Retinoid-induced limb defects 2: involvement of TGF-beta 2 in retinoid-induced inhibition of limb bud development.
We previously demonstrated that retinoid-induced inhibition of chondrogenesis in the forelimb bud may be mediated by TGF-beta2 (1). The present study was conducted to examine whether TGF-beta2 is involved in the inhibition of forelimb bud development caused by all-trans-retinoic acid (RA). Expression of TGF-beta2 was examined immunohistochemically in forelimb buds of embryos 24 h after dosing to the mother on Day 12 of gestation in the rat. In the control and 50 mg/kg group, TGF-beta2 was expressed in the epithelium and prechondrogenic area around dead cells in the forelimb bud. In the 100 mg/kg group, a dose at which RA caused reduction defects of forearm bones, TGF-beta2 expression was observed in the distal margin of forelimb buds, in which no expression was observed in the control and 50 mg/kg group. Immunohistologic studies also indicated that in the 100 mg/kg group, the expression of TGF-beta2 was enhanced in forearm-bone prechondrocytes around the dead cells. In a whole embryo culture system, exposure to RA for 24 h reduced the proximodistal length and protein content in forelimb buds at concentrations of 3 microg/mL or more. The whole embryo culture system also showed that the expression of TGF-beta2 was induced at the concentration of 3 microg/mL in the same region as found in forelimb buds of embryos from dams administered a teratogenic dosage of RA in vivo. Local application of TGF-beta2 to the distal margin of the forelimb bud in Day 12 embryos reduced proximodistal growth and protein content in forelimb buds for 24 h in culture even without RA treatment. We also found that exogenous TGF-beta2 inhibited DNA synthesis of forelimb bud cells in culture in a concentration-dependent manner. Neutralization of TGF-beta2 with its antibody in the distal margin of forelimb buds partially prevented the RA-induced inhibition of forelimb bud growth in the whole embryo culture system. These results suggest that RA-induced TGF-beta2 in the distal margin of forelimb buds may be involved in RA-induced inhibition of forelimb bud growth via reduction of cell proliferation in the distal margin, and RA-induced TGF-beta2 in the prechondrogenic area may inhibit chondrogenesis in the future forearm bones, followed by reduction defects of the forearm bones.
Tsuiki
H
H
Developmental Research Laboratories, Shionogi & Co., Toyonaka, Osaka, Japan. hisashi.tsuiki@shionogi.co.jp
Kishi
K
K
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000633:toxicity*; D046508:Culture Techniques; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004622:Embryo, Mammalian; D005260:Female; D005552:Forelimb / Q000002:abnormalities* / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism; D007150:Immunohistochemistry; D018878:Limb Buds; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000276:immunology / Q000502:physiology*; D014212:Tretinoin / Q000633:toxicity*
D000906:Antibodies; D000970:Antineoplastic Agents; D016212:Transforming Growth Factor beta; D014212:Tretinoin; D004247:DNA