Identification of Genetic Mechanisms that Control Limb Bone Proportions During Mammalian Evolution

The remarkable diversity of limb shapes and proportions allows kangaroos to hop, bats to fly, and humans to walk upright. Despite our knowledge of genes necessary for the elongation of all limb bones, the genetic mechanisms that make some bones long and others short within a skeleton or between homologous elements of different species remain unclear. Did diverse skeletal proportions emerge by modifying a common bone elongation “toolkit” and/or by novel genetic strategies acting in each skeletal element? To address this question, our work leverages the extraordinarily divergent hindlimb skeleton of a bipedal desert rodent and its close phylogenetic relationship to the laboratory mouse. The three‐toed jerboa ( Jaculus jaculus ) has greatly elongated hindlimbs with disproportionately long feet and forelimbs that are similar to the mouse. The similarity of forelimb development therefore provides an “internal control”; genes that are equivalently differentially expressed at both anatomical locations are unlikely to explain the accelerated jerboa metatarsal growth rate. Indeed, differential RNA‐Seq analysis of neonatal jerboa and mouse growth cartilage revealed 59% of all orthologous transcripts are differentially expressed between jerboa and mouse metatarsals, but 83% of these are equivalently different between forearm elements (radius/ulna). These include a striking majority of the most highly differentially expressed transcripts. After removing these from consideration, 1,755 genes remain that are more or exclusively differentially expressed between jerboa and mouse metatarsals. Within this controlled but otherwise unbiased dataset, we find that evolutionarily accelerated hindlimb elongation in the jerboa likely results from mechanisms that both promote and de‐repress growth. Some of these genes have known functions in skeletal elongation (e.g. the Wnt signaling inhibitor, Sfrp2, and the short stature homeobox transcription factor, Shox2 ), but many others have no known function in the growth plate. These include inhibitors of the retinoic acid and Bone Morphogenic Protein signaling pathways ( Crabp1 and Mab21L2, respectively) and are therefore predicted to alter skeletal growth rate. We are currently testing the sufficiency of these genes to promote or inhibit skeletal elongation in vivo using a retroviral misexpression system in chicken embryos. In addition to providing the opportunity to identify new molecular mechanisms of skeletal growth control, this expression dataset is a valuable resource to focus efforts to identify regulatory mechanisms that ultimately explain the genetic basis of differential bone elongation during mammalian evolution. We have begun to apply the ATAC‐Seq chromatin profiling approach to identify differentially accessible sites with a long‐term goal to connect genotype to phenotype by replicating evolutionary events in the laboratory mouse. Support or Funding Information We are grateful to the University of California, San Diego, the Searle Scholars Program, the Pew Biomedical Scholars Program and Packard Fellowship for Science and Engineering for funding and support.(A) An adult jerboa. Skeletal reconstructions of an adult bipedal jerboa (B) and quadrupedal mouse (C). Homologous metatarsals (MT) in the two species are highlighted in red and radius/ulna (R/U) in blue.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .