ENHANCing the limb: from micro- to macro-evolution

Abstract Understanding the molecular basis of the diverse morphological forms found within and across species is a longstanding goal in evolutionary biology. One especially relevant class of cis-regulatory elements are enhancers. This is because mutations affecting enhancers tend to be tissue- or stage-specific, which allows adaptation to proceed with relatively less harmful side effects in other organs or tissues. In Chapter 2 I explore how enhancers help drive morphological selection response within-species. We scanned the genomes of the Longshanks mice, which are mice selectively bred over 20 generations, for a 13% increase in tibiae. Against a backdrop of polygenic response, we found the bone repressor Nkx3-2, and specifically its enhancers, to be among the strongest contributor towards increased tibia length. I used transgenics to compare the enhancer activity of the F0 and F17 alleles at 3 candidate enhancers (two near the Nkx3-2 gene; and one near the limb developmental regulator gene, Gli3). We found that both loss-of-function (Nkx3-2) and gain-of-function (Gli3) alleles contributed to the selection response. In Chapter 3, we explored an approach to study macro-evolutionary variations across species. One of the major barriers to such study is the inability to perform direct genetic crosses due to hybrid sterility. We tackle the species barrier problem by inducing mitotic recombination in vitro in hybrid embryonic stem cells (including cross-species hybrids between Mus musculus and Mus spretus). This was achieved via Blm inhibition by the small molecule ML216. We further show, that the resultant mitotic recombinant cells can be used for genetic mapping by connecting tioguanine drug resistance to variations at the Hprt locus. Furthermore, in vitro recombinant stem cells can be used for rederivation of animals through laser-assisted morula injection, thus allowing the acquisition of morphological data. Here, through a multidisciplinary approach, we show that enhancer modulation contributes to morphological diversity and selection response within-species and provide a new methodology for enhancer study across-species, thus enabling the study of evolutionary developmental variations in genetic backgrounds that would otherwise be challenging to obtain. Overall, these studies highlight the relevance of enhancers in morphological diversification and provide new tools for their study