Final Report Summary - SMAD4 LIMB SKELETON (The Role of SMAD4-mediated BMP Signal Transduction in Determination of Digit Identities, Initiation of Chondrogenesis, and Skeletal Development)
Bones are formed by mineralization of a cartilage template that is laid down by cartilage-specific, differentiated cells called chondrocytes. Before this happens, cells of the embryonic mesenchyme have to initiate the chondrogenic differentiation program. BMP signaling is essential for the differentiation of both cartilage and bone cells during embryonic development. However, the precise mechanism(s) by which BMPs initiates chondrogenesis remained unknown. BMP signals are transduced through two main intracellular branches: SMAD-dependent and p38 MAPK-dependent pathways. We have used mouse genetics to specifically inactivate Smad4 in the developing limb bud mesenchyme SMAD4 is a so-called core-SMAD, which is required to transduce the bulk of SMAD-mediated BMP/TGFß signaling. Therefore, the genetic inactivation Smad4 should block SMAD-dependent BMP signaling and bypass redundancy at the level of ligands and receptors. This analysis revealed the critical role of SMAD4 in initiating the aggregation of the Sox9-positive chondrogenic progenitors, which hallmarks the onset of chondrogenic differentiation in limb buds. Therefore, the transcriptome of wild-type and Smad4-deficient limb buds was comparatively analyzed to identify the molecular alterations underlying the complete disruption in initiating chondrogenic differentiation. Analysis of limb buds and cell in culture provided evidence that regulation of the actin cytoskeletal dynamics is significantly altered in Smad4-deficient mesenchymal progenitors.
In Smad4-deficient limb buds, formation of the Sox9-positive digit primordia and the initiation of chondrogenic differentiation of all skeletal elements is disrupted. In contrast to the aggregation and condensation of wild-type chondrogenic progenitors, which prefigures the skeletal primordia, Smad4-deficient progenitors remain loose and fail to initiate the expression of chondrogenic differentiation markers such as collagen type II. The loose mutant cells divert progressively toward a general connective tissue fate in vivo, which shows that SMAD4 is required to maintain chondrogenic fate. mesenchymal condensation is critical to subsequent deposition of the cartilage matrix and shaping of the skeletal primordia. During the onset, the mesenchymal progenitors compact and establish numerous cadherin-based junctions with neighboring cells as cartilaginous differentiation is initiated. These cadherin complexes in turn depend on the actin cytoskeleton for stability - processes which are regulated at multiple levels different signals. Several of these are severely altered in Smad4-deficient cells such as e.g. the Semaphorin ligands their Plexin receptors. Semaphorins are known to regulate actin cytoskeletal dynamics during e.g. axonal outgrowth in the nervous system.
Furthermore, the BMP antagonist GREM1 also serves as an antagonist to Slit. Based on the observed down-regulation of Grem1 in Smad4-deficient limb buds, we hypothesize that Slit signaling through Robo is such increased that it might disrupt cadherin complexes. These and the other observed alterations in cell-cell signaling likely underlie the disruption of cellular aggregation and the actin cytoskeletal dynamics, but further analysis is required to understand the functional significance of these alterations with respect to disrupting the onset of chondrogenesis in Smad4-deficient limb buds.
Finally, we have isolated and characterized a population of mesenchymal stromal cells (MSCs)/progenitor cells from the adult mouse bone marrow together These cells have shown chondrogenic capacity; however, the unique aim of this study is to stimulate endochondral ossification in a spatiotemporally controlled manner in order to recapitulate developmental processes. In order to study similarities and differences between limb bud mesenchymal progenitor and the engineering of cartilage (and bone) from adult mesenchymal cells, limb bud mesenchymal cells will be directly compared to MSCs. These studies are ongoing as part of a Sinergia network grant and will likely have implications for human health and disease. In light of an aging population, there is great need for novel strategies to engineer cartilage and bone replacements, which will enormously profit from understanding of the molecular systems interactions that underlie the progressive differentiation of embryonic mesenchymal progenitors into cartilage and bone.
In Smad4-deficient limb buds, formation of the Sox9-positive digit primordia and the initiation of chondrogenic differentiation of all skeletal elements is disrupted. In contrast to the aggregation and condensation of wild-type chondrogenic progenitors, which prefigures the skeletal primordia, Smad4-deficient progenitors remain loose and fail to initiate the expression of chondrogenic differentiation markers such as collagen type II. The loose mutant cells divert progressively toward a general connective tissue fate in vivo, which shows that SMAD4 is required to maintain chondrogenic fate. mesenchymal condensation is critical to subsequent deposition of the cartilage matrix and shaping of the skeletal primordia. During the onset, the mesenchymal progenitors compact and establish numerous cadherin-based junctions with neighboring cells as cartilaginous differentiation is initiated. These cadherin complexes in turn depend on the actin cytoskeleton for stability - processes which are regulated at multiple levels different signals. Several of these are severely altered in Smad4-deficient cells such as e.g. the Semaphorin ligands their Plexin receptors. Semaphorins are known to regulate actin cytoskeletal dynamics during e.g. axonal outgrowth in the nervous system.
Furthermore, the BMP antagonist GREM1 also serves as an antagonist to Slit. Based on the observed down-regulation of Grem1 in Smad4-deficient limb buds, we hypothesize that Slit signaling through Robo is such increased that it might disrupt cadherin complexes. These and the other observed alterations in cell-cell signaling likely underlie the disruption of cellular aggregation and the actin cytoskeletal dynamics, but further analysis is required to understand the functional significance of these alterations with respect to disrupting the onset of chondrogenesis in Smad4-deficient limb buds.
Finally, we have isolated and characterized a population of mesenchymal stromal cells (MSCs)/progenitor cells from the adult mouse bone marrow together These cells have shown chondrogenic capacity; however, the unique aim of this study is to stimulate endochondral ossification in a spatiotemporally controlled manner in order to recapitulate developmental processes. In order to study similarities and differences between limb bud mesenchymal progenitor and the engineering of cartilage (and bone) from adult mesenchymal cells, limb bud mesenchymal cells will be directly compared to MSCs. These studies are ongoing as part of a Sinergia network grant and will likely have implications for human health and disease. In light of an aging population, there is great need for novel strategies to engineer cartilage and bone replacements, which will enormously profit from understanding of the molecular systems interactions that underlie the progressive differentiation of embryonic mesenchymal progenitors into cartilage and bone.