Irx1 and Irx2 Are Coordinately Expressed and Regulated by Retinoic Acid, TGFβ and FGF Signaling during Chick Hindlimb Development

Martha Elena Díaz-Hernández,Marcia Bustamante,Jesús Chimal-Monroy,Claudio Iván Galván-Hernández,Michael Schubert

doi:10.1371/journal.pone.0058549

Martha Elena Díaz-Hernández, Marcia Bustamante + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0058549

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Journal: PloS one	Publication Date: Mar 11, 2013
Citations: 22	License type: CC BY 4.0

Affiliation: Universidad Nacional Autónoma de México

Abstract

The Iroquois homeobox (Irx) genes play a crucial role in the regionalization and patterning of tissues and organs during metazoan development. The Irx1 and Irx2 gene expression pattern during hindlimb development has been investigated in different species, but its regulation during hindlimb morphogenesis has not been explored yet. The aim of this study was to evaluate the gene expression pattern of Irx1 and Irx2 as well as their regulation by important regulators of hindlimb development such as retinoic acid (RA), transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signaling during chick hindlimb development. Irx1 and Irx2 were coordinately expressed in the interdigital tissue, digital primordia, joints and in the boundary between cartilage and non-cartilage tissue. Down-regulation of Irx1 and Irx2 expression at the interdigital tissue coincided with the onset of cell death. RA was found to down-regulate their expression by a bone morphogenetic protein-independent mechanism before any evidence of cell death. Furthermore, TGFβ protein regulated Irx1 and Irx2 in a stage-dependent manner at the interdigital tissue, it inhibited their expression when it was administered to the interdigital tissue at developing stages before their normal down-regulation. TGFβ administered to the interdigital tissue at developing stages after normal down-regulation of Irx1 and Irx2 evidenced that expression of these genes marked the boundary between cartilage tissue and non-cartilage tissue. It was also found that at early stages of hindlimb development FGF signaling inhibited the expression of Irx2. In conclusion, the present study demonstrates that Irx1 and Irx2 are coordinately expressed and regulated during chick embryo hindlimb development as occurs in other species of vertebrates supporting the notion that the genomic architecture of Irx clusters is conserved in vertebrates.

Highlights

In vertebrate limb development, the outgrowth and patterning of the proximo-distal axis is controlled by the apical ectodermal ridge (AER), a thickened epithelium rimming the distal tip of the growing limb, which secretes fibroblast growth factor (FGF) to mesenchymal cells located beneath the AER, giving rise to the undifferentiated region of the limb [1,2]
The present study shows that Irx1 and Irx2 have comparable gene expression patterns during chick hindlimb development, and that they are coordinately expressed in the interdigital tissue and digital primordia, joints and in the boundary between cartilage and non-cartilage tissue
Because in this study Irx1 and Irx2 are coordinately expressed and regulated by retinoic acid (RA) and transforming growth factor b (TGFb), we solely evaluated the expression of Irx2 in the undifferentiated zone beneath AER region to determine whether non-expression of Irx2 is a consequence of inhibition by FGF signaling

Summary

Introduction

The outgrowth and patterning of the proximo-distal axis is controlled by the apical ectodermal ridge (AER), a thickened epithelium rimming the distal tip of the growing limb, which secretes FGF to mesenchymal cells located beneath the AER, giving rise to the undifferentiated region of the limb [1,2]. During the hand plate development, the TGFb/ Activin signaling pathway recruits undifferentiated mesenchymal cells to the cartilage lineage, prefiguring digital rays [3]. TGFb/ Activin signaling induces the formation of an ectopic digit by promoting activation of a chondrogenic molecular cascade in which Sox expression is induced [4,5,6,7]. If undifferentiated mesenchymal cells are not recruited to form digital rays, they develop into interdigital tissue. In species with free digits, the loss of FGF signaling from AER promotes interdigital cell death [8,9]. The inhibition of Fgf expression in the AER is under the control of BMP signaling leading to apoptosis [9]. RA a pro-apoptotic factor, antagonizes FGF8 function inhibiting Fgf receptor-1 gene expression [8]

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