Valproic acid (VPA) is an anti-epileptic drug known to cause congenital craniofacial abnormalities, including orofacial clefts (OFC). The exact mechanisms by which VPA leads to craniofacial skeletal malformations are poorly understood. In this study, we investigated the effects of VPA on cartilage and bone formation in the zebrafish larval head during 1-13 hpf (early) and 25-37 hpf (late) development in which cranial neural crest cells (CNCCs) arise and then proliferate and differentiate, respectively. Double-staining for cartilage and bone at 5 dpf revealed that VPA reduced cartilage and bone formation in a dose-dependent manner after both early or late exposure. Several different CNCC-derived cartilage and bone elements were affected in both groups. In the early group (100 μM VPA), the posterior head length and the ethmoid plate were reduced in length (both p < 0.01), while mineralization of 4 out of 9 bone elements was often lacking (all p < 0.01). In the late group (100 μM VPA), also the posterior head length was reduced as well as the length of the ceratohyals (both p < 0.01). Similar to early exposure, mineralization of 3 out of 9 bone elements was often lacking (all p < 0.01). These results indicate that both CNCC formation (early) and differentiation (late) are hampered by VPA treatment, of which the consequences for bone and cartilage formation are persistent at 5 dpf. Indeed, we also found that the expression of several genes related to cartilage and bone was upregulated at 5 dpf. These data indicate a compensatory reaction to the lack of cartilage and bone. Altogether, VPA seems to induce craniofacial malformations via disturbed CNCC function leading to defects in cartilage and bone formation.

中文翻译：

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抗癫痫药物丙戊酸导致斑马鱼幼虫发育中的颅面骨骼畸形

丙戊酸 (VPA) 是一种抗癫痫药物，已知会导致先天性颅面畸形，包括口面部裂隙 (OFC)。VPA 导致颅面骨骼畸形的确切机制知之甚少。在这项研究中，我们研究了 VPA 在 1-13 hpf（早期）和 25-37 hpf（晚期）发育期间对斑马鱼幼虫头部软骨和骨骼形成的影响，其中颅神经嵴细胞 (CNCC) 出现然后增殖和分别。5 dpf 的软骨和骨骼双染色显示，VPA 在早期或晚期暴露后以剂量依赖性方式减少软骨和骨骼形成。两组中几种不同的 CNCC 衍生的软骨和骨元素均受到影响。在早期组（100 μM VPA）中，后头部长度和筛板长度减少（均 p < 0.01），而 9 种骨元素中的 4 种经常缺乏矿化（所有 p < 0.01）。在晚期组 (100 μM VPA) 中，后头部长度以及角状玻璃的长度也减少了 (p < 0.01)。与早期暴露类似，9 种骨元素中的 3 种通常缺乏矿化（所有 p < 0.01）。这些结果表明 CNCC 形成（早期）和分化（晚期）都受到 VPA 处理的阻碍，其中骨和软骨形成的后果在 5 dpf 时持续存在。事实上，我们还发现与软骨和骨骼相关的几个基因的表达在 5 dpf 时上调。这些数据表明缺乏软骨和骨骼的代偿反应。共，