Species-specific sensitivity to TGFβ signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution
- Department of Orthopaedic Surgery, University of California, San Francisco, United States
Figures
Figure 1 with 1 supplement
Species-specific differences in spatial domains and levels of TRAP, MMP13, Tgfβ1, and Tgfβr1.
Sections of lower jaws stained with trichrome (osteoid matrix of bone is blue) in (A) quail (n=4) versus (B) duck (n=4) at HH40. (C) Adjacent sections stained for TRAP activity (red) reveal more …
Figure 1—figure supplement 1
Levels of TRAP activity, MMP13, Tgfβ1, and Tgfβr1 expression in chick.
Adjacent sections of lower jaws in chick (n=4), stained for trichrome, TRAP (red), MMP13 (pink), Tgfβ1 (yellow), and Tgfβr1 (white). (A) Adjacent sections through the more proximal angular bone …
Figure 2 with 4 supplements
Relative mRNA and protein levels of TGFβ pathway members and targets in the developing lower jaws of chick, quail, and duck.
(A) qPCR analyses show that Tgfβ1 mRNA increases in chick (blue circles, n=8) and in quail (red squares, n=8) at HH37 and does not change in duck (yellow triangles, n=10) in later developmental …
Figure 2—figure supplement 1
Relative mRNA levels of TGFβ pathway members and targets in lower jaws of chick, quail, and duck.
(A) qPCR analyses reveal Tgfβ2 expression does not change in chick (blue circles; n=8) and quail (red squares; n=8) over time; however, in duck (yellow triangles; n=10), there is a decrease at HH37 …
Figure 2—figure supplement 2
Representative western blot images.
Protein levels for (A) phosphorylated (p) SMAD3 (50 kDa) and (B) MMP13 (54 kDa) in chick (n=9), quail (n=12), and duck (n=12) jaws from stages HH31 to HH40 with β-Actin (42 kDa) as a loading …
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Figure 2—figure supplement 2—source data 1
Western blot images for phosphorylated SMAD and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig2-figsupp2-data1-v2.pdf
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Figure 2—figure supplement 2—source data 2
Western blot images for MMP13 and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig2-figsupp2-data2-v2.pdf
Figure 2—figure supplement 3
Relative mRNA levels for markers of bone remodeling in the developing lower jaws of chick, quail, and duck.
(A) qPCR analyses reveal Mmp14 expression does not change in chick (n=8) and quail (n=8), or duck (n=10). (B) Ctsk increases in chick and quail at HH37. There is no change in duck. There is higher …
Figure 2—figure supplement 4
RNA sequencing analysis of TGFβ pathway members in the lower jaws of chick, quail, and duck at HH37.
Normalized read counts for (A) Tgfβ1, which is higher in quail (n=2) compared to chick (n=2) and duck (n=2). (B) Tgfβ2 is not different among chick, quail, and duck. (C) Tgfβ3 is higher in chick …
Figure 3 with 3 supplements
Sensitivity of chick and duck cells to rTGFβ1 and effects of TGFβR1 and SMAD3 inhibition on RUNX2 and MMP13.
(A) pSMAD3 protein levels in cells treated for 2 hr with 5 ng/ml rTGFβ1 (dark gray) in chick (blue) and duck (yellow) cells show a significant induction in chick (n=8). (B) Chick and duck cells …
Figure 3—figure supplement 1
Representative western blot images.
Protein levels for (A) phosphorylated (p) SMAD3 (50 kDa) and (B) MMP13 (54 kDa) in chick (DF-1) and duck (CCL-141) cells treated with 5 ng/ml rTGFβ1 for 2 hr. β-Actin (42 kDa) is used as a loading …
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Figure 3—figure supplement 1—source data 1
Western blot images for pSMAD and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig3-figsupp1-data1-v2.pdf
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Figure 3—figure supplement 1—source data 2
Western blot images for MMP13 and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig3-figsupp1-data2-v2.pdf
Figure 3—figure supplement 2
Sensitivity of chick and duck cells to rTGFβ1 and effects on TGFβ target genes.
A) Pai1 in chick (blue) and duck (yellow) cells treated for 1–24 hr with rTGFβ1 (dark gray). rTGFβ1 induces chick Pai1 mRNA at 24 hr, while duck Pai1 increases at 1 hr and 24 hr (n=8). (B) Mmp2 …
Figure 3—figure supplement 3
Representative western blot images.
Protein levels for (A) RUNX2 (55 kDa) and (B) MMP13 (54 kDa) in chick cells treated for 24 hr with rTGFβ1, TGFβR1 inhibitor, a combination of both rTGFβ1 and TGFβR1 inhibitor, SMAD3 inhibitor, and a …
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Figure 3—figure supplement 3—source data 1
Western blot images for RUNX2 and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig3-figsupp3-data1-v2.pdf
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Figure 3—figure supplement 3—source data 2
Western blot images for MMP13 and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig3-figsupp3-data2-v2.pdf
Figure 4 with 2 supplements
Sensitivity of chick, quail, and duck lower jaws to rTGFβ1 and the effects on RUNX2 and MMP13.
