Doctoral Dissertation Improvement: Effects of temperature on growth plate physiology in an experimentally-induced mouse model of "Allen's Rule"

已发表的数据表明，在不同温度下饲养的同一窝哺乳动物的成年肢体和身体比例与生活在极端气候下的自然种群相似。在寒冷环境中饲养的动物身体粗壮，四肢和四肢缩短，而在温暖环境中饲养的动物则表现出外围附属物的伸长。这些变化类似于“艾伦法则”所预测的结果。“艾伦法则”是一种长期以来被认为是体温调节适应性机制的生态地理学原理。虽然通常被认为是遗传性质的，但其中一些表型变化似乎是对温度胁迫的个体发生反应；然而，迄今为止仅报道了描述性研究，这些变化背后的机制尚不清楚，尚未进行调查。温度可以直接影响细胞动力学，因此生长速度在温暖的温度下会增加，而在寒冷的温度下会减慢。如果正确的话，那么这些变化应该表现在软骨生长板上，即出生后发育过程中纵向骨快速生长的部位。受损的血管系统还可以通过减少必需的氧气、营养物质和激素的数量来限制生长速度，这些氧气、营养物质和激素可以通过冷诱导的血管收缩来发育骨骼。目前没有经验数据支持这些观点，并且在整个个体发育过程中长期暴露于极端温度的动物的生长板形态仍未被探索。本研究的目的是验证低温通过降低生长板动力学和/或血管供应来限制骨生长的假设，而温暖的温度会促进这些过程。研究气候引起的骨骼变化的模型将通过在小鼠活跃生长期的不同温度（7、20和27摄氏度）下饲养小鼠来建立，如先前文献所述。在为期九周的实验期间，将记录基本的生长数据，并创建一个个体发生系列，以检查温度对不同发育阶段的骨骼组织学和血流的影响。长骨生长板的细胞增殖和凋亡（程序性死亡）将通过免疫组织化学进行量化，并在光镜下研究详细的形态学。血管供应将使用免疫组织化学方法检测新血管形成和荧光微球技术测量区域骨血流量的组合来分析。科学价值：这项研究将为理解物种如何改变骨骼比例以应对环境压力提供必要的信息，并可能因此深刻地影响地理线中种内和种间变化的感知方式。通过区分温度对骨骼生长和血液供应的相对影响，这项工作可以进一步阐明化石记录中观察到的形态变化，也可能有助于解决跨学科的主要系统发育争议。更广泛的影响：这些结果将加强对参与调节正常纵向骨生长的复杂因素的理解。这里要检查的参数与骨骼损伤和疾病的各种生物医学研究模型有关，特别是那些涉及骨骼生长和脉管系统受损的模型。通过整合人类学、生态学、分子生物学和生理学的思想，该项目鼓励来自不同背景和专业的学生和高级研究人员之间的合作。

Published data demonstrate that mammals from the same litter reared at different temperatures have different adult limb and body proportions in parallel with those of natural populations living at climatic extremes. Animals raised in cold conditions have stout bodies with shortened limbs and extremities, while those housed at warmer temperatures exhibit peripheral appendage elongation. These changes resemble those predicted by "Allen's rule," the ecogeographical principle long recognized to be an adaptive mechanism for thermoregulation. Although typically thought to be genetic in nature, some of these phenotypic changes appear to be ontogenetic responses to temperature stress; however, only descriptive studies have been reported to date and the mechanisms underlying such changes are unknown and have not yet been investigated. Temperature may directly influence cellular kinetics such that growth rate is increased at warm temperatures but retarded in the cold. If correct, then these changes should be manifested in cartilaginous growth plates, sites of rapid longitudinal bone growth during postnatal development. Impaired vasculature could also limit growth rate by reducing the amount of essential oxygen, nutrients, and hormones available to developing bones via cold-induced vasoconstriction. There are currently no empirical data to support these ideas, and growth plate morphology in animals chronically exposed to extreme temperatures throughout ontogeny remains unexplored. The objective of this study is to test the hypothesis that cold temperature limits bone growth by reducing growth plate kinetics and/or vascular supply, and that warm temperature enhances these processes. A model for investigating climate-induced skeletal changes will be established by housing mice at different temperatures (7, 20, and 27 degrees C) during their active growth period as previously described in the literature. Basic growth data will be recorded throughout a nine week experimental period and an ontogenetic series will be created in order to examine the effects of temperature on bone histology and blood flow at different stages of development. Cell proliferation and apoptosis (programmed death) in long bone growth plates will be quantified using immunohistochemistry, and detailed morphology will be studied under light microscopy. Vascular supply will be analyzed using a combination of immunohistochemical methods to detect new vessel formation and fluorescent microsphere techniques to measure regional bone blood flow. SCIENTIFIC MERIT: This study will provide information essential to understanding how species modify skeletal proportions in response to environmental pressures and could thereby profoundly impact the manner in which intra- and inter-specific variation in geographical clines is perceived. By discriminating the relative influence of temperature on bone growth and blood supply, this work can further elucidate the morphological variation observed in the fossil record and might also help resolve major phylogenetic controversies across disciplines. BROADER IMPACTS: These results will enhance understanding of the complex factors involved in regulating normal longitudinal bone growth. The parameters to be examined here are relevant to a variety of biomedical research models of skeletal injury and disease, especially those involving impaired bone growth and vasculature. By integrating ideas from anthropology, ecology, molecular biology, and physiology, this project encourages collaboration between students and senior researchers from diverse backgrounds and specialties.