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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Otsuji
Miwako
M
Department of Orthopaedic Surgery, Yamagata University School of Medicine, 2-2-2 Iida Nishi, Yamagata 990-9585, Japan.
Takahara
Masatoshi
M
Naruse
Takuji
T
Guan
Dehong
D
Harada
Mikio
M
Zhe
Piao
P
Takagi
Michiaki
M
Ogino
Toshihiko
T
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D001973:Bromodeoxyuridine; D002066:Busulfan / Q000633:toxicity*; D002454:Cell Differentiation; D002455:Cell Division; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors / Q000235:genetics; D053823:Hedgehog Proteins; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051381:Rats; D013723:Teratogens / Q000633:toxicity*; D013977:Tibia / Q000187:drug effects* / Q000196:embryology; D015534:Trans-Activators / Q000235:genetics
D018906:Antineoplastic Agents, Alkylating; C525811:Bmp4 protein, rat; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; C496458:Fgf8 protein, rat; D053823:Hedgehog Proteins; D013723:Teratogens; D015534:Trans-Activators; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors; D002066:Busulfan; D001973:Bromodeoxyuridine