The subsequent distal-anterior expansion of the Grem1 expression domain depends largely on its positive regulation by AER-FGF and SHH signaling. Concurrently, BMP4 triggers expression of the BMP antagonist Grem1 in the posterior limb bud mesenchyme, which causes rapid lowering of mesenchymal BMP activity ( Nissim et al., 2006 Bénazet et al., 2009). During initiation of limb bud outgrowth, the SHH signaling centre is established and restricted to the posterior mesenchyme under the influence of BMP signaling ( Bastida et al., 2004).
During the onset of limb bud development, BMP4 is first required in the mesenchyme and BMPR1A in the ectoderm for formation of the AER, as inactivation of these molecules disrupts establishment of the AER-FGF signaling centre, which results in limb truncations and loss of dorsoventral polarity ( Ahn et al., 2001 Pizette et al., 2001 Pajni-Underwood et al., 2007 Bénazet et al., 2009). Genetic and functional analysis of mouse and chicken limb bud development has uncovered important morphoregulatory functions of the BMP pathway in the establishment and functioning of the two limb bud signaling centers, namely sonic hedgehog (SHH) signaling by the posterior mesenchymal organizer and fibroblast growth factor (FGF) signaling by the apical ectodermal ridge (AER).ĭuring limb bud outgrowth, BMPs and the BMP antagonist gremlin 1 (GREM1) control the epithelial-mesenchymal signaling interactions that coordinate outgrowth with patterning and determination of digit identities (reviewed by Zeller et al., 2009). These SMAD complexes translocate to the nucleus and regulate the expression of target genes in concert with other transcriptional regulators ( Feng and Derynck, 2005). These in turn form a complex with SMAD4, an essential mediator of both canonical BMP and TGFβ signal transduction ( Yang et al., 2002). BMP ligands interact with two types of BMP receptors (BMPR1 and BMPR2), which in turn trigger intracellular signal transduction by phosphorylation of the receptor-associated SMAD1, -5 and -8 proteins. In summary, our analysis reveals that SMAD4 is required to initiate: (1) formation of the Sox9-positive digit ray primordia and (2) aggregation and chondrogenic differentiation of all limb skeletal elements.īone morphogenetic proteins (BMPs) are ligands belonging to the transforming growth factor beta (TGFβ) superfamily, which control inductive processes in early embryos and during organogenesis (reviewed by Zakin and De Robertis, 2010) and are required for chondrogenesis, growth and ossification of bones in vertebrates (reviewed by Wu et al., 2007). Conditional inactivation of Bmp2 and Bmp4 indicated that the loss of digit ray primordia and increase in connective tissue were predominantly a consequence of disrupting SMAD4-mediated BMP signal transduction. This pointed to a general loss of tissue organization and diversion of mutant cells toward non-specific connective tissue. The progressive loss of SOX9 due to disrupting digit ray primordia and chondrogenesis was paralleled by alterations in genes marking other lineages. At the cellular level, Smad4 deficiency blocked the aggregation of Sox9-positive progenitors, thereby preventing chondrogenic differentiation as revealed by absence of collagen type II. Specific inactivation of Smad4 during handplate development pointed to its differential requirement for posterior and anterior digit ray primordia.
While this Smad4 inactivation did not alter the early Sox9 distribution, prefiguring the chondrogenic primordia of the stylopod and zeugopod, it disrupted formation of all Sox9-positive digit ray primordia. SMAD4 is an essential mediator of canonical TGFβ/BMP signal transduction and we inactivated Smad4 in mouse limb buds from early stages onward to study its functions in the mesenchyme.
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