Suppressor of Fused Is Required for Determining Digit Number and Identity via Gli3/Fgfs/Gremlin.

Jianying Li,Yan Li,Ying Cui,Zunyi Zhang,Xiaoyun Zhang,Mengsheng Qiu,Qihui Wang,Ze Zhang,Xueqin Yang,Moises Mallo

doi:10.1371/journal.pone.0128006

Abstract

The anterior-posterior patterning of the vertebrate limb bud requires closely coordinated signaling interactions, including Sonic Hedgehog (Shh)-mediated counteraction of the Gli3 transcription factor in the distal and posterior mesenchyme of the limb bud. Suppressor of Fused (Sufu), an intracellular negative regulator of Shh signaling via Gli2 and Gli3, is implicated in early development of the mouse limb bud. However, how Sufu is involved in the genetic regulation of limb bud patterning still remains elusive. In this study, we show that the conditional deletion of Sufu in the mesenchyme of the early limb bud results in polydactyly with loss of digit identity and supernumerary bones in the wrist and the ankle. These pattern alterations are associated with anterior expansion of HoxD genes located at the 5’ end of the cluster. By focusing on gene expression analysis of Shh/Gremlin1/Fgf signaling critical for the establishment and maintenance of anterior-posterior patterning, we show that early response to loss of Sufu involves anterior prolongation of Fgf4 and Fgf8 expression in the apical ectodermal ridge at E10.5. We also reveal the anterior activation of Shh-dependent posterior markers Ptc1, Gli1 and Gremlin in limb buds lacking Sufu. Furthermore, we find that loss of Sufu leads to attenuated levels of repressor Gli2 and repressor Gli3 in the early limb bud. Moreover, expression of Hand2 is activated in the entire limb bud at the early outgrowth stage in the mutant lacking Sufu. Thus, we provide evidence that Sufu is involved in the genetic network that restricts the posterior expression of Gli2/3/Hand2 and Gremlin/Fgf in limb bud patterning.

Highlights

Anterior-posterior (A-P) patterning and outgrowth of the vertebrate limb bud are coordinated by two critical centers during limb bud development [1,2,3]: the zone of polarizing activity (ZPA) [4, 5] in the posterior mesenchyme and the apical ectodermal ridge (AER) in the distal ectoderm [6]
Gremlin, the secreted antagonist for Bone Morphogenetic Proteins (BMPs), relays Sonic hedgehog (Shh) signaling from the posterior mesenchyme and positively regulates the expression of Fgfs (Fgf4 and Fgf8) in the AER [7, 8]
We further demonstrate that the Shh signaling/Gremlin/Fgfs are ectopically expanded to the anterior limb bud in the Suppressor of Fused (Sufu) mutant

Summary

Introduction

Anterior-posterior (A-P) patterning and outgrowth of the vertebrate limb bud are coordinated by two critical centers during limb bud development [1,2,3]: the zone of polarizing activity (ZPA) [4, 5] in the posterior mesenchyme and the apical ectodermal ridge (AER) in the distal ectoderm [6]. The anterior expansion of Fgf4 and up-regulation of Fgf8 in the AER reflects the expanded AER in SufuDermo1Cre. Our data revealed that in contrast to the wild type forelimb bud, where the Shh was undetectable from E11.5 and on, in the Sufu mutant, Shh was still detectable in the posterior forelimb bud at E11.5 (S3 Fig).

Results

Conclusion