Developmental abnormalities in rat embryos leading to tibial ray deficiencies induced by busulfan


  • This study was carried out in accordance with the Guide for Animal Experimentation, Yamagata University School of Medicine and Japanese Governmental Law (No.105).

  • Presented at the 48th Annual Meeting of the Orthopaedic Research Society, February 10–13, 2002, Dallas, Texas; 58th Annual Meeting of the American Society for Surgery of the Hand, September 18–20, 2003, Chicago, Illinois; 46th Annual Meeting of the Japanese Society for Surgery of the Hand, April 18–19, 2003, Aichi, Japan; and 18th Annual Research Meeting of the Japanese Orthopaedic Association, October 16–17, 2003, Fukuoka, Japan.



Little is known about the developmental changes associated with tibial ray deficiencies. The aim of this study was to detect cell death, proliferation, and gene expression that result in tibial ray deficiencies.


We induced tibial ray deficiencies in rat embryos using a teratogenic agent (busulfan) and observed the developmental changes in 1126 hindlimbs. We performed Nile blue staining, whole mount in situ hybridization for fibroblast growth factor 8 (Fgf8), bone morphogenetic protein 4 (Bmp4) and Sonic hedgehog (Shh), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and assessment of cell proliferation by 5-bromo-2′-deoxy-uridine (BrdU)/anti-BrdU immunohistochemistry.


In situ hybridization showed reductions in Fgf8 and Bmp4 expression. Histological examination showed a delay of mesenchymal condensation, increased mesenchymal cell death, decreased mesenchymal cell proliferation, and a reduction in the number of mesenchymal cells. These abnormalities may cause hypoplasia of the limb. Bmp4 expression was markedly reduced in the anterior mesenchyme. Shh was expressed in the posterior mesenchyme. We suggest that the posterior skeletal elements may be fully formed owing to Shh expression, but the anterior skeletal elements may be underdeveloped owing to an intense reduction of Bmp4 expression in the anterior mesenchyme, causing hypoplasia of the tibial ray.


The combined effects of increased cell death, decreased cell proliferation, reduction of Fgf8 expression, and intense reduction of Bmp4 expression in the anterior mesenchyme may play an important role in the development of tibial ray deficiency induced by busulfan. Birth Defects Research (Part A), 2005. © 2005 Wiley-Liss, Inc.