Threespine stickleback fish offer a powerful system to dissect the genetic basis of morphological evolution in nature. Marine sticklebacks have repeatedly invaded and adapted to numerous freshwater environments throughout the Northern hemisphere. In response to new diets in freshwater habitats, changes in craniofacial morphology, including heritable increases in tooth number, have evolved in derived freshwater populations. Using a combination of quantitative genetics and genome resequencing, here we fine-mapped a quantitative trait locus (QTL) regulating evolved tooth gain to a cluster of ten QTL-associated single nucleotide variants, all within intron four of Bone Morphogenetic Protein 6 (Bmp6). Transgenic reporter assays revealed this intronic region contains a tooth enhancer. We induced mutations in Bmp6, revealing required roles for survival, growth, and tooth patterning. Transcriptional profiling of Bmp6 mutant dental tissues identified significant downregulation of a set of genes whose orthologs were previously shown to be expressed in quiescent mouse hair stem cells. Collectively these data support a model where mutations within a Bmp6 intronic tooth enhancer contribute to evolved tooth gain, and suggest that ancient shared genetic circuitry regulates the regeneration of diverse vertebrate epithelial appendages including mammalian hair and fish teeth.
The ligands of the Bone Morphogenetic Protein (BMP) family of developmental signaling molecules are often under the control of complex cis-regulatory modules and play diverse roles in vertebrate development and evolution. Here, we investigated the cis-regulatory control of stickleback Bmp6. We identified a 190 bp enhancer ~2.5 kilobases 5’ of the Bmp6 gene that recapitulates expression in developing teeth and fins, with a core 72 bp sequence that is sufficient for both domains. By testing orthologous enhancers with varying degrees of sequence conservation from outgroup teleosts in transgenic reporter gene assays in sticklebacks and zebrafish, we found that the function of this regulatory element appears to have been conserved for over 250 million years of teleost evolution. We show that a predicted binding site for the TGFβ effector Smad3 in this enhancer is required for enhancer function and that pharmacological inhibition of TGFβ signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. Finally, we used TALENs to disrupt the enhancer in vivo and find that Bmp6 expression is dramatically reduced in teeth and fins, suggesting this enhancer is necessary for expression of the Bmp6 locus. This work identifies a relatively short regulatory sequence that is required for expression in multiple tissues and, combined with previous work, suggests that shared regulatory networks control limb and tooth development.
Changes in developmental gene regulatory networks enable evolved changes in morphology. These changes can be in cis regulatory elements that act in an allele-specific manner, or changes to the overall trans regulatory environment that interacts with cis regulatory sequences. Here we address several questions about the evolution of gene expression accompanying a convergently evolved constructive morphological trait, increases in tooth number in two independently derived freshwater populations of threespine stickleback fish (Gasterosteus aculeatus). Are convergently evolved cis and/or trans changes in gene expression associated with convergently evolved morphological evolution? Do cis or trans regulatory changes contribute more to gene expression changes accompanying an evolved morphological gain trait? Transcriptome data from dental tissue of ancestral low-toothed and two independently derived high-toothed stickleback populations revealed significantly shared gene expression changes that have convergently evolved in the two high-toothed populations. Comparing cis and trans regulatory changes using phased gene expression data from F1 hybrids, we found that trans regulatory changes were predominant and more likely to be shared among both high-toothed populations. In contrast, while cis regulatory changes have evolved in both high-toothed populations, overall these changes were distinct and not shared among high-toothed populations. Together these data suggest that a convergently evolved trait can occur through genetically distinct regulatory changes that converge on similar trans regulatory environments.
25Changes in developmental gene regulatory networks enable evolved changes in morphology. 26These changes can be in cis regulatory elements that act in an allele-specific manner, or 27 changes to the overall trans regulatory environment that interacts with cis regulatory 28 sequences. Here we address several questions about the evolution of gene expression 29 accompanying a convergently evolved constructive morphological trait, increases in tooth 30 number in two independently derived freshwater populations of threespine stickleback fish 31 (Gasterosteus aculeatus). Are convergently evolved cis and/or trans changes in gene 32 expression associated with convergently evolved morphological evolution? Do cis or trans 33 regulatory changes contribute more to the evolutionary gain of a morphological trait? 34Transcriptome data from dental tissue of ancestral low-toothed and two independently derived 35 high-toothed stickleback populations revealed significantly shared gene expression changes 36 that have convergently evolved in the two high-toothed populations. Comparing cis and trans 37 regulatory changes using phased gene expression data from F1 hybrids, we found that trans 38 regulatory changes were predominant and more likely to be shared among both high-toothed 39 populations. In contrast, while cis regulatory changes have evolved in both high-toothed 40 populations, overall these changes were distinct and not shared among high-toothed 41 populations. Together these data suggest that a convergently evolved trait can occur through 42 genetically distinct regulatory changes that converge on similar trans regulatory 43
In nature, multiple adaptive phenotypes often coevolve and can be controlled by tightly linked genetic loci known as supergenes. Dissecting the genetic basis of these linked phenotypes is a major challenge in evolutionary genetics. Multiple freshwater populations of threespine stickleback fish () have convergently evolved two constructive craniofacial traits, longer branchial bones and increased pharyngeal tooth number, likely as adaptations to dietary differences between marine and freshwater environments. Prior QTL mapping showed that both traits are partially controlled by overlapping genomic regions on chromosome 21 and that a regulatory change in likely underlies the tooth number QTL. Here, we mapped the branchial bone length QTL to a 155 kb, eight-gene interval tightly linked to, but excluding the coding regions of and containing the candidate gene Further recombinant mapping revealed this bone length QTL is separable into at least two loci. During embryonic and larval development, was expressed in the branchial bone primordia, where allele specific expression assays revealed the freshwater allele of was expressed at lower levels relative to the marine allele in hybrid fish. Induced loss-of-function mutations in revealed an essential role in stickleback craniofacial development and show that bone length is sensitive to dosage in heterozygotes. Combined, these results suggest that closely linked but genetically separable changes in and contribute to a supergene underlying evolved skeletal gain in multiple freshwater stickleback populations.
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