Limb and other postcranial skeletal defects induced by amniotic sac puncture in the mouse.

A mouse model for studying the teratogenic effects of amniotic sac puncture has recently been established (MacIntyre et al. 1995), and the anomalies encountered included cleft palate, limb and tail abnormalities, and postural deformities. In order to investigate the underlying mechanism(s) involved, mouse embryos subjected to amniotic sac puncture on day 13 of gestation were examined externally on day 19, then 'cleared' and bulk stained with alizarin red S and Alcian blue to reveal, respectively, their bony and cartilaginous elements. This procedure allowed a comparison to be made between the incidence of soft tissue anomalies of the distal parts of the limbs and the appearance of the underlying skeletal elements. Despite a high incidence of soft tissue (principally digital) anomalies, relatively few skeletal anomalies were encountered. Measurements of intact long bones, and regions of ossification, were made in the major long bones of 'nonexperimental' and 'internal' controls and 'experimental' fetuses that displayed external morphological malformations to establish whether the experimental procedure had a greater effect on the proximal or distal components of the limb skeleton. No significant difference was observed when the ratios of proximal: distal length were compared with those obtained from the control series. The degree of severity of 'clubhand' and 'clubfoot' deformity was determined, and a comparison made between the severity observed on the right and left sides. This analysis revealed that for 'clubhand', the left forelimb was more severely affected than the right forelimb. The incidence of 'clubfoot' deformity was similar between the 2 sides. A possible explanation for this asymmetric effect is provided. An unexpected finding was the abnormal pattern of ossification seen in the sternum of two thirds of the 'experimental' fetuses that displayed external morphological malformations. This abnormal pattern was seen in none of the controls nor in fetuses in the 'experimental' series that displayed no external morphological malformations.