Targeting canonical BMP signaling : SMAD4 in limb patterning and differentiation

The developing limb is an ideal genetic model to investigate basic developmental mechanisms and signaling networks. The Bone Morphogenetic Protein (BMP) signaling pathway has been associated with a number of context-specific functions during limb development, including establishment of the limb signalling domains, regulation of cell proliferation and cell death, digit patterning, differentiation of the endochondral skeleton and the soft tissue.
The present work aimed at providing insights into the roles of canonical BMP signaling in mouse limb bud patterning and tissue differentiation. The canonical BMP pathway includes numerous components, which are often functionally redundant. Conversely, the non-redundant intracellular transducer SMAD4 is essential for gastrulation, such that its inactivation results in an early lethal phenotype and prevents the analysis of its functions during limb development. For the purpose of our investigation, we used the conditional inactivation of the Smad4 gene to generate time- and space-restricted loss-of-function models during limb development.
This approach allowed us to show that mesenchymal SMAD4 is dispensable for establishment of the Apical Ectodermal Ridge (AER), which is an ectodermal source of the Fibroblast Growth Factor (FGF) signalling factors that contribute to proximo-distal (P-D) limb axis extension. However, mesenchymal SMAD4 contributes to the establishment the SHH/GREM1/AER-FGFs feedback loop that controls limb outgrowth and patterning.
Most importantly, we observed a discrete temporal requirement of SMAD4 for the specification of digit primordia during a developmental period when high BMP activity is essential to initiate chondrogenesis. Specific inactivation of SMAD4 in the limb mesenchyme at this stage is sufficient to inhibit the initiation of mesenchymal condensations, which represent the first structures committed to endochondral bone formation. In fact, the Smad4 deficiency results in the absence of any limb skeletal elements. Molecular evidence indicates that the discrete pattern of genes that normally specify the chondrogenic fate is replaced by wide-spread up-regulation of genes relevant to tendon and joint development in Smad4 deficient limb bud, but no ectopic tendons or joints are formed. These observations suggest a role for SMAD4 in cell fate restriction and differentiation of lateral plate mesoderm-derived tissues in the limb.
To further analyze the rapid changes in BMP activity during limb development, we sought to generate a mouse model which senses BMP activity in a specific and dynamic fashion. For the purpose of this project, I have established the aggregation chimera technique to allow for the rapid investigation of cis-regulatory elements in the context of the Gt(ROSA)26Sor locus.