Creb5 controls its own expression and directly induces the joint interzone regulatory program

Abstract

Prior studies have indicated that the transcription factor Creb5 is expressed in the joint interzone, which contains the progenitors for all synovial joint tissues in both mouse and human embryos. In the absence of Creb5 function, most synovial joint interzones fail to form and the cartilage templates in the long bones remain fused. This earlier work did not clarify whether Creb5 initiates a cascade of signaling molecules, such as growth and differentiation factor 5 (Gdf5) and Wnt-family members, that in turn induce the formation of the joint interzone, or instead directly activates the expression of joint interzone markers. In the present study, an integrative analysis of the transcriptome, chromatin accessibility, and Creb5-occupancy in joint progenitors revealed that Creb5 directly binds to both its own two promoters and to the regulatory regions of Gdf5 and Sfrp2, each of whose expression in the joint interzone is Creb5-dependent. Functional enhancer analysis indicated that Creb5 binding sites in either the two Creb5 promoters, or in Gdf5 and Sfrp2 regulatory elements are necessary for these sequences to drive transgene expression in the developing synovial joints. While Creb5 directly drives Gdf5 and Sfrp2 expression in the inner joint interzone, Creb5 activates Barx1 expression specifically in the outer joint interzone. Our findings indicate that Creb5 initiates a regulatory network that both promotes the formation of synovial joints, and subsequently activates distinct transcriptional targets in the inner versus the outer regions of the joint interzone, thus regionalizing gene expression in the developing joint.

Keywords: Barx1; Creb5; Gdf5; Sfrp2; synovial joint interzone.

Fig. 1.

Differential gene expression in Creb5-expressing cells isolated from the knees of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Sox9^iresEGFP/+ embryos. (A) Diagram of targeting vehicle and correctly targeted Creb5^{3XHA-P2A-tdTomato} allele. (B) Direct fluorescence of optically cleared E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Sox9^iresEGFP/+ embryos and their dissected hindlimbs. Scale bar equals 2 mm in Top images; and 1 mm in Bottom images. (C and D) Direct fluorescence of cryosections of the developing knees of E14.5 and E18.5 Creb5^{3XHA-P2A-tdTomato/+}; Sox9^iresEGFP/+ embryos. Joint interzone (arrowhead), peripheral tissues adjacent to the joint interzone (arrows), Femur (Fe), Tibia (Ti), and Fibula (Fi) are indicated. Scale bar equals 100 microns in (C) and 200 microns in (D). (E) FACS profile of cells isolated from the developing knees of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Sox9^iresEGFP/+ embryos, which express either Creb5HA-tdTomato, Sox9-ires-EGFP, or both Creb5HA-tdTomato and Sox9-ires-EGFP. (F) Principal-component analysis (PCA) of the transcriptomes expressed in either Creb5- Sox9+ cells, Creb5+ Sox9+ cells, or Creb5+ Sox9- cells. (G) Violin plots of the normalized expression of Creb5, Gdf5, Sox9, and Col2a1 in bulk RNA-Seq of Creb5+ Sox9- cells, Creb5+ Sox9+ cells, or Creb5- Sox9+ cells. (H and I) Volcano plots of differentially expressed genes (DEGs) in Creb5+Sox9+ cells relative to Creb5-Sox9+ cells (H); or DEGs in Creb5+Sox9- cells relative to Creb5-Sox9+ cells (I). Joint interzone markers are indicated in red. In (H) a total of 2,780 genes were differentially expressed in Creb5+Sox9+ cells versus Creb5-Sox9+ cells; in Creb5+Sox9+ cells, expression levels of 1,620 genes were enriched and expression levels of 1,160 genes were decreased. In (I), a total of 4,751 genes were differentially expressed in Creb5+Sox9- cells versus Creb5-Sox9+ cells; in Creb5+Sox9- cells, expression levels of 2,569 genes were enriched and expression levels of 2,182 genes were decreased.

Fig. 2.

