EvolvedBmp6enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks

Abstract

Mutations in enhancers have been shown to often underlie natural variation but the evolved differences in enhancer activity can be difficult to identify in vivo. Threespine sticklebacks (Gasterosteus aculeatus) are a robust system for studying enhancer evolution due to abundant natural genetic variation, a diversity of evolved phenotypes between ancestral marine and derived freshwater forms, and the tractability of transgenic techniques. Previous work identified a series of polymorphisms within an intronic enhancer of the Bone morphogenetic protein 6 (Bmp6) gene that are associated with evolved tooth gain, a derived increase in freshwater tooth number that arises late in development. Here we use a bicistronic reporter construct containing a genetic insulator and a pair of reciprocal two-color transgenic reporter lines to compare enhancer activity of marine and freshwater alleles of this enhancer. In older fish the two alleles drive partially overlapping expression in both mesenchyme and epithelium of developing teeth, but the freshwater enhancer drives a reduced mesenchymal domain and a larger epithelial domain relative to the marine enhancer. In younger fish these spatial shifts in enhancer activity are less pronounced. Comparing Bmp6 expression by in situ hybridization in developing teeth of marine and freshwater fish reveals similar evolved spatial shifts in gene expression. Together, these data support a model in which the polymorphisms within this enhancer underlie evolved tooth gain by shifting the spatial expression of Bmp6 during tooth development, and provide a general strategy to identify spatial differences in enhancer activity in vivo.

Site pleiotropy of a stickleback tooth and fin enhancer

“…1F). Thus, Foxc1 gene expression overlaps with activity of the Bmp6 intronic enhancer in teeth (Stepaniak et al, 2021). (bottom).…”

“…This enhancer drives robust expression in all developing pharyngeal and oral teeth, as well as in the distal margins of the pectoral and median fins (Fig. 1A,B) (Cleves et al, 2018; Stepaniak et al, 2021). The sequence of this intronic enhancer is evolutionarily conserved in teleosts, with clear homology detectable in medaka and zebrafish, as well as to gar, an outgroup to teleosts (Fig.…”

“…This site pleiotropy is reminiscent of a previously described enhancer 5’ to the stickleback Bmp6 gene, where a conserved predicted SMAD3 binding site is required for enhancer activity in teeth and fins (Erickson et al, 2015). While both marine and freshwater intron four Bmp6 enhancers drive strong expression in the distal edges of embryonic fins, only the freshwater enhancer was detected in intersegmental joints in pectoral and caudal fins (Stepaniak et al, 2021). The intact enhancer tested here was the freshwater allele and drove robust fin ray joints as previously reported.…”

“…Tol2 plasmid transgenesis was performed using pT2HE plasmid backbone as previously described (Erickson et al, 2016; O’brown et al, 2015). To generate GFP reporter constructs, PCR-based site-directed mutagenesis was done on plasmids containing the ∼1300 bp Paxton benthic (PAXB) high-tooth-associated intron four enhancer allele (Cleves et al, 2018; Stepaniak et al, 2021). Primers for site-directed mutagenesis were designed using the Quickchange tool and PCR was carried out according to Erickson et al (Erickson et al, 2015) https://www.agilent.com/store/primerDesignProgram.jsp.…”

“…A study on chromatin from a subset of human tissues found that at least 1% of identified cis -regulatory elements were active in at least two spatially distinct domains (Singh & Yi, 2021). In vivo studies in fish and mice have additionally identified enhancer sequences that drive gene expression in more than one tissue type (Cleves et al, 2018; Erickson et al, 2015; Jackman & Stock, 2006; Jumlongras et al, 2012; Stepaniak et al, 2021). Two different described mechanisms of enhancer pleiotropy are (1) tightly clustered, but different, transcription factor binding sites (TFBS) that drive expression in different tissues and (2) site pleiotropy, where a single TFBS drives expression in multiple tissues (Preger-Ben Noon et al, 2018).…”