(A) HH34 chick, (B) quail, and (C) duck heads in ventral view showing the boundaries (dashed line) of the lower jaws dissected for analyses and culture experiments. (D) Chick (blue), quail (red), …
Figure 4—figure supplement 1
Representative western blot images.
Protein levels for (A) pSMAD3 (50 kDa) and (B) MMP13 (54 kDa) in chick, quail, and duck lower jaws treated in culture with 10, 25, and 50 ng/ml of rTGFβ1 for 6 hr or 24 hr, respectively (n=12). …
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Figure 4—figure supplement 1—source data 1
Western blot images for pSMAD and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig4-figsupp1-data1-v2.pdf
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Figure 4—figure supplement 1—source data 2
Western blot images for MMP13 and β-Actin.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig4-figsupp1-data2-v2.pdf
Figure 4—figure supplement 2
Sensitivity of chick, quail, and duck lower jaws to rTGFβ1 and effects on TGFβ target genes and Tgfβr expression.
(A) Chick (blue), quail (red), and duck (yellow) lower jaws treated in culture with 25 ng/ml (dark gray) of rTGFβ1 for 24 hr. Tgfβr1 does not change with rTGFβ1 in any species (n=10). (B) Tgfβr2 …
Figure 5 with 1 supplement
Effects of TGFβR1, SMAD3, and MMP13 inhibition on TRAP staining and effects of MMP13 overexpression (OE) on jaw length.
Quail lower jaws harvested at HH35 and placed in culture with control beads (left side) and treatment beads (right side) soaked in (A) TGFβR1 inhibitor (TGFβi, n=5), (B) SMAD3 inhibitor (SMAD3i, …
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Figure 5—source data 1
Effects of TGFβR1, SMAD3, and MMP13 inhibition on TRAP staining in quail embryos.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig5-data1-v2.pdf
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Figure 5—source data 2
Effects of MMP13 overexpression on jaw length in duck embryos as visualized by µCT.
- https://cdn.elifesciences.org/articles/66005/elife-66005-fig5-data2-v2.pdf
Figure 5—figure supplement 1
Enzymatic activity and morphometrics following Mmp13 overexpression (OE).
(A) Functional assay to validate the enzymatic activity of overexpressed duck MMP13. HEK293 cells were transfected with pPIDNB (control) or pPIDNB-Mmp13 (Mmp13 OE), and the enzymatic activity of the …
Figure 6 with 1 supplement
Regulation of Mmp13 via species-specific promoter elements.
(A) Schematic of a –2 kb region of the chick, quail, and duck Mmp13 promoter upstream from the transcriptional start site (ATG). There are four SMAD-binding elements in chick, three in quail, and …
Figure 6—figure supplement 1
Sequence logos for RUNX2 and SMADs.
Graphical representation of the binding profiles of the position weight matrices taken from the JASPAR 2020 database for (A) RUNX2, (B) SMAD2/3 heterodimer, (C) SMAD3, and (D) SMAD4. The relative …
Figure 7 with 1 supplement
Response of the Mmp13 promoter to rTGFβ1 in relation to species-specific single nucleotide polymorphisms (SNPs) and RUNX2 and SMAD-binding elements.
(A) Schematic of the proximal region of the chick/quail (–184 bp) and duck (–181 bp) promoters. Chick/quail contain two SNPs (i.e. adenine and guanine (‘AG’)) adjacent to a RUNX2-binding element …
Figure 7—figure supplement 1
SMAD4 and RUNX2 interactions with the chick, quail, and duck Mmp13 promoter.
Electrophoretic mobility shift assay analysis of the chick, quail, and duck Mmp13 biotinylated promoter (i.e. –184 or –181 bp). (A) The chick/quail Mmp13 promoter interaction with increasing …
Additional files
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Supplementary file 1
Materials, reagents, equipment, supplies, and software used in this study.
- https://cdn.elifesciences.org/articles/66005/elife-66005-supp1-v2.docx
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Supplementary file 2
MMP13 antigen sequence.
- https://cdn.elifesciences.org/articles/66005/elife-66005-supp2-v2.docx
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Supplementary file 3
Primer sequences used for PCR and qPCR analysis.
- https://cdn.elifesciences.org/articles/66005/elife-66005-supp3-v2.docx
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Supplementary file 4
Potential binding elements within the Mmp13 promoter predicted via the JASPAR 2020 database.
- https://cdn.elifesciences.org/articles/66005/elife-66005-supp4-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66005/elife-66005-transrepform1-v2.docx
