A 190 base pair, TGF-β responsive tooth and fin enhancer is required for stickleback Bmp6 expression

Abstract

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.

Morphological Evolution Repeatedly Caused by Mutations in Signaling Ligand Genes

“…Another locus, encoding the bone morphogenetic protein 6 (BMP6) ligand, was found to cause tooth gain in freshwater stickleback population (Cleves et al 2014;Erickson et al 2015) (Fig. 4.2b).…”

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

“…Ten clutches were injected with a 190 bp stickleback Bmp6 enhancer that drives pectoral fin and median fin expression at 5 dpf 20 . From each clutch, at least 20 embryos were scored for having tissue-specific (pectoral and/or median fin) and/or nonspecific (all other tissues) GFP expression.…”

“…In addition to manipulating their coding sequences, the roles of candidate genes can be assessed by studying their cis -regulatory sequences and by functionally increasing, decreasing, or eliminating expression of the candidate gene. Microinjection and transgenesis methods in sticklebacks are well established 7,8,20 and were initially developed using a meganuclease-mediated method 21 first described in medaka 22 . The modified microinjection method presented here has been optimized for both Tol2-mediated transgenesis and recently developed genome editing reagents including TALENs and CRISPRs.…”

“…For studies in fish, Tol2 sites can be added to the BAC to facilitate genomic integration 32,33 . In later stages of development when in situ hybridization is technically challenging, the fluorescent readout of the BAC can be used to study patterns of gene expression, as has been shown for stickleback Bone morphogenetic protein 6 ( Bmp6 ) 20 . Additionally, fluorescent expression patterns in an individual can be tracked over time, which cannot be accomplished with in situ hybridization.…”

“…The subsequent repair of the double strand break created by both TALENs and CRISPRs often leaves behind a small insertion or deletion, which can disrupt the function of the target sequence 35–37 . In sticklebacks, TALENs have been used to disrupt gene expression by targeting an enhancer 20 , and both TALENs and CRISPRs have successfully produced mutations in coding sequences (unpublished data). A detailed protocol for the generation of CRISPRs for use in zebrafish can be used as a guideline to develop CRISPRs for sticklebacks 38 .…”