Attempting genetic inference from directional asymmetry during convergent hindlimb reduction in squamates

Loss and reduction of paired appendages is common in vertebrate evolution. How often does such convergent evolution depend on similar developmental and genetic pathways? For example, many populations of the Threespine Stickleback and Ninespine Stickleback (Gasterosteidae) have independently evolved pelvic reduction, usually based on independent mutations that caused reduced Pitx1 expression. Reduced Pitx1 expression has also been implicated in pelvic reduction in manatees. Thus, hind limb reduction stemming from reduced Pitx1 expression has arisen independently in groups that diverged tens to hundreds of millions of years ago, suggesting a potential for repeated use of Pitx1 across vertebrates. Notably, hindlimb reduction based on reduction of Pitx1 expression produces left-larger directional asymmetry in the vestiges. We used this phenotypic signature as a genetic proxy, testing for hindlimb directional asymmetry in six genera of squamate reptiles that independently evolved hindlimb reduction and for which genetic and developmental tools are not yet developed: Agamodon anguliceps, Bachia intermedia, Chalcides sepsoides, Indotyphlops braminus, Ophisaurus attenuatuas and O. ventralis, and Teius teyou. Significant asymmetry occurred in one taxon, Chalcides sepsoides, whose left-side pelvis and femur vestiges were 18% and 64% larger than right-side vestiges, respectively, suggesting modification of Pitx1 expression in that species. However, there was either right-larger asymmetry or no directional asymmetry in the other five taxa, suggesting multiple developmental genetic pathways to hindlimb reduction in squamates and vertebrates more generally.

Methods
Micro Computed Tomography (μ-CT) scanning. Each specimen was wrapped in plastic and scanned for 4 minutes using a Perkins-Elmer Quantum GX2 micro-CT Imaging System. To prevent beam hardening, an image artifact, we used either an Al 1.0 mm or an Al 0.5 mm + Cu 0.06 mm filter, as needed. Because individual size varied within and among taxa, we adjusted voltage, current, and voxel size for specimen size (Table 1). We used the smallest voxel size that still contained the pelvis and femurs (if present).

Image processing. We used the open-source program InVesalius v.3.1 to reconstruct 3D images from raw DICOM files. We applied a reconstruction threshold to reduce image noise. Thresholds varied by voxel size and tissue density and were adjusted by eye to create surfaces with reduced artifact, maximal bone quality, and high definition. Because these criteria are subjective, no single setting optimizes 3D surface reconstruction for all specimens. For consistency, the thresholds were determined by the same individual (S.S.) across all samples.

Landmarks. Homologous landmarks were placed on each 3D surface using open-source software MeshLab 2016. All landmarks were digitized by S.S. for consistency. Each image was rotated during landmark placement to minimize parallax, and the images were rotated after landmark placement to ensure visually that they had been placed correctly. We marked the anterior point of the pubis and the posterior point of the ilium on right and left sides to measure the pelvis (Fig. 1). In specimens with a less well-developed pubis, the anterior- medial point of the epipubis was used as the anterior landmark. In species without femurs, the pubis, ilium, and ischium bones of the pelvis were usually indistinguishable; we therefore landmarked the anterior and posterior points of the pelvic vestige.

For species with femurs, we placed landmarks on the proximal point of the femur head (at the hip) and the most distal point of the femur (at the knee; Fig. 1). CT resolution was not fine enough to distinguish distal limb bones in B. intermedia due to extensive reduction (Fig. 1B). Therefore, proximal landmarks were placed on femur head and distal landmarks were placed on the most distal skeletal point of the limb, which might include non-femoral elements. However, in mice, altered Pitx1 expression resulted in reduction in the tibia, fibula, and metatarsals, as well as the pelvis and femur (Lanctot et al. 1999; Szeto et al. 1999; Marcil et al. 2003). Thus, including elements distal to the femur should yield a suitable measure of hind limb length in B. intermedia, especially since our metric is asymmetry. We exported landmarks from MeshLab as picked_points.pp files.

