Fig 1.
Hes1 over-expression within limb bud mesenchyme induces preaxial polydactyly.
(A) Alcian Blue/Alizarin Red staining of WT and Prx1Cre;R26-Hes1f/f (Hes1 GOF) mutant E16.5 forelimbs. Red asterisk indicates extra digit. (B) Quantification of digit numbers for WT (N = 52) and HES1 GOF (N = 46) forelimbs (χ2 test) (p-value <0.0001). Y axis represents the number of digits whereas the x-axis represents the percent of the forelimbs. Intervals of 0.5 represent syndactylous digits (S1 Fig). (C-D) Representative X-ray and MicroCT images of 2-month old WT and HES1 GOF forelimbs. Red asterisks indicate the extra digit (N = 4) (E) Digit length analysis of WT and HES1 GOF at 2-months of age (N = 4).
Fig 2.
Hes1 over-expression within limb bud mesenchyme promotes cell proliferation.
(A) BrdU labeling of WT and HES1 GOF limb buds at E11.5 (N = 3) (B) Quantification of BrdU positive cells within the apical zone and anterior domain (N = 3). Asterisks indicate significance with a p-value < 0.05 (Student’s t-test). (C) Western blots of p27 and CCND1 utilizing protein isolated from E11.5 WT and HES1 GOF limb buds. LMNB and TUBULIN are loading controls. Quantification represented as fold change± SD relative to WT N = 3. (D) qPCR for Cdk2, Cdk4, Cdk6 and Cdkn1b using RNA isolated from the anterior halves of WT and HES1 GOF limb buds at E11.5 (N≥4). Each data point represents separate animals. Asterisks indicate significance with a p-value < 0.05 (Student’s t-test). (E) Densitometry measurements of ChIP PCR amplifications from the chromatin containing an E-Box within the Cdkn1b promoter of WT and HES1 GOF limb buds (N = 3; p-value < 0.05; Student’s t-test).
Fig 3.
Hes1 over-expression within limb bud mesenchyme delays chondrogenesis.
(A) WISH for Sox9 on WT and HES1 GOF E11.5 limb buds. ABH/OG staining of WT and HES1 GOF E12.5 limb bud sections. COL2A1 IHC staining of WT and HES1 GOF E12.5 limb bud sections. (B) qPCR for Sox9, Sox5, Sox9, and Acan utilizing RNA from anterior limb bud halves of E11.5 and E12.5 WT and HES1 GOF embryos (N≥ 3; p-value < 0.05; Student’s t-test) Each data point represents separate animals (C) Densitometry measurements of ChIP PCR amplifications from the chromatin containing a N-Box within the Col2a1 enhancer of WT and HES1 GOF limb buds (N = 3; p-value < 0.05; Student’s t-test).
Fig 4.
Hes1 over-expression within limb bud mesenchyme alters the expression of digit patterning factors.
(A) WISH for Pax9, Alx4, Fgf8 and Bmp4 on E11.5 WT and HES1 GOF limb buds. (B) qPCR performed on RNA from E11.5 WT and HES1 GOF limb buds (N≥4). Each data point represents separate animals. Asterisks indicate significance with a p-value < 0.05 (Student’s t-test).
Fig 5.
Hes1 over-expression within limb bud mesenchyme enhances Shh expression, but not SHH signaling.
(A) WISH for Shh, Grem1, and Ptch1 on E11.5 WT and HES1 GOF limb buds. (B) qPCR for Shh, Gli1, Ptch1, and Grem1 on RNA isolated from the anterior halves of E11.5 WT and HES1 GOF limb buds (N = 4). Asterisks indicate significance with a p-value < 0.05 (Student’s t-test). (C) Western blots performed on protein extracted from E11.5 WT and HES1 GOF limb buds for GLI3, HES1, and TUBULIN (N = 4). Band intensities were quantified and normalized to WT for each antibody.
Fig 6.
Hes1 over-expression within limb bud mesenchyme is sufficient to overcome the digit loss observed in SHH LOF limbs.
