2 −/− ; β-catenin −/+ embryos in which otic cup closure was partially rescued is indicated. j Average anterior-posterior lengths. Only the subset of Spry1 −/− ; Spry2 −/− ; β-catenin −/+ embryos in which Foxi2 expression domains appeared more similar to normal were selected for length measurement. Scale bar (a – f), (g – i), 100 μm

References [+] :

Abelló, Independent regulation of Sox3 and Lmx1b by FGF and BMP signaling influences the neurogenic and non-neurogenic domains in the chick otic placode. 2010, Pubmed