成对附肢的丢失与退化在脊椎动物演化过程中极为常见。这类趋同演化究竟在多大程度上依赖于相似的发育与遗传通路？例如，三棘刺鱼（Threespine Stickleback）与九棘刺鱼（Ninespine Stickleback，Gasterosteidae）的多个种群已独立演化出骨盆退化性状，其背后通常是独立发生的突变导致Pitx1基因表达量下调。海牛的骨盆退化也被证实与Pitx1表达降低相关。由此可见，由Pitx1表达下调引发的后肢退化，在距今数千万至数亿年分化的类群中已独立出现多次，这提示脊椎动物演化中可能反复动用Pitx1基因通路。值得注意的是，由Pitx1表达下调导致的后肢退化，其残留结构会呈现左侧偏大的方向不对称性。我们以此表型特征作为遗传替代标记，对6个独立演化出后肢退化性状的有鳞爬行动物属开展后肢方向不对称性检测；这些类群暂未建立成熟的遗传与发育研究工具，分别为：角隐盲蛇（Agamodon anguliceps）、中型南折肢蜥（Bachia intermedia）、多肢石龙子（Chalcides sepsoides）、印度盲蛇（Indotyphlops braminus）、细蛇蜥（Ophisaurus attenuatuas）、腹斑蛇蜥（Ophisaurus ventralis）以及泰氏蜥（Teius teyou）。检测结果显示，仅多肢石龙子（Chalcides sepsoides）呈现出显著的不对称性：其左侧骨盆与股骨残留结构分别比右侧大18%与64%，提示该类群的Pitx1表达发生了调控改变。但其余5个类群均表现为右侧偏大的不对称性或无方向不对称性，这表明有鳞爬行动物乃至脊椎动物的后肢退化可能存在多种发育遗传通路。

方法

显微计算机断层扫描（Micro Computed Tomography, μ-CT）
将每份标本以塑料包裹后，使用Perkins-Elmer Quantum GX2显微CT成像系统进行4分钟扫描。为防止线束硬化这一图像伪影，我们根据需要使用1.0mm铝滤片，或0.5mm铝+0.06mm铜的复合滤片。由于类群内部及类群间的个体体型存在差异，我们根据标本尺寸调整电压、电流与体素大小（详见表1）。我们选用可完整覆盖骨盆与股骨（若存在）的最小体素尺寸。

图像处理
我们使用开源软件InVesalius v.3.1从原始DICOM文件中重建三维图像，并通过设置重建阈值以降低图像噪声。阈值需根据体素大小与组织密度进行调整，通过人工目视调节以获得伪影最少、骨质显示最佳且细节清晰的表面模型。由于该调整标准具有主观性，不存在适用于所有标本的统一最优参数。为保证实验一致性，所有样本的阈值均由同一操作者（S.S.）确定。

地标点采集
我们使用开源软件MeshLab 2016在每份三维表面模型上放置同源地标点，所有地标点均由S.S.手动数字化以保证一致性。在地标点采集过程中，我们会旋转图像以最小化视差误差，并在采集完成后再次旋转图像以目视确认地标点位置正确。针对骨盆的测量，我们在左右两侧分别标记耻骨前缘与髂骨后缘（图1）。若耻骨发育较差，则以耻骨前内侧点作为前侧地标点。对于无股骨的物种，骨盆的耻骨、髂骨与坐骨通常难以区分，因此我们以骨盆残留结构的前后端点作为地标点。

对于保留股骨的物种，我们在股骨头近端（髋关节处）与股骨最远端（膝关节处）放置地标点（图1）。由于中型南折肢蜥（Bachia intermedia）的附肢退化程度极高，CT分辨率不足以区分其远端肢骨（图1B），因此我们将近端地标点设于股骨头，远端地标点设于肢体最远端的骨骼结构点，该点可能包含非股骨的骨骼成分。不过，已有研究显示，在小鼠中，Pitx1表达异常会导致胫骨、腓骨、跖骨以及骨盆与股骨的退化（Lanctot et al. 1999; Szeto et al. 1999; Marcil et al. 2003）。因此，纳入股骨远端的结构应可适用于中型南折肢蜥的后肢长度测量，尤其是我们的测量指标为不对称性。我们将MeshLab采集的地标点导出为picked_points.pp格式文件。