Investigating the mechanisms involved in producing limb length variation in mice
Creators: name: Marchini, Marta
Publisher: name: Graduate Studies
Year: 2017
Subjects: subject: Anatomy
subject: Biology--Cell
subject: Genetics
subject: FOS: Biological sciences
subject: Zoology
subject: Longshanks
subject: Limb bone elongation
subject: Endochondral ossification
subject: Chondrocytes
subject: Artificial selection
subject: Comparative transcriptomics
subject: Skeletal dysplasia

description: The genetic and developmental mechanisms involved in outgrowth, patterning and elongation of the vertebrate limb during ontogeny are relatively well documented. However, how these mechanisms contribute to continuous variation in bone length remains unknown. Limb bone length is a complex quantitative trait, which involves the expression and interaction of many genes and gene pathways, as well as interactions with the environment. Understanding how phenotypic variation in limb bone length can be produced through development can help to understand how limb length diversity across mammal evolution. Limb bones are patterned during embryonic development, and in postnatal ontogeny they undergo elongation and remodeling to reach their adult size and shape. The elongation of the bone occurs by endochondral ossification within the growth plate, a cartilaginous structure in one or both ends of a long bone that is composed of distinct cellular zones reflecting different stages in the life cycle of chondrocytes: resting, proliferating and hypertrophic. This chondrocyte life cycle plays a key role in the ossification and elongation of long bones. Changes in the mechanisms of proliferation and/or hypertrophy can produce changes in bone apposition that cause variation in bone size and shape. Similarly, perturbation of either embryonic and/or postnatal development can cause severe effects on skeletal development, producing a range of disorders classified as skeletal dysplasias. Since 2010, our research group has selectively bred mice, known as Longshanks, for increases in relative tibia length, achieving a 15% increase at generation F20. Using artificial selection in mice in a controlled environment, and comparing selectively bred mice with unselected controls, provides the resolution necessary to study the developmental and genetic processes involved in producing continuous phenotypic differences in tibia length between individuals. Specifically, large differences in tibia length between selected and control mice can be used to identify where, when and how cell and molecular differences in growth plate chondrocyte function in the tibia contribute to continuous variation in long bone length. This dissertation aims to identify and quantify how cellular dynamics and gene expression in the growth plate contribute to limb bone length variation in Longshanks mice. We also investigate a novel dysmorphic phenotype identified during the Longshanks experiment that exhibits a severe but viable skeletal dysplasia-like phenotype (Shorty mice), in order to better understand how the Shorty mutation relates to broader categories of skeletal disorder. Using histomorphometry (Chapter 2) I discovered that Longshanks mice have a thicker proliferative zone with more chondrocytes per column compared to Controls, but the hypertrophic zone does not show differences in numbers and size of its chondrocytes. Proliferation assays further showed that there were no differences in the number of mitotic cells per proliferating chondrocytes in a column, suggesting that proliferative chondrocytes in Longshanks do not undergo mitosis faster than Control mice. Using RNAseq and qPCR (Chapter 3) I compared the epiphyseal and growth plate transcriptome between juvenile Longshanks mice and Control. My data suggest that the cellular differences we have found using histomorphometry (Chapter 2), may be due to the downregulation of Fxyd2, a γ-subunit of the NA+/K+ ATPase pump. Two other genes, Sox9 and Dlk1 may also play important roles in generating the underlying cellular differences between Longshanks and Control mice. In Chapter 3, I also investigate (Chapter 3) the role of the NA+/K+ ATPase pump in bone growth using embryonic limb and tibia culture using ouabain, a pharmacological inhibitor of the pump. The limbs and tibiae cultured with ouabain grew less than the controls. We also noticed a decrease of proliferative chondrocytes and using flow cytometry, and a parallel increase of programmed cell death in hypertrophic chondrocytes. These data suggest that regulation of the NA+/K+ ATPase pump is important in regulating chondrocyte differentiation and hypertrophic zone chondrocyte turnover. In Chapter 4, I studied the Shorty mice phenotype using µCT scanning and histology. My data suggest that the mutation that causes this unique skeletal dysplasia-like phenotype does not interfere with limb development in a manner comparable to common genetically-characterized skeletal dysplasias. The phenotype appears before embryonic stage E14, and may appear during limb outgrowth and patterning. This study provides a strong phenotypic foundation for the study of the underlying genetic causes of the Shorty mutation, and identifies a novel variant that may provide insight into underappreciated aspects of the limb development program. Overall, the research presented in this dissertation provides important insight into aspects of limb development and growth plate biology that have been previously difficult to identify. In particular, this dissertation used a novel approach to answer one of evolutionary developmental biology’s long-standing questions: the developmental origins of phenotypic variation.