The ability of Creb5 promoters to drive transgene expression requires intact Creb5 binding sites; forced expression of bovine Creb5 induces ectopic expression of endogenous mouse Creb5. (A) Creb5 binds to several differentially accessible regulatory regions surrounding the Creb5 locus. Integrated Genome View (IGV) of RNA-Seq, ATAC-Seq, and Cut&Run-Seq over the Creb5 locus, performed with sorted cells isolated from either the knees of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Sox9^iresEGFP/+ embryos or the limbs of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Hoxa11^EGFP/+ embryos. Creb5 promoters are indicated by gray shadows. CpG islands and evolutionary conservation in 60 vertebrate genomes (mm10.60way.phyloP60way.bw) is displayed. (B, C) Schematics display either Creb5 Promoter 1 wild-type (B) or Creb5 Promoter 2 wild-type (C). In all schematics within this figure, CRE motifs (rectangles), TRE motifs (ovals), Creb5 Electrophoretic Mobility Shifts (red), and mutant CREs (X) containing “double T to A” nucleotide substitutions are indicated. Images below promoter diagrams display X-GAL staining (of both the outside and inside) of a bisected E14.5 embryo that had been injected with a LacZ reporter driven by either Creb5 Promoter 1 wild-type (B) or Creb5 Promoter 2 wild-type (C). Number of embryos which displayed a similar pattern of LacZ staining/total number of transgenic embryos is indicated. Scale bars equal 2mm. (D, E) Dissected limbs of embryos injected with LacZ reporter driven by either Creb5 Promoter 1 wild-type (D) or Creb5 Promoter 2 wild-type (E). Scale bars equal 1mm. Arrows point to shoulder and elbow joints in forelimbs; femoral head and knee joint in hindlimbs. (F, G) Schematics display either Creb5 Promoter 1 CRE-mutant (F) or Creb5 Promoter 2 CRE-mutant (G). Images below promoter diagrams display X-GAL staining (of both the outside and inside) of a bisected E14.5 embryo that had been injected with a LacZ reporter driven by either Creb5 Promoter 1 CRE-mutant (F) or Creb5 Promoter 2 CRE-mutant (G). Number of embryos which displayed a similar pattern of LacZ staining/total number of transgenic embryos is indicated. Scale bars equal 2mm. (H and I) Luciferase activity in MLB14 cells transfected with either a control expression vehicle (gray) or a Creb5 expression vehicle (red) plus a Firefly luciferase reporter driven by either Creb5 Promoter 1 (H) or Creb5 Promoter 2 (I) and a control CMV-Renilla luciferase reporter. Relative expression of Firefly to Renilla Luciferase is displayed. n = 4; ** (P < 0.01) (J) MLB14 cells were infected with a lentivirus encoding doxycycline-inducible bovine Creb5 and cultured for the indicated number of days in either the absence (gray) or presence (red) of doxycycline. Relative expression of murine Creb5 was assayed by RT-qPCR (employing primers that amplify the murine Creb5 Exon10-3′UTR, but not the viral encoded bovine Creb5 transcript). n = 3; (P < 0.001) (ordinary two-way ANOVA) (K) Mating scheme for (L and M): Prrx1-Cre male mice were mated to Rosa26^{LSL-bovine-Creb5-ires-GFP/LSL-tdTomato} female mice, yielding E14.5 embryos which express either bovine iCreb5HA-GFP^(Prrx1-Cre) or itdTomato^(Prrx1-Cre) in their limb bud mesenchyme. Image created in BioRender. Zhang, C. (2025) https://BioRender.com/q57w462. (L) Bulk RNA-Seq was performed on the isolated bovine iCreb5HA-EGFP^(Prrx1-Cre)-expressing or itdTomato^(Prrx1-Cre)-expressing limb bud mesenchymal cells. The normalized expression of either the exogenous bovine Creb5 transgene or endogenous murine Creb5 in either itdTomato^(Prrx1-Cre)-expressing cells (red dots) or bovine iCreb5HA-EGFP^(Prrx1-Cre)-expressing cells (green dots) from 6 independent matings is displayed (gene expression in litters 1 to 3 are identified). (M) IGV of ATAC-Seq and Creb5-Cut&Run Seq over the Creb5 locus. ATAC-Seq was performed with sorted cells isolated from either itdTomato^(Prrx1-Cre)-expressing cells (red tracks) or bovine iCreb5HA-EGFP^(Prrx1-Cre)-expressing cells (green tracks) from litters 1 to 3 (whose gene expression is displayed in L). Creb5 ATAC-Peaks 1 & 5 are indicated by gray shadows. (N) RNAscope of both Col2a1 and murine Creb5, employing an RNAScope probe that detects the murine Creb5 3′UTR (which is absent in the bovine iCreb5-transgene), in the forelimb of either an E14.5 Prrx1-Cre; Rosa26^{LSL-bovine-Creb5-ires-GFP/+} embryo or control littermate (similar results were obtained in two embryos). Scale bar equals 200 microns.

