Attenuation of bone morphogenetic protein signaling during amphibian limb development results in the generation of stage‐specific defects

Abstract

The vertebrate limb is one of the most intensively studied organs in the field of developmental biology. Limb development in tetrapod vertebrates is highly conserved and dependent on the interaction of several important molecular pathways. The bone morphogenetic protein (BMP) signaling cascade is one of these pathways and has been shown to be crucial for several aspects of limb development. Here, we have used a Xenopus laevis transgenic line, in which expression of the inhibitor Noggin is under the control of the heatshock promoter hsp70 to examine the effects of attenuation of BMP signaling at different stages of limb development. Remarkably different phenotypes were produced at different stages, illustrating the varied roles of BMP in development of the limb. Very early limb buds appeared to be refractory to the effects of BMP attenuation, developing normally in most cases. Ectopic limbs were produced by overexpression of Noggin corresponding to a brief window of limb development at about stage 49/50, as recently described by Christen et al. (2012). Attenuation of BMP signaling in stage 51 or 52 tadpoles lead to a reduction in the number of digits formed, resulting in hypodactyly or ectrodactyly, as well as occasional defects in the more proximal tibiafibula. Finally, inhibition at stage 54 (paddle stage) led to the formation of dramatically shortened digits resulting from loss of distal phalanges. Transcriptome analysis has revealed the possibility that more Nogginsensitive members of the BMP family could be involved in limb development than previously suspected. Our analysis demonstrates the usefulness of heat-shock-driven gene expression as an effective method for inhibiting a developmental pathway at different times during limb development.

Formation of a New Limb Bud at the Boundary Between a Transplanted Limb Bud and the Tail Surface ofXenopusTadpoles

“…Since BMPs (2, 4, and 7) and retinoic acid (RA)-synthesizing enzyme (Raldh2) were shown to be expressed at the base of the stage 51 Xenopus limb bud (Jones et al, 2013;McEwan et al, 2011), Raldh2 (or RA) together with the BMPs in the proximal part of the stage 51 limb buds might work for transplantation-induced new limb bud formation, though further experimental data is needed to confirm this tentative theory.…”

“…1). If this kind of manipulation causes the attenuation of BMP expression at the specific cut surface of the graft (limb bud), the "self-duplication" of a limb bud might occur, similar to how noggin-induced BMP attenuation causes extra limbs (Christen et al, 2012;Jones et al, 2013). On the other hand, the grafting procedure also involves cutting of the tail skin.…”

“…As described above, the most basal part of the stage 51 limb bud expresses Raldh2 and BMPs (2, 4, and 7) (McEwan et al, 2011;Jones et al, 2013). The grafting procedure in the present study involves cutting the basal part tissues of the limb bud (Fig.…”

“…Furthermore, RA is known to indirectly induce wnt2ba, the earliest limb inducer, which leads to the mesenchymal fgf10 expression (Mercader et al, 2006;Neto et al, 2012). More recently, bone morphogenetic protein (BMP) signaling was shown to be involved in the RAmediated limb field initiation by experiments with temporal overexpression under the control of the heat shock promoter of noggin (BMP inhibitor) using transgenic X. laevis tadpoles (Christen et al, 2012;Jones, 2013). In these experiments, BMP attenuation by the heatshock treatment of tadpoles for 7 days (during stage 49-51 or stage 50-52) caused the production of extra limbs.…”

The origins, scaling and loss of tetrapod digits

“…Therefore, convergent miniaturization of embryos is often accompanied by digit loss. Although the molecular mechanisms that drive amphibian digit reduction and loss remain less clear, blocking SHH signalling with cyclopamine in salamanders and frogs [121,122] and inhibiting BMP signalling during late frog development [123] can both cause digit loss, indicating mechanisms associated with miniaturization may function by limiting the range of signalling pathways common to the development of all tetrapod limbs.…”