Animal Models for Understanding Human Skeletal Defects.

Skeletal defects, such as cleft palate, scoliosis, and shortening of the limb bones are common in the human population. Animal models have been essential for characterizing the molecular and cellular mechanisms that underlie these and other skeletal disorders. This chapter will explore the cellular origins of the vertebrate skeleton and introduce a selection of animal models for human disorders of the skull and facial bones, spinal column, and limbs. The common genetic pathways that build the skeleton of various vertebrate species and how these similarities facilitate the study of human developmental processes in laboratory animals will be a focus of discussion. This chapter will also highlight how current genome editing technologies can be applied to model various perturbations of human chromatin structure in laboratory animals.

An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice.

Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci tending to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response.

Identification of a limb enhancer that is removed by pathogenic deletions downstream of the SHOX gene.

Haploinsufficiency of the human SHOX gene causes Léri-Weill dyschondrosteosis (LWD), characterized by shortening of the middle segments of the limbs and Madelung deformity of the wrist. As many as 35% of LWD cases are caused by deletions of non-coding sequences downstream of SHOX that presumably remove an enhancer or enhancers necessary for SHOX expression in developing limbs. We searched for these active sequences using a transgenic mouse assay and identified a 563 basepair (bp) enhancer with specific activity in the limb regions where SHOX functions. This enhancer has previously escaped notice because of its poor evolutionary conservation, although it does contain 100 bp that are conserved in non-rodent mammals. A primary cell luciferase assay confirmed the enhancer activity of the conserved core sequence and demonstrated that putative HOX binding sites are required for its activity. This enhancer is removed in most non-coding deletions that cause LWD. However, we did not identify any likely pathogenic variants of the enhancer in a screen of 124 LWD individuals for whom no causative mutation had been found, suggesting that only larger deletions in the region commonly cause LWD. We hypothesize that loss of this enhancer contributes to the pathogenicity of deletions downstream of SHOX.