(A) Alcian Blue/Alizarin Red staining of WT, Prx1Cre; R26-Hes1f/f; Shhf/+ (HES1 GOF), Prx1Cre; Shhf/f (SHH LOF), and Prx1Cre; R26-Hes1f/f; Shhf/f (SHH LOF/HES1 GOF) double mutant forelimbs. Red asterisks indicate HES1-induced digits (B) Quantification of digit numbers for WT (n = 60), HES1 GOF (n = 44), SHH LOF (N = 16), and SHH LOF/HES1 GOF (n = 54) forelimbs (χ2 test) (p-value <0.0001). Y axis represents the number of digits whereas the x-asix represents the percent of the forelimbs. Intervals of 0.5 represent syndactylous digits. (C) qPCR for Hes1, Ptch1, Gli1, Pax9, and Cdkn1b performed on RNA from Shhf/f (Control), Prx1Cre; Shh f/f (SHH LOF), R26-Hes1f/f; Shhf/f (Control), and Prx1Cre; R26-Hes1f/f; Shhf/f (SHH LOF/HES1 GOF) limb buds at E11.5 (N = 3). Each data point represents separate animals. Asterisks indicate significance with a p-value < 0.05 (One-way ANOVA).
Fig 7.
HES1 and SHH/GLI3 signaling cooperatively regulate digit number.
(A) Alcian Blue/Alizarin Red staining of WT, Prx1Cre; R26-Hes1f/f (HES1 GOF), R26-Hes1f/f; Gli3xt/+ (GLI3 HET), and Prx1Cre; R26-Hes1f/f; Gli3xt/+ (HES1 GOF/GLI3 HET) double mutant forelimbs. Red asterisks indicate extra digits (B) Quantification of digit numbers for WT (N = 26), HES1 GOF (N = 28), GLI3 HET (N = 20), and HES1 GOF/GLI3 HET (N = 16) forelimbs (χ2 test) (p-value <0.0001).). Y axis represents the number of digits whereas the x-asix represents the percent of the forelimbs. Intervals of 0.5 represent syndactylous digits. (C) qPCR for Hes1 and Pax9 on RNA from E11.5 WT, Prx1Cre; R26-Hes1f/f (HES1 GOF), R26-Hes1f/f; Gli3xt/+ (GLI3 HET), and Prx1Cre; R26-Hes1f/f; Gli3xt/+ (HES1 GOF/GLI3 HET) double mutant limb buds (N = 3). Each data point represents separate animals. Asterisks indicate significance with a p-value < 0.05 (One-way ANOVA). (D) Densitometry measurements of ChIP PCR amplifications from the chromatin containing an E-Box within the Pax9 promoter of WT and HES1 GOF limb buds (N = 3; p-value < 0.05; Student’s t-test).
Fig 8.
HES1 is a critical effector of SHH-induced PPD.
(A) Alcian Blue/Alizarin Red staining of WT, Hes1 f/f; Gli3xt/+ (GLI3 HET), and Prx1Cre; Hes1f/f; Gli3xt/+ (HES1 LOF/GLI3 HET) double mutant forelimbs. Red asterisk indicates syndactylous digit. (B) Quantification of digit numbers for WT, GLI3 HET (N = 66) and HES1 LOF/GLI3 HET (N = 36) double mutant forelimbs (χ2 test) (p-value <0.0001). Y axis represents the number of digits whereas the x-axis represents the percent of the forelimbs. Intervals of 0.5 represent syndactylous digits. (C) qPCR for Hes1, Pax9, and Cdkn1b on RNA from E11.5 WT, Prx1Cre; Hes1f/f (HES1 LOF), Hes1 f/f; Gli3xt/+ (GLI3 HET), and Prx1Cre; Hes1f/f; Gli3xt/+ (HES1 LOF/GLI3 HET) double mutant limb buds (N = 3). Each data point represents separate animals. Asterisks indicate significance with a p-value < 0.05 (One-way ANOVA).
Fig 9.
Proposed model for SHH/GLI3/HES1 regulation of digit number.
HES1 functions downstream of SHH/GLI3 signaling to regulate digit number via: 1) a HES1-mediated transcriptional regulation of Cdkn1b expression to coordinate mesenchymal cell proliferation, 2) a HES1-mediated transcriptional regulation of Pax9 to establish anterior boundaries of digit chondrogenesis, and 3) a HES1-mediated and BMP/GREM1 signaling independent transcriptional regulation that coordinates the timing of digit chondrogenesis. Red lines and arrows indicate novel findings in the SHH/GLI3/HES1-mediated regulation of digit number.