Fig. 3.

Creb5 (CRE) binding sites are required for Gdf5 ATAC-Peak 4 to drive joint interzone–specific transgene expression. (A) Creb5 binds to several differentially accessible regulatory regions surrounding the Gdf5 locus. IGV of RNA-Seq, ATAC-Seq, and Cut&Run-Seq over the Gdf5 locus, performed with sorted cells isolated from either the knees of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Sox9^iresEGFP/+ embryos or the limbs of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Hoxa11^EGFP/+ embryos. Evolutionary conservation in 60 vertebrate genomes is displayed. The 10 Gdf5 ATAC-Peaks (gray shadows) include the evolutionarily conserved noncoding regions R1-R5 (32). Two Bacterial Artificial Chromosomes (BACs) which contain regulatory information sufficient to drive transgene expression in joint interzones (32) are indicated below the IGV. (B, C) Diagrams of either the Gdf5 ATAC-Peak 4 wild-type (B) [containing the Gdf5 R4 enhancer (32)] or Gdf5 ATAC-Peak 4 mutant CRE 3 (C). In all schematics within this figure, CRE motifs (rectangles), TRE motifs (ovals), Creb5 Electrophoretic Mobility Shifts (red), and mutant CREs (X) containing “double T to A” nucleotide substitutions are indicated. TREs 5, 6, and 7 are not displayed. Images below promoter diagrams display X-GAL staining (of both the outside and inside) of a bisected E14.5 embryo that had been injected with a LacZ reporter driven by either Gdf5 ATAC-Peak 4 wild-type (B) or Gdf5 ATAC-Peak 4 mutant CRE 3 (C). Number of embryos which displayed a similar pattern of LacZ staining/total number of transgenic embryos is indicated. Scale bars equal 2mm. (D, E) Dissected limbs of embryos injected with LacZ reporter driven by either Gdf5 ATAC-Peak 4 wild-type (D) or Gdf5 ATAC-Peak 4 mutant CRE 3 (E). Scale bars equal 1mm. (F, G) Sections of the elbow and knee, which were also processed for Col2a1 expression (displayed in pink) by RNAScope, of E14.5 embryos that had been injected with a LacZ reporter driven by either Gdf5 ATAC-Peak 4 wild-type (F) or Gdf5 ATAC-Peak 4 mutant CRE3 (G). Scale bars equal 100 microns. (H) Diagram of Gdf5 ATAC-Peak 4 mutant CRE 1, 3, 4. Images display X-GAL staining (of both the outside and inside) of a bisected E14.5 embryo that had been injected with a LacZ reporter driven by Gdf5 ATAC-Peak 4 mutant CRE 1, 3, 4. Number of embryos which displayed a similar pattern of LacZ staining/total number of transgenic embryos is indicated. Scale bars equal 2mm. (I) Dissected limbs of embryos injected with LacZ reporter driven by Gdf5 ATAC-Peak 4 mutant CRE 1, 3, 4. Scale bars equal 1mm. Images of additional transgenic embryos injected with a LacZ reporter driven by Gdf5 ATAC-Peak 4 wild-type are displayed in SI Appendix, Fig. S6C.

Fig. 4.

