Analysis of transcription factors expressed at the anterior mouse limb bud.

Shigetoshi Yokoyama,Hiroshi Asahara,Yoshiaki Ito,Takeshi Saito,Soichi Furukawa,Shoya Kitada,Tempei Sato,Juan Carlos Izpisua Belmonte,Masaki Mori,Koichi Kawakami,Yasuhiko Kawakami,Michael Schubert

doi:10.1371/journal.pone.0175673

Abstract

Limb bud patterning, outgrowth, and differentiation are precisely regulated in a spatio-temporal manner through integrated networks of transcription factors, signaling molecules, and downstream genes. However, the exact mechanisms that orchestrate morphogenesis of the limb remain to be elucidated. Previously, we have established EMBRYS, a whole-mount in situ hybridization database of transcription factors. Based on the findings from EMBRYS, we focused our expression pattern analysis on a selection of transcription factor genes that exhibit spatially localized and temporally dynamic expression patterns with respect to the anterior-posterior axis in the E9.5–E11.5 limb bud. Among these genes, Irx3 showed a posteriorly expanded expression domain in Shh-/- limb buds and an anteriorly reduced expression domain in Gli3-/- limb buds, suggesting their importance in anterior-posterior patterning. To assess the stepwise EMBRYS-based screening system for anterior regulators, we generated Irx3 transgenic mice in which Irx3 was expressed in the entire limb mesenchyme under the Prrx1 regulatory element. The Irx3 gain-of-function model displayed complex phenotypes in the autopods, including digit loss, radial flexion, and fusion of the metacarpal bones, suggesting that Irx3 may contribute to the regulation of limb patterning, especially in the autopods. Our results demonstrate that gene expression analysis based on EMBRYS could contribute to the identification of genes that play a role in patterning of the limb mesenchyme.

Highlights

Developmental morphogenesis advances through a set of rules and interweaving gene interactions that regulate specification, proliferation, and differentiation [1, 2]
Initial EMBRYS-based screening for candidate genes involved in AP patterning based on spatial localizations and temporal expression changes of 691 transcription-associated factors in the mouse limb bud Previously, we established a whole-mount in situ hybridization (WISH) database of transcription factors that are present in mouse embryos termed EMBRYS [19]
691 transcription-associated factors expressed in the mouse limb bud were selected and classified into categories based on their spatial expression patterns [26] (Figs 1 and 2)

Summary

Introduction

Developmental morphogenesis advances through a set of rules and interweaving gene interactions that regulate specification, proliferation, and differentiation [1, 2]. The morphogenesis of appendages, such as limb buds, progresses through well-established mechanisms involving axis formation, patterning, outgrowth, and differentiation. From this viewpoint, the limb bud acts as an excellent developmental model for studying the formation of the vertebrate body plan. Sonic hedgehog (SHH) in the ZPA [12] was identified as the morphogen responsible for controlling the AP polarity of the limb bud This observation could not explain all the published research findings from genetic experiments and called for further analysis to postulate a theory that complied with experimental findings and integrated novel viewpoints [1, 2]

Methods

Results

Conclusion