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

公布的数据表明，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条件下的自然种群的成年肢体和身体比例不同。在寒冷条件下饲养的动物身体粗壮，四肢缩短，而在较温暖的环境中饲养的动物外周附肢延长。这些变化类似于“艾伦规则”所预测的那些，这一生态地理学原理长期以来被认为是一种适应体温调节的机制。虽然通常被认为是遗传的，但这些表型变化中的一些似乎是个体发育对温度胁迫的反应；然而，到目前为止，只有描述性研究被报道，这种变化的机制尚不清楚，也尚未被研究。温度可能直接影响细胞的动力学，因此在温暖的温度下生长速度加快，而在寒冷的温度下生长速度减缓。如果正确，那么这些变化应该在软骨生长板上表现出来，软骨生长板是出生后发育过程中骨骼快速纵向生长的部位。血管系统受损也可能会限制生长速度，因为冷诱导的血管收缩会减少骨骼发育所需的必要氧气、营养物质和激素的数量。目前还没有经验数据支持这些观点，在整个个体发育过程中长期暴露在极端温度下的动物的生长板形态仍未被探索。本研究的目的是验证一种假说，即低温通过减少生长板动力学和/或血管供应来限制骨的生长，而温暖的温度促进了这些过程。一个研究气候引起的骨骼变化的模型将建立在不同温度(7、20和27摄氏度)下的小鼠在它们活跃的生长期内，正如先前文献中所描述的那样。在为期9周的实验期内，将记录基本的生长数据，并将创建个体发育系列，以检查温度对骨组织学和不同发育阶段血液流动的影响。长骨生长板中的细胞增殖和凋亡(程序性死亡)将通过免疫组织化学进行量化，详细的形态将在光学显微镜下进行研究。将结合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局部骨血流量来分析血管供应。科学价值：这项研究将提供必要的信息，以了解物种如何改变骨骼比例以响应环境压力，从而可能深刻影响感知地理斜坡的种内和种间差异的方式。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，并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：这些结果将加强对调节正常纵向骨生长所涉及的复杂因素的理解。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血管系统受损的模型。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.