Tgfβ2 and 3 are coexpressed with their extracellular regulator Ltbp1 in the early limb bud and modulate mesodermal outgrowth and BMP signaling in chicken embryos

<p>Abstract</p> <p>Background</p> <p>Transforming growth factor β proteins (Tgfβs) are secreted cytokines with well-defined functions in the differentiation of the musculoskeletal system of the developing limb. Here we have studied in chicken embryos, whether these cytokines are implicated in the development of the embryonic limb bud at stages preceding tissue differentiation.</p> <p>Results</p> <p>Immunohistochemical detection of phosphorylated Smad2 and Smad3 indicates that signaling by this pathway is active in the undifferentiated mesoderm and AER. Gene expression analysis shows that transcripts of <it>tgfβ2 </it>and <it>tgfβ3 </it>but not <it>tgfβ1 </it>are abundant in the growing undifferentiated limb mesoderm. Transcripts of <it>tgfβ2 </it>are also found in the AER, which is the signaling center responsible for limb outgrowth. Furthermore, we show that Latent Tgfβ Binding protein 1 (LTBP1), which is a key extracellular modulator of Tgfβ ligand bioavailability, is coexpressed with <it>Tgfβs </it>in the early limb bud. Administration of exogenous Tgfβs to limb buds growing in explant cultures provides evidence of these cytokines playing a role in the regulation of mesodermal limb proliferation. In addition, analysis of gene regulation in these experiments revealed that Tgfβ signaling has no effect on the expression of master genes of musculoskeletal tissue differentiation but negatively regulates the expression of the BMP-antagonist Gremlin.</p> <p>Conclusion</p> <p>We propose the occurrence of an interplay between Tgfβ and BMP signaling functionally associated with the regulation of early limb outgrowth by modulating limb mesenchymal cell proliferation.</p

Defining the Earliest Transcriptional Steps of Chondrogenic Progenitor Specification during the Formation of the Digits in the Embryonic Limb

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

In situ hybridization on transverse sections of the proximal segment of the autopod of embryos after 7 and 7,5 days of incubation, to show the expression domains of BMP modulators in the zone of hypertrophic differentiation of the metatarsal and in the maturing tendons at the zone close to the myotendinous junction.

<p><i>Noggin</i> (A), <i>Fstl- 5</i> (B), <i>Chd</i> (C), <i>BMPER</i> (D), <i>Chdl-2</i> (E), <i>Sost</i> (F), <i>Tll1</i> (G), and <i>Tsg</i> (H). Note that at this stage of differentiation <i>Tll1</i> transcripts are present in the tendon blastemas (arrow heads in G). Note also that the expression domains in the diaphysis of the metatarsal are specifically located in the inner cellular layer of the perichondrium in the interface with the hypertrophic cartilage.</p

Regulation of Cartilage Markers.

<p>Regulation of 3 selected markers of cartilage in 2 days Micromass cultures of digit progenitors treated for 24 hr with recombinant proteins. As indicated in the table (o/e), in the cases of TSG, DAN, and BMPER gain-of-function experiments were also performed by transfection of mesodermal cells with expression vectors.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.001.</p

Regulation of Joint Markers.

<p>Regulation of 3 selected markers of fibrous and tendon tissue in 2 days Micromass cultures of digit progenitors treated for 24 hr with recombinant proteins. As indicated in the table (o/e), in the cases of TSG, DAN and BMPER gain-of-function experiments were also performed by transfection of mesodermal cells with expression vectors.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.001.</p

Regulation of BMP modulators.

<p>Regulation of BMP modulators in 48 hr Micromass cultures of digit progenitors treated for 6 hr with BMP2, FGF2, all-trans-retinoic acid, TGFβ1, ACTIVIN A, and WNT 5A.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.001.</p

In situ hybridizations in longitudinal (A–G, M–P) and transverse (H–L; Q–T) vibratome sections of the autopod showing the expression of <i>Noggin</i> (A), <i>Chd</i> (B), <i>Chdl-1</i> (C), <i>Chdl-2</i> (D), <i>Tsg</i> (E and I), <i>Dan</i> (F and J), <i>BMPER</i> (G and K), <i>Sost</i> (H), <i>Sostdc1</i> (L), <i>Tll1</i> (M and Q), <i>Fst</i> (N and R), <i>Fstl-1</i> (O and S), and <i>Fstl-5</i> (P and T).

<p>All the specimens are at day 6,5 of incubation (stage 30HH) except H, which is at 7,5 days of incubation (stage 32HH).</p

Immunolabeling for p-Smad 1/5/8 (green, A–D) and p-c-Jun (green, E), to show the zones of active BMP signaling in the course of digit formation.

<p>Note in A, the absence of labeling in the tendon blastema (tb) at 7,5 days of incubation. In addition, zones of intense labeling paralleling the expression of BMPER of are observed, (arrows, compared with Fig. 1K). B is a higher magnification image of A to show the labeling in the differentiating cartilage except in the most peripheral region subjacent to the perichondrium (p). C shows positive labeling in a proximal region of the tendons corresponding with the myotendinous junction (day 8 of incubation). D is a longitudinal section of digit 3 at day 6 of incubation showing intense labeling in the growing tip (dt) and reduced labeling in the zones of joint formation (arrow heads). E, is a longitudinal section of digit 3, showing intense labeling for p-c-Jun in the joint forming region (arrow heads). In A and B the actin cytoskeleton was labeled in red with Phalloidin-TRITC. F–M, are longitudinal (F, G, I, K, and M) and transverse (H, J, and L) sections through digit 3 showing the expression of type 1 BMP receptors in the autopod. <i>Bmpr1b</i> (F–H) is highly expressed at RNA (F–H) and protein (G) levels in the early chondrogenic digit ray with zones of reduced expression in the joint forming region. In the non-chondrogenic domains of the autopod, the expression of <i>Bmpr1b</i> is predominant in the subectodermal mesenchyme and interdigital regions. Expression of <i>Bmpr1a</i> (I–J) and <i>Alk2</i> (K–L) is poor in the chondrogenic rays, but transcripts are abundant in the tissues around the digit cartilage including the perichondrium, the interdigital mesenchyme. Transcripts of <i>Alk2</i>, are also appreciated in the developing joints (arrow in K). <i>Alk 1</i> was only appreciated in the peridigital blood vessels (M).</p

Semi-quantitative association of gene expression intensity in the autopodial tissues.

<p>Table summarizing the expression intensity of the BMP modulators in the autopodial tissues.</p