How new communication behaviors evolve: Androgens as modifiers of neuromotor structure and function in foot-flagging frogs

How diverse animal communication signals have arisen is a question that has fascinated many. Xenopus frogs have been a model system used for three decades to reveal insights into the neuroendocrine mechanisms and evolution of vocal diversity. Due to the ease of studying central nervous system control of the laryngeal muscles in vitro, Xenopus has helped us understand how variation in communication signals between sexes and between species is produced at the molecular, cellular, and systems levels. Yet, it is becoming easier to make similar advances in non-model organisms. Here, we summarize our research on a group of frog species that have evolved a novel hind limb signal known as ‘foot flagging.’ We have shown that the evolution of foot flagging in multiple species is accompanied by the evolution of higher androgen hormone sensitivity in the leg muscles and an increased density of spinal interneurons in the neuromotor system that controls the hind limb. Comparing this work to prior wor..., Data on androgen receptor (AR) expression was generated using radioactive in situ hybridization. See manuscript supplemental files for complete methods. Data were collected from photomicrographs. Tissue slides were developed in photographic emulsion and silver grains were developed over cells that contained androgen receptor mRNA. AR expression data were collected from photomicrographs taken on a light microscope. Tissue sections were counterstained with Nissl stain so we could also see the cells. We quantified the number and area of developed silver grains in each image, then subtracted the number of background silver grains outside for the target area, and divided by the total number of identified neurons in the image/field of view. Thus, AR expression data can be expressed as the total area covered by AR grains, percent area covered by AR grains, number of grains above the background, and number of grains above the background per cell. Additionally, we can average the values for each..., , # How new communication behaviors evolve: Androgens as modifiers of neuromotor structure and function in foot-flagging frogs --- ## Description of the data and file structure ### Filename: ARData Each row in the data file represents one observation. There were multiple measurements of AR and cells from a single frog (grouped by SubjectID). Columns represent each of the independent variables (species, spinal cord segment) and dependent variables (AR grains above background, cell counts). Column names: * \"MotorNuc\" = spinal cord segment that data were taken from, L= lumbar, B=brachial * \"Sample\" = denotes how many image samples were taken from each subject, with 1=most anterior location in the spinal cord * \"MNCount\" = number of motoneurons counted in each photomicrograph; \"INCount\" = number of interneurons counted in each photomicrograph * \"TotalCells\" = MNCount+INCount * \"TotAreaGrains\" = area covered by silver grains in square pixels * \"PercAreaGrains\" = percen...动物通讯信号的多样性起源始终是令学界着迷的核心问题。三十余年来，爪蟾属（Xenopus）青蛙一直作为经典模式系统，为揭示发声多样性的神经内分泌机制与演化历程提供了重要洞见。得益于体外（in vitro）研究中枢神经系统对喉肌调控的便捷性，爪蟾属（Xenopus）帮助我们阐明了：两性间以及物种间通讯信号的差异是如何在分子、细胞及系统层面产生的。然而如今，在非模式生物中取得类似研究进展已愈发可行。本文综述了我们针对一类演化出新型后肢通讯信号——“扬足动作（foot flagging）”——的蛙类开展的研究工作。我们的研究证实，多个物种中扬足动作的演化，伴随着腿部肌肉雄激素敏感性的提升，以及调控后肢的神经运动系统中脊髓中间神经元密度的增加。相较于此前的研究[...]，本研究中雄激素受体（androgen receptor, AR）的表达数据通过放射性原位杂交（radioactive in situ hybridization）实验获得，完整实验方法详见论文补充材料。实验数据通过显微照片采集获得：组织切片经感光乳剂显影后，携带雄激素受体mRNA的细胞表面会形成银粒（silver grains）。本研究的AR表达数据均采集自光镜下拍摄的显微照片；组织切片经尼氏染色（Nissl stain）复染，以便同时观察细胞形态。我们对每张图像中显影银粒的数量与面积进行定量分析，随后扣除目标区域外的背景银粒数，再除以该图像/视野内已识别的神经元总数。因此，AR表达数据可通过以下指标表征：AR银粒总覆盖面积、AR银粒覆盖面积百分比、背景校正后的银粒数，以及每细胞背景校正银粒数。此外，我们还可针对每个[...]计算平均值。 # 新型通讯行为的演化：扬足示警蛙类中雄激素作为神经运动系统结构与功能的调控因子 --- ## 数据与文件结构说明 ### 文件名：ARData 数据文件中的每一行对应一个观测值。 单只青蛙（按SubjectID分组）的AR与细胞测量数据存在多个重复样本。 数据列包含各类自变量（物种、脊髓节段）与因变量（背景校正后AR银粒数、细胞计数）。 列名说明： * "MotorNuc"：数据采集对应的脊髓节段，L代表腰段（lumbar），B代表臂段（brachial） * "Sample"：标记从每个受试个体采集的图像样本序号，1对应脊髓最前端位置 * "MNCount"：每张显微照片中计数的运动神经元数量；"INCount"：每张显微照片中计数的中间神经元数量 * "TotalCells"：MNCount与INCount之和 * "TotAreaGrains"：银粒覆盖面积，单位为平方像素 * "PercAreaGrains"：百分比[...]