The length of each skeletal element changes independently during development and evolution to
transform an embryonic skeleton with similar sized cartilages into a diverse array of adult forms and functions.
Loss of function mutations of many genes produce proportionately dwarfed skeletons that suggest a common
“toolkit” is required for elongation of all of the long bones. Far less well understood, however, are the
mechanisms that establish the specific rate and duration of elongation at each growth plate, which together
determine adult limb skeletal proportion. What are the genes that define skeletal proportion? Is differential
growth controlled by modular enhancers that locally tune expression of genes common to all growth
plates and/or by genes that function only in subsets of growth plates?
Our laboratory is positioned to answer these profoundly important questions about how vertebrate limbs
acquire form and function using two uniquely suitable species: the laboratory mouse and the lesser Egyptian
jerboa. Among the nearest mouse relatives, the jerboa has the most extremely different hindlimbs with
extraordinarily long feet, but its forelimbs are similar to the mouse. These similarities and differences coupled
with high genome sequence homology enable the identification of genetic mechanisms that locally control
skeletal growth rate. RNA-Seq analysis of mouse and jerboa forelimb and hindlimb elements revealed that
10% of orthologous genes are differentially expressed correlating with relative growth rates within and between
species. These include 40 genes with strong evidence for enhancer modularity in both species. Aim 1 will
implement comparative ATAC-Seq and mouse transgenesis to identify and functionally test modular enhancers
in the mouse and jerboa genomes. We predict that some of these 40 genes are controlled by radius/ulna
enhancers that are conserved between species and by distinct metatarsal enhancers that functionally diverged
in jerboa and allowed the uncoupled evolution of jerboa hindlimb proportion.
Our expression data also provides a valuable opportunity to fill critical gaps in our understanding of the
genes that regulate limb skeletal growth and proportion in all vertebrates. We previously showed that IGF1
signaling is required in mice for hypertrophic chondrocyte size differences in growth plates that elongate at
different rates. Although IGF1 has a well-established role in whole organism and organ growth, it is unclear
how the pathway is locally regulated to modulate differential growth. In Aim 2, we will biochemically test the
hypothesis that elevated protease expression in rapidly elongating skeletal elements cleaves IGF binding
proteins thus freeing bioactive IGF1 protein for signaling to accelerate growth. Although six other high priority
candidate genes are also expected to be critical regulators of skeletal growth, they have not yet been attributed
growth plate functions. Aim 3 will implement a powerful overexpression approach in chicken embryos to test
the hypothesis that each of these genes is sufficient to accelerate or inhibit limb growth rate.