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

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 tgfβ2 and tgfβ3 but not tgfβ1 are abundant in the growing undifferentiated limb mesoderm. Transcripts of tgfβ2 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 Tgfβs 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.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.Tgfβs constitute a subfamily formed in birds and mammals by 3 isoforms of secreted cytokines (Tgfβ1; Tgfβ2; Tgfβ3), which gives the name to the large Tgfβ superfamily made up of more than 30 structurally related proteins that comprises Activins, BMPs and GDFs. Tgfβs are multifunctional factors with important regulatory roles in adult and embryonic systems. During development Tgfβs are able to regulate almost all basic cellular processes including migration, proliferation, apoptosis and differentiation (reviewed by [1]). Their effects are mediated by binding to specific cell surface transmembrane receptors with serine/threonine kinase activity that trigger an intracellular cascade which regulates the expression of target genes and the biogenesis of specific microRNAs (reviewed