Defining the earliest transcriptional steps of chondrogenic progenitor specification during the formation of the digits in the embryonic limb

Abstract

The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The "pre-condensation stage", occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the "condensation stage" is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the "pre-cartilage period", precedes the formation of molecularly identifiable cartilage by 2-3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo.

Figure 1. Characterization of chondrogenesis induced by interdigital application of a heparin bead incubated in 2 µg/ml Tgfβ 1.

A, Morphological appearance of the extra digit 3 days after the interdigital implantation of a Tgfβ bead. B, PNA positive labeling of the interdigital mesoderm 10 hr after the implantation of a Tgfβ bead. C, Alcian blue-positive cartilage 22 hr after the implantation of a Tgfβ bead. D, presence of an ectopic aggrecan gene expression domain 12 hr after the implantation of a Tgfβ bead. E, Presence of an ectopic collagen 2 alpha 1 expression domain 17 hr after the implantation of a Tgfβ bead. (*) Tgfβ bead; (D3) digit 3; (D4) digit4. F-F′ charts representing cell proliferation of control interdigits (F) and of those treated with a Tgf-β bead (F′) as measured by flow cytometry. The percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle are shown.

Figure 2. Actin cytoskeleton in digit chondrogenic induction.

A, Low magnification view of a phalloidin-stained section of the autopod at 3 hr ABI to show increased staining around the bead (*) and at the digit tip of neighboring digit 3 (d3). B, Detailed view of the actin cytoskeleton of p-Smad2-positive (green nuclear labeling) cells located around the Tgfβ bead (*) 3 hr ABI. Note the increased protrusive actin-based filaments in the p-Smad2-positive cells. Digit 3 (d3), digit4 (d4). C–D, Morphology of the ectopic cartilage (arrows), 2 days after combined treatment with a Tgfβ bead and a Cytochalasin D bead (experimental, D) or a DMSO bead (control, C). Cytochalasin D (D) and DMSO (C) beads are indicated by arrowheads.

Figure 3. Expression of Ccn1 (A–C), Ccn2 (D–F), and Ccn3 (G–I), in the developing digits at days 5,5–6,5 (A,D,G), 7–8 (B,E,H) and 8,5–9 (C, F, I) of incubation.

A–C, Intense expression of Ccn1 is first seen in the digit blastemas (A), and in the course of development, expression becomes restricted to the digit tips (arrow), joints and tendons (B). Note in C the disappearance of the digit tip domains once all of the phalanxes have formed. D–F, Expression of Ccn2 is first associated with the zones of hypertrophic differentiation of digit cartilage (D) followed by the appearance of joint domains (arrowheads, B) and then tendons domains (arrows, E). G–I, Expression of Ccn3 first appears in the developing tendons (G,H) and then extends to the developing joints (I).

Figure 4. Regulation of chondrogenic factors by CCN proteins.

Charts showing the regulation of Sox9, Scleraxis, and CG-8 (A); and Tenascin C, Activin βA and alpha 5 Integrin (B) in the interdigital mesoderm 6 hr after the implantation of beads bearing PBS (first column, control), Tgfβ (second column), CCN1 (third column), CCN2 (fourth column) and CCN3 (fifth column). (*) p-value≤0.05; (**) p-value≤0.01.

Figure 5. Chart showing the regulation of the Ccn1, Ccn2, and Ccn3 genes in the interdigital mesoderm 6 hr after the implantation of beads bearing PBS (control), CCN1, CCN2 or CCN3.

(*) p-value≤0.05; (***) p-value≤0.001.