FGF16 suppresses natal down growth and thickens the epithelium through

From the above transcriptome analysis, I noted that FGF10, SNAI1 and TWIST2, which belong to the GO category of epithelial development, showed a higher expression level in AD skin than in PD skin in zebra finch embryos (Cluster L, Figure 10), but the homologs of these genes in chicken showed little differential expression in our

quantitative PCR data (Figure 11). The same comment applies to FGF16, which is in the GO category of the MAPK signaling pathway (Cluster N, Figure 10).

Among the four candidate genes (FGF10, FGF16, SNAI1 and TWIST2), SNAI1 and TWIST2 were two highly expressed genes in the transcriptome and so were selected for whole mount in situ hybridization in zebra finch embryos. By whole mount in situ hybridization in E9 zebra finch, I found that the expression of TWIST2 was throughout the dermis of Type I feather buds (Figure 13A,B,D), but was restricted to the anterior proximal dermis of Type II feather buds (Figure 13A,C,E). A similar expression profile was detected in SNAI1 (Type I feather buds: Figure 13F,G,I; Type II feather buds:

Figure 13F,H,J), which is a zinc finger transcription factor for regulating epithelial to mesenchymal transition (EMT) during embryonic development (Paznekas, et al. 1999).

These data support the association between our predicted genes and the feather bud growth suppression.

In feather development, the MAPK signaling pathway was shown to be the major downstream pathway in response to FGFs (Lin, et al. 2009). Previous knockout of the key component of the MAPK pathway reduced epithelium thickness in mouse (Scholl, et al. 2007). Thus, it appears that the FGF/MAPK pathway participates in the natal down growth suppression. FGF16 is a known upstream signal of SNAI1 in promoting

ovarian cancer cell invasion through activation of the MAPK signaling pathway (Basu, et al. 2014). Therefore, I hypothesized that the up-regulation of FGF16 in AD skin suppresses natal down growth and increases epithelium thickness.

To test this hypothesis, I utilized the RCAS retrovirus to overexpress the FGF16 gene in chicken embryos. Because injecting FGF16 cDNA into the chicken AD skin region caused high lethality (data not shown), I injected it into the legs instead. In each chicken embryo, one leg was injected with the virus carrying the FGF16 cDNA, while the other leg was used as the control. We found that FGF16 overexpressed legs

exhibited a similar phenotype of the zebra finch AD skin region: periodic feather buds were formed, but natal down elongation was suppressed (Figure 14A,C,E). The natal down elongation in the control leg was normal (Figure 14B,D,F). Moreover, bone formation was also influenced by FGF16 overexpression (Figure 14A), supporting a previous prediction (Laurell, et al. 2014). In the paraffin sections of the skin, both the H&E stain and the immunostaining with CDH1 showed thicker epithelia in the FGF16 overexpressed leg skin than in the control leg skin (Figure 14E,G vs. F,H; statistics in Figure 14J). Four independent experiments were conducted for FGF16 overexpression and three individuals with suppressed natal down were shown in Appendix data (Figure A3).

To understand how FGF16 suppresses natal down elongation, I studied the

of FGFR1 in the AD than in PD skin of E9 zebra finch (Table S4). Moreover, SNAI1 and TWIST2 were also up-regulated in the FGF16 overexpressed skin (Figure 15A), although the differences were not statistically significant. Interestingly, FGF10 was up-regulated, while SHH was down-regulated in the FGF16 overexpressed skin (Figure 15A).

To test the relationship between FGF10 and FGF16, FGF10 was overexpressed in the dorsal skins of chicken embryos, resulting in the suppression of the natal down formation (Figure 16A,C vs. B,D), but the expression of FGF16 was not affected (Figure 16E). These observations suggest that FGF10 is not a regulator but a target of FGF16. Thus, I conclude that FGF16 suppresses the natal down elongation through the FGF/MAPK pathway (FGF10, FGFR1, SNAI1 and TWIST2) and the down-regulation of SHH (Figure 15B).

In addtion to FGF16 and FGF10, I also injected several candidate regulators that expressed higher in AD skin than in PD skin in E8 zebra finch embryos to the dorsal region of chicken; however, only RCAS-FGF16 and FGF10 suppressed the natal down elongation. All the tested genes and the microinjection results are shown in the

Appendix data (Figure A4).

Figure 13. Whole mount in situ hybridization of TWIST2 (A-E) and SNAI1 (F-J) in E9 zebra finch and the paraffin sections. (B, C) The enlargements of the dotted-line square regions in (A). (G, H) The enlargements of the dotted-line square regions in (F).

(D, E) the paraffin sections of feather buds of B and C, respectively. (I, J) the paraffin sections of feather buds of G and H, respectively. Red arrows indicate the restrictive

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Figure 14. FGF16 overexpression suppressed the natal down growth, reduced the bone length, and increased the epithelial thickness in E12 chicken. (A, B) The chicken embryo was microinjected with RCAS-FGF16 in one leg, and the other leg was used as the control. (C, D) The enlargement images of Figures A and B, respectively. (E, F) H&E stains of the paraffin sections of FGF16 overexpressed and control skins. (G, H) Immunochemical stain with CDH1 in the paraffin sections of the FGF16 overexpressed and the control skins. (I) AMV-3C2 staining of adjacent sections showing the RCAS virus infected regions. (J) Quantification of the epithelium thickness between the FGF16 overexpressed (white bar) and the control epithelia (black bar). Ep: epithelium;

me: mesenchyme; fb: feather bud. **: p < 0.01 (Student’s t-test). Yellow scale bar: 1 cm, black scale bar: 100 μm.

Figure 15. The quantification of the candidate genes for natal down growth

suppression, and the summary diagram of Type I and Type II feather formations.

(A) The gene expressions in the FGF16 overexpressed (white bar) and control (black bar) skins in chicken were compared by quantitative PCR. Relative expression values were given in mean ± SD from at least three independent experiments. *: p < 0.05; **: p

< 0.01 (Student’s t-test). (B) The summary diagram of Type I and Type II feather formations, and the involved phenotypes (black words) and molecular regulators (pink words).

Figure 16. Overexpression of FGF10 suppressed the natal down growth but had no