Fig. 5.

Evolution of a unique enhancer controlling csf1ra expression in pigmentation and its variation. (A) Schematic of different mechanistic hypotheses of evolution of csf1r regulation. Duplication of an ancestral csf1r gene has led to two lineages in vertebrates within teleost fishes having two copies, ‘a’ and ‘b’, and tetrapods having one copy. The functional difference between ‘a’ and ‘b’ could be a consequence of neo-functionalization, where ‘a’ gained functionality in pigment cells; or sub-functionalization, where ‘a’ and ‘b’ retained partial functionality of the ancestral gene; yellow box represents a hypothetical enhancer regulating expression in pigment precursors. (B) Identification of a conserved non-coding element (CNE) among fishes within csf1ra, not found in csf1rb or tetrapod csf1r sequences; Dr, Danio rerio; Hs, Homo sapiens; yellow box represents the position of the CNE. (C) Schematic and alignment of putative enhancer. Within Danios a broad 150 bp sequence is conserved with a ∼27 bp core sequence showing a 100% identity with all other teleost fishes (ostariophysans and acanthomorphs). The numbers of bp in each region are indicated. (D) Schematic of expression construct containing a 50 bp conserved region (50bp_Csf1raEN). (E-G) Representative clone of pigment cells observed in injected adult animals and in 1 dpf and adult individuals in the F1 generation; overlay of brightfield and fluorescence (E).