Reorganization of HoxD regulatory landscapes during the evolution of a snake-like body plan (4C-seq)

While the vertebrate body plan is highly conserved amongst all species of this taxon, extreme variations thereof can be documented in snakes, which display both an absence of limbs and an unusually elongated trunk. As Hox genes are strong candidates both for the making and the evolution of this body plan, their comparative study in such a morphologically diverged group is informative regarding their potential causative importance in these processes. In this work we use an interspecies comparative approach where different aspects of regulation at the HoxD locus are investigated. We find that although spatial collinearity and associated epigenetic mark dynamics are conserved in the corn snake, other regulatory modalities have been largely restructured. A BAC transgenic approach indeed revealed that, while the majority of mesodermal enhancers in vertebrates appear to be mostly located outside of the cluster, the corn snake contains most mesodermal trunk enhancers within the HoxD cluster. We also find that, despite the absence of limbs and an altered Hoxd gene regulation in external genitalia, the bimodal chromatin structure at the corn snake HoxD locus is maintained. The analysis of particular enhancer sequences initially defined in the mouse and further isolated at the snake orthologous locus showed differences in their specificities for the limb and genital bud expression. Of particular interest, a snake counterpart of a mouse limb-only enhancer sequence evolved into a genital-only enhancer. Such a regulatory exaptation suggests that enhancer versatility may have been an important factor to accompany the transition towards the snake body plan. These results show that vertebrate morphological evolution is likely to have been associated with extensive reorganization at the HoxD regulatory landscapes while respecting a very conserved general regulatory framework. Overall design: 4C-seq analysis of mouse and snake whole embryo and genital bud tissue using viewpoints contained within the cluster

尽管该类群所有物种的脊椎动物躯体构型（vertebrate body plan）都高度保守，但蛇类却展现出极端变异：它们既无四肢，躯干也异常修长。同源框基因（Hox genes）是塑造该躯体构型及其演化的核心候选调控因子，因此针对这类形态分化显著的类群开展比较研究，有助于揭示其在躯体构型形成与演化过程中的潜在关键调控作用。本研究采用种间比较分析方法，对HoxD基因座（HoxD locus）的多种调控机制展开研究。我们发现，尽管玉米蛇（corn snake）的Hox基因空间共线性及其相关表观遗传标记动态均保持保守，但其他调控模式已发生大范围重构。细菌人工染色体（BAC）转基因实验证实：尽管脊椎动物的绝大多数中胚层增强子均位于基因簇之外，但玉米蛇的大部分中胚层躯干增强子都位于HoxD基因簇内部。我们还发现，尽管蛇类无四肢且外生殖器中的Hoxd基因调控发生改变，但玉米蛇HoxD基因座的双峰染色质结构仍得以保留。对最初在小鼠中定义、后续在蛇类同源基因座中分离得到的特定增强子序列进行分析后发现，这些增强子在肢体与生殖芽表达中的特异性存在差异。尤为值得关注的是，小鼠仅在肢体中表达的增强子序列的蛇类同源序列，已演化成为仅在生殖器中表达的增强子。这种调控性外适应现象表明，增强子的多功能性可能是推动躯体构型向蛇类演化的重要因素之一。本研究结果表明，脊椎动物形态演化很可能伴随着HoxD调控景观的大规模重构，同时又保留了高度保守的整体调控框架。实验整体设计：利用基因簇内的锚定区域，对小鼠与蛇类的全胚胎及生殖芽组织开展环形染色体构象捕获测序（4C-seq）分析。