1 CPS 49-induced neurotoxicity does not cause limb patterning anomalies in 2 developing chicken embryos 3 4 5 6

49 Thalidomide notoriously caused severe birth defects, particularly to the limbs, in 50 those exposed in utero following maternal use of the drug to treat morning sickness. 51 How the drug caused these birth defects remains unclear. Many theories have been 52 proposed including actions on the forming blood vessels. However, thalidomide 53 survivors also have altered nerve patterns and the drug is known for its neurotoxic 54 actions in adults following prolonged use. We have previously shown that CPS49, an 55 antiangiogenic analog of thalidomide, causes a range of limb malformations in a time 56 sensitive manner in chicken embryos. Here we investigated if CPS49 also is 57 neurotoxic and if effects on nerve development impact upon limb development. We 58 found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal 59 loss but only in late developing chicken limbs when the limb is already innervated. 60 However, CPS49 exposure does not cause defects in limb size when added to late 61 developing chicken limbs. In contrast, in early limb buds which are not innervated, 62 CPS49 exposure affects limb area significantly. To investigate in more detail the role 63 of neurotoxicity and its impact on chicken limb development we inhibited nerve 64 innervation at a range of developmental timepoints through using β-bungarotoxin. 65 We found that neuronal inhibition or ablation before, during or after limb outgrowth 66 and innervation does not result in obvious limb cartilage patterning or number 67 changes. We conclude that while CPS49 is neurotoxic, given the late innervation of 68 the developing limb, and that neuronal inhibition/ablation throughout limb 69 development does not cause similar limb patterning anomalies to those seen in 70 thalidomide survivors, nerve defects are not the primary underlying cause of the 71 severe limb patterning defects induced by CPS49/thalidomide. 72 73 Page 2 of 21 Journal of Anatomy