RA Acts in a Coherent Feed-Forward Mechanism with Tbx5 to Control Limb Bud Induction and Initiation

Abstract

SummaryThe retinoic acid (RA)- and β-catenin-signaling pathways regulate limb bud induction and initiation; however, their mechanisms of action are not understood and have been disputed. We demonstrate that both pathways are essential and that RA and β-catenin/TCF/LEF signaling act cooperatively with Hox gene inputs to directly regulate Tbx5 expression. Furthermore, in contrast to previous models, we show that Tbx5 and Tbx4 expression in forelimb and hindlimb, respectively, are not sufficient for limb outgrowth and that input from RA is required. Collectively, our data indicate that RA signaling and Tbx genes act in a coherent feed-forward loop to regulate Fgf10 expression and, as a result, establish a positive feedback loop of FGF signaling between the limb mesenchyme and ectoderm. Our results incorporate RA-, β-catenin/TCF/LEF-, and FGF-signaling pathways into a regulatory network acting to recruit cells of the embryo flank to become limb precursors.

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

“…A BMS 493 bead placed in the hindlimb LPM at stage 10 (see protocol 3.4.1.3) downregulates Tbx4 expression (Figures 2H and 2I), whereas control beads soaked in DMSO do not affect Tbx4 expression (Figures 2J and 2K). Together, these results support a model that an RA signal from axial tissues regulates limb induction by positively regulating Tbx4 in the hindlimb LPM 7 .…”

“…The protocol detailed above describes the methods employed in Nishimoto et al, 2015. The paper studies limb bud induction and initiation in both the fore and hindlimb of the chick embryo.…”

“…More recently mouse and fish genetics have taken the study of vertebrate limb initiation into a new era (reviewed in 7 ). There is good evidence that RA present in the somites prior to limb bud initiation is the essential diffusible signal required by the LPM to initiate limb budding.…”

Subdivision of the lateral plate mesoderm and specification of the forelimb and hindlimb forming domains

“…The analysis of the cis-regulatory element of Tbx5 has identified a number of upstream regulators, including Hox genes, ␤-catenin and retinoic acid (RA) signalling and this has revealed the molecular network that determines the forelimb LPM [2,32,33].…”

“…1), following an inductive signal from the paraxial mesoderm [2]. Responding to this axial signal, cells in distinct subdomains of the LPM activate genes required to initiate limb outgrowth.…”