Dynamics of BMP signaling in limb bud mesenchyme and polydactyly

Abstract

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

“…We reasoned that if BMP4 activity was increased in Prmt5 cKO hindlimb buds, it might rescue the hindlimb polydactyly caused by reduced BMP levels. We did not expect to rescue the forelimb outgrowth defect in Prx1Cre +/− ; Rosa Gremlin/+ embryos, as this is caused by an early failure in limb outgrowth that would precede Prmt5 inactivation (Norrie et al, 2014). As expected, control and Prmt5 cKO littermates had five hindlimb digits, whereas Rosa Gremlin/+ embryos had polydactyly with an average of 6.8 digits.…”

“…As Bmp4 expression is negatively regulated by BMP activity, the observed increase in gene expression does not in itself imply enhanced activity (Khokha et al, 2003;Michos et al, 2004;Norrie et al, 2014). The BMP target gene Msx2 Pizette and Niswander, 1999), although slightly upregulated in independent qRT-PCR experiments (Fig.…”

“…Together, these results suggest that there are no substantial alterations in responses to canonical BMP signaling in Prmt5 cKOs. As BMP4 activity is regulated on several levels, including negative transcriptional feedback by GREM1 and proteolytic processing of the pro-protein, the increased Bmp4 expression does not imply an increase in BMP activity (Benazet et al, 2009;Bragdon et al, 2011;Capdevila et al, 1999;Michos et al, 2004;Norrie et al, 2014). To establish if there is a biologically relevant increase in BMP activity in Prmt5 cKO hindlimbs, we crossed…”

“…GREM1 binds to and sequesters BMP ligands, resulting in a reduction in overall BMP activity by over 50% (Hsu et al, 1998;Norrie et al, 2014). As a consequence of reduced BMP activity, there is an early failure of forelimb initiation, while the hindlimbs have fully penetrant polydactyly with decreased apoptosis (Norrie et al, 2014).…”

“…Blocking BMP ligand activity will result in inhibition of both canonical and non-canonical BMP signaling. As forelimbs of Rosa Gremlin embryos have an early defect in AER formation that results in a near complete block to limb outgrowth (Norrie et al, 2014), the later BMP4 upregulation in Prmt5 cKO forelimbs would occur after the early defect and would therefore be incapable of rescuing the early phenotype.…”