Knockdown of the Intraflagellar Transport Protein IFT46 Stimulates Selective Gene Expression in Mouse Chondrocytes and Affects Early Development in Zebrafish

Jérôme Gouttenoire,Ulrich Valcourt,Carole Bougault,Elisabeth Aubert-Foucher,Estelle Arnaud,Lionel Giraud,Frédéric Mallein-Gerin

doi:10.1074/jbc.m705730200

Abstract

Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.

Highlights

IntroductionOssification begins with the invasion of the calcified cartilage by blood capillaries
As an approach to attaining better understanding of the molecular mechanisms involved in chondrocyte differentiation and maturation, we examined differentially expressed genes in mouse chondrocytes cultured in the presence or absence of bone morphogenetic proteins (BMPs)-2, by using the differential display reverse transcriptionPCR technique
In these conditions we previously reported that BMP-2 favors chondrogenic expression and promotes hypertrophic maturation and osteoblastic-like differentiation of chondrocytes [16]

Summary

Introduction

Ossification begins with the invasion of the calcified cartilage by blood capillaries This neo-vascularization of the growth plate is concomitant with apoptosis of most hypertrophic chondrocytes. Among the members of the transforming growth factor-␤ superfamily, bone morphogenetic proteins (BMPs) play an essential role during the formation of cartilage molds for long bones, vertebrae, and ribs, and in their transition to bone during endochondral ossification (reviewed in Ref. 2). The fact that Bmp-2 mRNA is expressed in hypertrophic chondrocytes of the growth plate in mouse embryos [18] strongly suggests that BMP-2 plays a physiological role during endochondral ossification. Our results taken together indicate that the ciliary protein IFT46 has a specific function both in chondrocytes and during the early development of vertebrates

Results

Discussion

Conclusion