Conditional Ablation of the Heparan Sulfate-synthesizing Enzyme Ext1 Leads to Dysregulation of Bone Morphogenic Protein Signaling and Severe Skeletal Defects

Yoshihiro Matsumoto,Kazu Matsumoto,Fumitoshi Irie,Jun-Ichi Fukushi,William B Stallcup,Yu Yamaguchi

doi:10.1074/jbc.m110.105338

Yoshihiro Matsumoto, Kazu Matsumoto + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.105338

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Abstract

Increasing evidence indicates that heparan sulfate (HS) is an integral component of many morphogen signaling pathways. However, its mechanisms of action appear to be diverse, depending on the type of morphogen and the developmental contexts. To define the function of HS in skeletal development, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS synthesis, in limb bud mesenchyme using the Prx1-Cre transgene. These conditional Ext1 mutant mice display severe limb skeletal defects, including shortened and malformed limb bones, oligodactyly, and fusion of joints. In developing limb buds of mutant mice, chondrogenic differentiation of mesenchymal condensations is delayed and impaired, whereas the area of differentiation is diffusely expanded. Correspondingly, the distribution of both bone morphogenic protein (BMP) signaling domains and BMP2 immunoreactivity in the mutant limb mesenchyme is broadened and diffuse. In micromass cultures, chondrogenic differentiation of mutant chondrocytes is delayed, and the responsiveness to exogenous BMPs is attenuated. Moreover, the segregation of the pSmad1/5/8-expressing chondrocytes and fibronectin-expressing perichondrium-like cells surrounding chondrocyte nodules is disrupted in mutant micromass cultures. Together, our results show that HS is essential for patterning of limb skeletal elements and that BMP signaling is one of the major targets for the regulatory role of HS in this developmental context.

Highlights

T intensities similar to those in wild-type mice (Fig. 2, G–J), indicating that the program for chondrogenic differentiation is initiated relatively normally in mutants
At E11.5, Sox[9] and Col2a1 were both expressed in mutant limbs at Scapula Humerus Radius/ulna Carpal bones Metacarpal bones Phalanges
At E12.5, Col2a1 expression levels in wild-type mice increased greatly, and Col2a1 expression domains were separated into multiple subdomains corresponding to individual cartilage templates (Fig. 2K)

Summary

Introduction

T intensities similar to those in wild-type mice (Fig. 2, G–J), indicating that the program for chondrogenic differentiation is initiated relatively normally in mutants. The spatial patterns of Sox[9] and Col2a1 expression in mutants were altered; both Sox[9] and Col2a1 expression domains were enlarged in mutants (Fig. 2, H and J). At E12.5, Col2a1 expression levels in wild-type mice increased greatly, and Col2a1 expression domains were separated into multiple subdomains corresponding to individual cartilage templates (Fig. 2K). Col[2] expression in mutant mice was diffuse, lacking distinct subdomains (Fig. 2L)

Results

Discussion

Conclusion