Quantitative evaluation in vivo of the degree of differentiation of hindlimb cartilage in a rat clubfoot model

Abstract

The Limb Morphogenetic Differentiation Scoring system introduced by Neubert and Barrach in 1977 has been used in drug testing as a measure of the degree of cartilage growth inhibition especially for forelimb in vitro. There is no scoring system to quantify the degree of hindlimb bud cartilage differentiation in vivo. A total of 60 female Sprague-Dawley rats weighing 220-250 g were assigned at random to six control groups and six experimental groups on day 0 of pregnancy. The experimental groups were treated with all-trans-retinoic acid (ATRA). A new limb morphogenetic differentiation scoring system was developed and used to quantify the degree of development of the hindlimb buds from the fetuses at embryonic days E13 to E18. The differentiation of cartilages assessed by the new scoring system showed a statistically significant difference between the experimental group and the control group from E13 to E18 (T-test, p < 0.05). Cartilage growth (the proximodistal length) in the control group increased gradually from E14, reaching its peak at E17, but in the experimental group the growth at E13, E16, E17, and E18 was significantly shorter (p < 0.05). In conclusion, the new limb morphogenetic differentiation scoring system described here can be used to quantify the degree of inhibition of the hindlimb bud development by teratogenic drugs or materials, and morphogenetic differentiation in vivo.

All-trans-retinoic acid inhibits chondrogenesis of rat embryo hindlimb bud mesenchymal cells by downregulating p53 expression

“…Additionally, it has been proposed that the teratogenic effects of ATRA, leading to limb defects, are based on increased cellular apoptosis in the developing limb ( 33 , 34 ). Previous studies by our group have demonstrated that a rat embryo CCF-like model may be established via a single intragastric dose of ATRA, which exhibits hindlimb congenital malformations, impaired chondrogenesis and increased cellular apoptosis ( 3 , 4 , 35 ). These findings suggested that defective chondrogenesis of the primary hindlimb bud mesenchymal cells may contribute to CCF.…”

“…Delgado-Baeza et al ( 2 ) have established a fetal rat clubfoot model using a single intragastric dose of RA on day 10 of gestation. Our group previously demonstrated that inhibition of hindlimb cartilage development is a crucial factor for ATRA-induced congenital clubfoot (CCF) in a rat model ( 3 , 4 ). Limb patterning and growth are carefully regulated through a complex network of transcription factors and signaling molecules ( 5 , 6 ).…”

Genotype-phenotype correlation in clubfoot (talipes equinovarus)

“…Unfortunately, difficulties with genetic manipulations excluded the model from clubfoot studies. Nonetheless, several studies have shown that administration of ATRA during pregnancy-induced the clubfoot deformity in rat embryos 71 97 98. The ATRA treatment may lead to unilateral or bilateral clubfoot phenotype, together with other malformations, such as spina bifida, anal atresia, rima oculi and cranial deformation 75.…”

All-trans-retinoic acid suppresses rat embryo hindlimb bud mesenchymal chondrogenesis by modulating HoxD9 expression

“…Under the action of high dose of ATRA, growth and morphogenesis of the bone will be strongly inhibited, and the bone resorption function of osteoclasts will be stimulated [ 19 ]. In our previous studies, we used all-trans-retinoic acid (ATRA) to establish a CCF-like model in vivo by inhibiting the development of hindlimb cartilage [ 20 ]. This model was used to investigate the effects of ATRA on the chondrogenesis of rat embryo hindlimb bud mesenchymal cells (rEHBMCs) and their specific underlying cellular mechanisms.…”