GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis

Abstract

Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G(1)-S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation.

Figure 1. Preaxial Polydactyly and Conditional Inactivation of Gli3 during Mouse Autopod Development

(A) Shh-Cre-mediated activation of a LacZ transgene inserted into the mouse Rosa26 locus was used to map Shh descendants in wild-type and Gli3-deficient forelimbs. Wild-type (Wt) genotype, Shh^Cre/+, R26^LacZ/+; Gli3^XtJ/XtJ, Gli3-deficient embryo carrying the Shh^Cre/+ and R26^LacZ/+ alleles. (B) Forelimb skeletons at E16.5. The mineralization of metacarpal bones (red; cartilage appears blue) in combination with the number and length of phalanges allows identification of posterior digits in wild-type (Wt) and Gli3-deficient (Gli3^XtJ/XtJ) embryos (see also Figure S1). (C) Scheme depicting the Gli3 locus and Gli3 conditional allele. Deletion results in complete loss of function identical to the Gli3^XtJ null allele. Black triangles indicate loxP sites (see also Figure S1). (D) Inactivation of Gli3 in the limb bud mesenchyme from early stages onward using the Prx1-Cre transgene (P1-Cre) results in fore- and hindlimb polydactylies indistinguishable from Gli3^XtJ/XtJ limbs. (E) Upper left panels show the Hoxa13-Cre transgene recombines the R26^LacZ/+ reporter specifically in the distal part of the forming autopod. Upper right and lower panels illustrate the clearing of Gli3 transcripts that was assessed by RNA in situ hybridization. Wt and Gli3^Δ/Δ embryos were used as positive and negative controls (upper right panels). Hoxa13^Cre/+-mediated recombination of the Gli3^f allele produces the Gli3^Δc allele. (F) Immunoblot analysis of full-length (GLI3FL) and processed GLI3 repressor (GLI3R) protein in forelimb hand plates at E11.75. Protein extracts were normalized for Vinculin content (VCL). (G) Immunoblot analysis of GLI3 protein in Gli3^Δ/Δc forelimb hand plates dissected into proximal (P) and distal (D) portions. For digit nomenclatures in (A), (B), (D), and all subsequent figures, normal digits with respect to position and morphology are indicated in black. Duplicated and/or additional digits in Gli3-deficient forelimbs are indicated in red. Red asterisks indicate hypomorphic digits or digits with uncertain identity. *s, “split” digit due to duplication of distal phalanges.

Figure 2. Hoxa13-Cre-Mediated Inactivation of Gli3 Uncouples Preaxial Polydactyly from the Loss of AP Axis Specification

(A) Analysis of forelimb skeletons at E16.5. (B–E) Analysis of the spatial distribution of Gli1 (B), Hoxd13 (C), Hoxd12 (D), and AER-Fgf8 transcripts (E) in forelimb buds (E11.75, 50–52 somites). Ectopic (white arrowheads) and normal (black arrowheads) expression domains of Gli1 and Fgf8 are indicated. Broken lines mark the expanded (white) and normal (black) expression of 5′Hoxd genes in Gli3^Δ/Δ and Gli3^Δ/Δc forelimb buds. All forelimb buds analyzed carried one Hoxa13^Cre/+ allele to exclude possible phenotypic variation due to heterozygosity for Hoxa13 (see also Figure S2).

Figure 3. The G₁–S Transition of the Cell Cycle Is Altered in the Anterior Mesenchyme of Both Types of Gli3-Deficient Forelimb Buds

(A) Anterior forelimb autopods dissected for transcriptome analysis at E11.75. Lower panel shows the clearance of Gli3 RNAs from the dissected anterior regions in Gli3^Δ/Δc mutants. (B) Hierarchical clustering of the significant transcriptome alterations (p ≤ 0.05 in all paired comparisons using two-way ANOVA tests). Red, genes upregulated; blue, genes downregulated. (C) Ingenuity Pathway Analysis was used to uncover the transcriptional alterations shared by Gli3^Δ/Δ and Gli3^Δ/Δc anterior forelimbs. The core module that regulates the G₁–S transition of the cell cycle is indicated by a broken line. Orange-red labeling of Ccnd1 and Cdk6 indicates significantly increased expression. Cdk1, Mycn, increased expression in only one Gli3-deficiency. Myc, alteration was not confirmed by qPCR. Blue labeling of Cdkn2c indicates significantly decreased expression. (D) Validation of the alterations in Cdk6, Ccnd1, and Cdkn2c transcript levels by qPCR (n = 8, E11.75, ~52 somites). Statistically significant changes are indicated in blue (downregulation) and orange-red (upregulation). All results are represented as mean ± SD; p ≤ 0.01. (E) The spatial distribution of Cdk6 and Ccnd1 transcripts was analyzed by RNA in situ hybridization in combination with optical projection tomography (OPT), which results in improved spatial resolution of low and/or widely expressed genes. Limb bud morphology is shown in the blue channel, whereas transcript distribution is shown in the red channel. Broken white lines indicate the anterior domains with expanded expression. All forelimb buds analyzed were heterozygous for the Hoxa13^Cre/+ allele.