Creb5 (TRE) binding sites are required for Sfrp2 ATAC-Peak 7 to drive joint interzone–specific transgene expression. (A) Creb5 binds to several differentially accessible regulatory regions surrounding the Sfrp2 locus. IGV of RNA-Seq, ATAC-Seq, and Cut&Run-Seq over the Sfrp2 locus, performed with sorted cells isolated from either the knees of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Sox9^iresEGFP/+embryos or the limbs of E14.5 Creb5^{3XHA-P2A-tdTomato/+}; Hoxa11^EGFP/+ embryos. 7 ATAC-Peaks (gray shadows) in the Sfp2 locus, which displayed both greater accessibility in Creb5-expressing cells and contained Creb5-Cut&Run peaks are indicated. Evolutionary conservation in 60 vertebrate genomes is displayed. (B and C) Diagrams of either wild-type (B) or TRE-mutant (C) Sfrp2 ATAC-Peak 7 with CRE motifs (rectangles), TRE motifs (ovals), Creb5 Electrophoretic Mobility Shifts (red), and mutant TREs (X) containing double T to A nucleotide substitutions displayed. Images display X-GAL staining of either the outside or inside of an entire bisected embryo (B and C); dissected limbs (D and E); or sections of the elbow or knee, which were also stained with nuclear fast red (F and G) of E14.5 embryos that had been injected with a LacZ reporter driven by either Sfrp2 ATAC-Peak 7 wild-type (B, D, and F) or Sfrp2 ATAC-Peak 7 mutant TRE (3L3R) (C, E, and G). Number of embryos which displayed a similar pattern of LacZ staining/total number of transgenic embryos is indicated. Scale bars equal either 2 mm (B and C), 1 mm (D and E), or 100 microns (F and G).

Fig. 5.

Creb5 is necessary to maintain expression of both itself and other interzone markers between the cartilage elements. (A–F) RNA-scope of the indicated genes displayed in the developing knee joints of either E14.5 Creb5^Δ9/Δ9 embryos or their control littermates. Femur (Fe), Tibia (Ti), and Fibula (Fi) are indicated. Scale bars equal 100 microns.

Fig. 6.

Forced expression of bovine Creb5 in limb bud mesenchyme boosts expression of different joint interzone markers in mesenchyme, perichondrium, and chondrocytes. (A and B) Prrx1-Cre male mice were mated to Rosa26^{LSL-bovine-Creb5-ires-GFP/LSL-tdTomato} female mice, yielding E14.5 embryos which express either bovine iCreb5HA-GFP^(Prrx1-Cre) or itdTomato^(Prrx1-Cre) in their limb bud mesenchyme. (A) Bulk RNA-Seq was performed on the isolated bovine iCreb5HA-EGFP^(Prrx1-Cre)-expressing or itdTomato^(Prrx1-Cre)-expressing limb bud mesenchymal cells. The normalized expression of either Gdf5, Osr2, Sfrp2, or Barx1 in either itdTomato^(Prrx1-Cre)-expressing cells (red dots) or bovine iCreb5HA-EGFP^(Prrx1-Cre)-expressing cells (green dots) from six independent matings is displayed (gene expression in litters 1 to 3 are identified). (B) IGV of ATAC-Seq and Creb5-Cut&Run Seq over the Gdf5 locus (Left) and the Sfrp2 locus (Right). ATAC-Seq was performed with sorted cells isolated from either itdTomato^(Prrx1-Cre)-expressing cells (red tracks) or bovine iCreb5HA-EGFP^(Prrx1-Cre)-expressing cells (green tracks) from litters 1 to 3 (whose gene expression is displayed in A). Gdf5 ATAC-Peaks 4 & 7 and Sfp2 ATAC-Peaks 3 & 7 are indicated by gray shadows. Evolutionary conservation in 60 vertebrate genomes is displayed. (C–F) RNAScope of Creb5 (using a probe that detects both the bovine iCreb5 transgene and murine Creb5) and other indicated genes in the hindlimbs of either E14.5 Prrx1-Cre; Rosa26^{LSL-bovine-Creb5-ires-GFP/+} embryos or control littermates (similar results were obtained in three embryos). Scale bars equal 200 microns. (G) Creb5 initiates a regulatory network that promotes both the formation and subsequent regionalization of the joint interzone. Creb5 both induces expression of Wnt ligands, which promote the dedifferentiation of Sox9+Col2a1+ cells to form the joint interzone, and subsequently activates distinct transcriptional targets in the inner versus the outer regions of the joint interzone, thus regionalizing gene expression in the developing joint. Creb5 directly binds to the regulatory regions of Creb5, Gdf5, and Sfrp2 (underlined).