Evolving New Skeletal Traits by cis-Regulatory Changes in Bone Morphogenetic Proteins

Abstract

Changes in bone size and shape are defining features of many vertebrates. Here we use genetic crosses and comparative genomics to identify specific regulatory DNA alterations controlling skeletal evolution. Armor bone-size differences in sticklebacks map to a major effect locus overlapping BMP family member GDF6. Freshwater fish express more GDF6 due in part to a transposon insertion, and transgenic overexpression of GDF6 phenocopies evolutionary changes in armor-plate size. The human GDF6 locus also has undergone distinctive regulatory evolution, including complete loss of an enhancer that is otherwise highly conserved between chimps and other mammals. Functional tests show that the ancestral enhancer drives expression in hindlimbs but not forelimbs, in locations that have been specifically modified during the human transition to bipedalism. Both gain and loss of regulatory elements can localize BMP changes to specific anatomical locations, providing a flexible regulatory basis for evolving species-specific changes in skeletal form.

Figure 1. Genetic, Sequence, and Morphological Evidence for Dual Loci Controlling Armor Plate Dimensions in Sticklebacks

(A) Two distinct, but closely-linked loci regulate armor plate height and width. Plate height (green) and width (red) were mapped in a large marine x benthic F2 cross using MapQTL. Likelihood-of-odds (LOD) scores and the percent variance explained (PVE) for peak markers for each trait are shown. The 2-LOD intervals for each QTL are shaded (green and red, overlap in yellow), and high significance cut offs (1000 permutations with MapQTL; p<0.001) are shown with dotted lines. Representative fish from the grandparental marine and benthic populations stained with Alizarin red are shown. (B) There are two major peaks of repeated sequence divergence between freshwater and marine fish in the plate size interval. Sequence divergence between pairs (n=11) of geographically proximal marine and freshwater sticklebacks are plotted (colored lines). Populations are listed in Table S2. The 2-LOD score intervals for plate height and width (black rectangles), the GDF6 locus, and the region cloned for testing for enhancer activity in Figure 2 are shown. (C and D) Geographic patterns in armor plate morphology match patterns of sequence divergence. Armor plate height (C) and width (D) were measured from adults (Table S2) from each of the twenty-one sequenced populations and were normalized for standard fish depth and length, respectively. Pacific basin freshwater (FW) populations are shown in black, Atlantic basin freshwater populations in gray, and Pacific and Atlantic basin marine populations in white. Two-sample Wilcoxon tests were used to examine the significances of plate size averages distribution differences between groups of fish populations (bracketed), and p-values are shown. Error bars represent SEM. See also Figure S1 and Tables S1, S2 and S3.

Figure 2. Identification of cis-Regulatory Changes in the Plate Size Interval

(A) The freshwater allele of GDF6 is expressed at higher level than the marine allele in F1 hybrids. RNA was extracted from the indicated region of F1 hybrid fish from a cross between a small-plated freshwater (FW) and large-plated marine fish, and the relative abundance of the freshwater and marine alleles was quantified by pyrosequencing. Significance of observed deviation from the fraction of freshwater allele of 0.5 (dashed line) was tested using one sample t-test, and “*” represents p<0.05. Error bars represent SEM. (B) Freshwater but not marine enhancer drives expression along the flanks of developing stickleback embryos. Marine and freshwater (FW) sequences were cloned from the regulatory region shown in Figure 1B into a GFP reporter vector with a minimal hsp70 promoter, and were then injected in fertilized one-cell stage embryos from marine fish. Pictures of the flanks of developing fry (dashed white lines) were taken at 4 dpf. (C) Map of the enhancer region and sequences tested in transgenic fish. Repeating vertical lines represent regions of alignment between marine (red) and freshwater (blue) sequence, with gaps shown in the region of a complex repeat and a L2 LINE transposable element (TE) present in the freshwater but not marine sequence. Tall vertical lines denote the edges of the region added or removed from the modified constructs also tested for enhancer activity. See also Figure S2 and Table S6.