Figure 4. Enhanced S Phase Entry Contributes to the Preaxial Polydactyly in Gli3-Deficient Forelimb Buds

(A) Flow cytometric cell-cycle analysis of anterior and posterior limb bud cells in representative control (Gli3^Δ/+) and Gli3^Δ/Δ autopod samples at E11.75. Limb bud cells were gated to define three populations in the G₀–G₁, S, or G₂–M phases of the cell cycle. (B) Analysis of several Gli3^Δ/Δ (n = 5) and control (Wt, n = 3; and Gli3^Δ/+, n = 4) samples to reveal cell-cycle alterations. Wt and Gli3^Δ/+ forelimb autopods were pooled as controls because no significant differences were detected by analyzing individual samples. All data are shown as mean ± SD. **p ≤ 0.01; *p ≤ 0.05. A, anterior limb bud; P, posterior limb bud. (C) ChIP-qPCR using GLI3 antibodies detects interactions of the endogenous GLI3 proteins with the Cdk6 locus. The blue region corresponds to a 3 kb fragment identified by Vokes et al. (2008) (mm9: chr5: 3,341,265–3,344,289) that includes the proximal promoter. Amplicons “2” and “3” are located in the critical region. All results are shown as mean ± SD (n ≥ 3; p ≤ 0.05). (D and E) Skeletal phenotypes resulting from additional inactivation of Cdk6 in Gli3^Δ/Δ and Gli3^Δ/Δc forelimb buds. All forelimb buds analyzed carried one Hoxa13^Cre/+ allele. See also Figure S3.

Figure 5. Loss of Gli3 Decreases BMP Activity in Anterior Forelimb Buds

(A) Msx2 expression in Gli3^Δ/Δ and Gli3^Δ/Δc forelimb buds (E11.75). (B) Alterations in the expression of the BMP antagonist Grem1 in forelimbs. Broken lines indicate the relevant anterior regions in wild-type (black) and Gli3-deficient forelimb buds (white). Open arrowheads point to Grem1 expression in the proximal interdigital mesenchyme, which does not contribute to the digit primordia. (C) qPCR analysis of two transcriptional BMP targets, Msx2 and Id1, and the BMP antagonist Grem1 (n = 8, E11.75). Statistically significant changes are indicated in blue (downregulation) and orange-red (upregulation). All results are represented as mean ± SD; p ≤ 0.01. (D) ChIP-qPCR using GLI3 antibodies detects interactions of the endogenous GLI3 proteins with a conserved element within the Grem1-Fmn1 regulatory landscape (indicated in green; Vokes et al., 2008) (mm9: chr2: 113,481,000–113,481,438). Amplicons “2” and “3” are located in the critical region. All results are shown as mean ± SD (n ≥ 3; p ≤ 0.05). All forelimb buds shown were heterozygous for the Hoxa13^Cre/+ allele. See also Figure S4.

Figure 6. The Exit of Proliferating Progenitors to Chondrogenesis Is Delayed in the Anterior of Gli3-Deficient Autopods

(A) Sox9 (red fluorescence) demarcates the forming digit primordia on sections of wild-type, Gli3^Δ/Δ, and Gli3^Δ/Δc autopods (E12.5, ~60 somites). (B) The Ki67 antigen (green fluorescence) marks proliferating cells, whereas the autofluorescent erythrocytes appear white. Cell nuclei appear blue due to counterstaining with Hoechst-33258. In the upper panels, dotted rectangles indicate the position of the enlargements. In the left and right lower panels, dotted white lines indicate the proximal limit of the mesenchymal zone with largely Ki67-positive cells. (C) Noggin and Col2a1 transcripts mark the ongoing chondrogenesis during digit formation at E12.5. (D) RNA in situ hybridization revealed the induction of Noggin (n = 13/16) and Col2a1 (n = 10/13) expression (right panels) following implantation of BMP4-loaded beads (0.5 mg/ml) into the anterior of Gli3^Δ/Δ forelimb buds at E12.0. Contralateral controls with no or PBS-soaked beads (left panels). All relevant forelimb buds in (A)–(C) carried one Hoxa13^Cre/+ allele.

Figure 7. Reduction and Promotion of Preaxial Polydactyly by Altering Grem1, Bmp4, and Gli3 Gene Dose

Comparative analysis of genetic alteration of BMP pathway activity in the context of both types of Gli3 deficiencies. Col2a1 expression at E12.5 detects mesenchymal condensations (upper panels), which is compared with the resulting skeletal pattern at E16.5 (lower panels). In the upper panels, arrowheads indicate the small condensation for digit 1; red asterisks point to reduced or forked digit primordia with uncertain identity. In the lower panels, black asterisks indicate postaxial condensations. Figure S5 shows the skeletal preparations of all genotypes analyzed. (A) Wild-type (i.e., Hoxa13^Cre/+) forelimbs compared to Gli3^Δ/Δ (n = 6) and Gli3^Δ/Δ, Grem1^Δ/+ forelimbs (n = 9). (B) Constitutive genetic inactivation of one Grem1 allele in Gli3^Δ/Δc forelimb buds. (C) Gli3^Δ/+forelimb buds carrying the Hoxa13^Cre/+ allele compared with Gli3^Δ/+, Bmp4^Δ/Δc forelimbs in which one Bmp4 allele was conditionally inactivated by Hoxa13-Cre. (D) Gli3^Δ/Δc, Bmp4^Δ/Δc forelimb buds. (E) Regulation of the proliferative expansion and chondrogenic exit of digit progenitors by GLI3R and SHH-dependent gene networks. Green indicates genetic interactions positively regulating proliferation; red shows genetic interactions restraining the cell cycle and promoting exit to chondrogenic differentiation.