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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 - BMC Dev. Biol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Children's Research Institute, Medical College of Wisconsin, Milwaukee, WI, 53226, USA. kwright@mcw.edu.

ABSTRACT

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 allele, Spry2 allele, β-catenin 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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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
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Fig5: 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

Mentions: The earliest otic phenotype that we observed in Spry-deficient embryos was an enlargement of the otic placode [18]. To visualize the otic placode in experimental, Spry-deficient, and heterozygous control embryos, we examined expression of Pax8, an early marker of the otic placode [20], in 9 – 11 s embryos. In experimental embryos, the domain of Pax8 expression in the otic placode looked just as enlarged as in Spry-deficient controls (compare Fig. 5c with Fig. 5a and b; n = 4 experimental embryos, n = 5 Spry-deficient control embryos, n = 7 heterozygous controls). Other genes whose expression patterns mark the otic placode were not suitable for our analysis: the expansion of the Pax2 expression domain in the otic placode in Spry-deficient embryos is incompletely penetrant [18]; expression of Hmx3 is reduced in Spry-deficient embryos [42]; and Dlx5 expression is tightly regulated by Wnt signaling [43]. Thus, we examined the expression pattern of Foxi2, which is expressed in cranial epidermis, but is excluded from the otic placode [44]. At 12 – 14 s, all Spry-deficient controls (n = 6) had enlarged Foxi2-negative domains, suggesting the presence of an enlarged otic placode (compare Fig. 5e to Fig. 5d; n = 18 heterozygous controls). In 57 % (4/7) of experimental embryos, the Foxi2-negative domains were more similar in size to heterozygous controls (compare Fig. 5f to Fig. 5d). Whereas in 43 % (3/7) of experimental embryos, the Foxi2-negative domains appeared more similar in size to Spry-deficient controls (data not shown). To directly examine whether the size of the otic placode was rescued in experimental embryos, we embedded and sectioned embryos to visualize the otic placode anatomically as a thickened epithelium that does not express Foxi2. We measured the anterior-posterior length of the otic placode – a metric that was increased in Spry-deficient embryos (see Fig. 4f) – by multiplying the number of sections containing an otic placode by the thickness of each section (10 μm). Only the subset of experimental embryos in which the Foxi2-negative domain appeared similar to heterozygous controls by whole-mount in situ hybridization was analyzed. Average anterior-posterior lengths of the otic placode in experimental embryos were comparable to lengths in heterozygous controls (Fig. 5j, n = 7 experimental placodes, n = 6 heterozygous placode, p = 0.12), indicating restoration of otic placode expansions in this subset of experimental embryos. In both experimental and heterozygous embryos, average anterior-posterior otic placode lengths were significantly smaller than lengths in Spry-deficient controls (Fig. 5j, n = 8 Spry-deficient placodes, p < 0.001 for both comparisons). Thus, by assessment of the Foxi2-negative domain and direct measurement of the otic epithelium in histological sections, the otic placode/cup size is partially rescued in experimental embryos by 12 – 14 s. The lack of rescue of the otic Pax8 expression domain in 9 – 11 s experimental embryos was not investigated further, but suggests the possibility that formation of the early otic placode is less sensitive to β-catenin gene dosage.Fig. 5


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

Wright KD, Mahoney Rogers AA, Zhang J, Shim K - BMC Dev. Biol. (2015)

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
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Fig5: 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
Mentions: The earliest otic phenotype that we observed in Spry-deficient embryos was an enlargement of the otic placode [18]. To visualize the otic placode in experimental, Spry-deficient, and heterozygous control embryos, we examined expression of Pax8, an early marker of the otic placode [20], in 9 – 11 s embryos. In experimental embryos, the domain of Pax8 expression in the otic placode looked just as enlarged as in Spry-deficient controls (compare Fig. 5c with Fig. 5a and b; n = 4 experimental embryos, n = 5 Spry-deficient control embryos, n = 7 heterozygous controls). Other genes whose expression patterns mark the otic placode were not suitable for our analysis: the expansion of the Pax2 expression domain in the otic placode in Spry-deficient embryos is incompletely penetrant [18]; expression of Hmx3 is reduced in Spry-deficient embryos [42]; and Dlx5 expression is tightly regulated by Wnt signaling [43]. Thus, we examined the expression pattern of Foxi2, which is expressed in cranial epidermis, but is excluded from the otic placode [44]. At 12 – 14 s, all Spry-deficient controls (n = 6) had enlarged Foxi2-negative domains, suggesting the presence of an enlarged otic placode (compare Fig. 5e to Fig. 5d; n = 18 heterozygous controls). In 57 % (4/7) of experimental embryos, the Foxi2-negative domains were more similar in size to heterozygous controls (compare Fig. 5f to Fig. 5d). Whereas in 43 % (3/7) of experimental embryos, the Foxi2-negative domains appeared more similar in size to Spry-deficient controls (data not shown). To directly examine whether the size of the otic placode was rescued in experimental embryos, we embedded and sectioned embryos to visualize the otic placode anatomically as a thickened epithelium that does not express Foxi2. We measured the anterior-posterior length of the otic placode – a metric that was increased in Spry-deficient embryos (see Fig. 4f) – by multiplying the number of sections containing an otic placode by the thickness of each section (10 μm). Only the subset of experimental embryos in which the Foxi2-negative domain appeared similar to heterozygous controls by whole-mount in situ hybridization was analyzed. Average anterior-posterior lengths of the otic placode in experimental embryos were comparable to lengths in heterozygous controls (Fig. 5j, n = 7 experimental placodes, n = 6 heterozygous placode, p = 0.12), indicating restoration of otic placode expansions in this subset of experimental embryos. In both experimental and heterozygous embryos, average anterior-posterior otic placode lengths were significantly smaller than lengths in Spry-deficient controls (Fig. 5j, n = 8 Spry-deficient placodes, p < 0.001 for both comparisons). Thus, by assessment of the Foxi2-negative domain and direct measurement of the otic epithelium in histological sections, the otic placode/cup size is partially rescued in experimental embryos by 12 – 14 s. The lack of rescue of the otic Pax8 expression domain in 9 – 11 s experimental embryos was not investigated further, but suggests the possibility that formation of the early otic placode is less sensitive to β-catenin gene dosage.Fig. 5

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Children's Research Institute, Medical College of Wisconsin, Milwaukee, WI, 53226, USA. kwright@mcw.edu.

ABSTRACT

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 allele, Spry2 allele, β-catenin 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.

Show MeSH
Related in: MedlinePlus