Figure 3. GDF6 Transgenic Fish have Armor Plate Phenotypes

(A) Schematic of the construct used for generating GDF6 transgenic fish. The flank enhancer in the height QTL (Figures 1B and 2B) was cloned upstream of a minimal hsp70 promoter and GDF6 cDNA flanked by Tol2 transposase recognition sites. The construct was then co-injected with mRNA encoding Tol2 transposase into one-cell marine stickleback embryos. (B) μCT-derived volumetric reconstructions of representative control (left) and GDF6 transgenic (right) fish at 219 dpf. The armored plates are colored in red. Note the absence of caudal plates from the left flank of the GDF6 transgenic fish. (C and D) Close up views of control (C) and GDF6 (D) transgenic fish pictured in (B). The last three plates remaining in the GDF6 transgenic fish are noticeably smaller than the corresponding plates in the control fish. Scale bars in (B) and (D), 1 mm. See also Figure S3.

Figure 4. A Chimpanzee GDF6 Enhancer Missing in Humans Drives Expression in the Posterior of Mouse Embryos

(A) There are two human-specific deletions of highly conserved chimpanzee sequences in the GDF6 locus. A 1.2 Mb region of the chimpanzee chromosome 8 is shown. Red bars show the positions of a 492 bp and a 5775 bp deletions in humans, hCONDEL.305 and hCONDEL.306, respectively (McLean et al, 2011). Below: multiple species comparison of the hCONDEL.306 region (red), showing sequences aligned between chimpanzee and other mammals. Blue bar represents the chimpanzee sequence tested for enhancer activity in transgenic mice. (B) hCONDEL.306 chimpanzee sequence drives consistent expression of lacZ reporter in the posterior of E12.5 mouse embryos. (C) Mice with the hCONDEL.306-hsp-CreER-T2 construct were bred to floxed-ROSA26 reporter mice. Tamoxifen was administered at E9.5 and embryos were lacZ stained at E12.5, showing consistent expression patterns in the posterior of the embryo. See also Figure S4 and Table S7.

Figure 5. The Chimpanzee GDF6 Enhancer Drives Expression in Posterior Digits of the Hindlimb

(A) hCONDEL.306-hsp-CreER-T2 transgenic mice treated with tamoxifen at E8.5 show consistent lacZ localization in the hindlimbs but not forelimbs of E16.5 embryos, with stronger labeling in the posterior foot digits compared to the digit 1. Foot is shown in plantar view, with first digit on the left. (B) The lacZ pattern in the developing foot is seen in transverse sections of the phalanges (outlined with white dashed line). Approximate section plane is indicated on the left (white line). See also Figure S5.

Figure 6. Gdf6 Mutant Mice Have Altered Flat Bones and Shorter Toes

(A) Average width and length of skull bones in wild type (n=12) and Gdf6−/− (n=16) mutant mice. Dimensions were measured for parietal bone width (PW), the combined length of the frontal and parietal bones (PL), nasal bone length (NL), nasal bone width (NW), and the relative length and width of the skulls. (B) Gdf6 mutant mice have shorter hindlimb digits. The lengths of the sums of the phalanges of digit I, digit III, and digit V from wild type (n=8) and Gdf6−/− (n=9) mice were normalized to femur length and averaged for the two hindlimbs. We used two sample t-tests to examine the significance of skull plate (A) and digits size (B) differences between the wild type and Gdf6−/− mice, and p-values are shown. Error bars represent SEM.

Figure 7. Region-Specific GDF6 Enhancer Compared to Limb-Specific Anatomical Modifications in Human Hindfeet and Forefeet

(A) Human hindlimbs show multiple changes related to evolution of bipedalism, including shortening of posterior digits of the foot (red), presence of a large toe aligned with other digits, and reduction of the abductor hallicus muscle (orange), which is prominent in other primates with grasping feet and an opposable first toe. In contrast, the homologous digits of the human hand are still long and mobile (dark grey), illustrating the region-specific anatomical changes that have evolved in human hands and feet (modified from Swindler and Wood (1973) and skeletal reproductions of chimpanzee and human feet (BoneClones)). Enhancer sequences in the GDF6 gene also show striking regional specificity during development. The enhancer deleted in the human lineage shows prominent hindlimb but not forelimb expression, and stronger expression in posterior digits within the hindlimb. Loss of this enhancer would not disrupt GDF6 functions in cranial or forelimb regions, but could reduce GDF6 activity in many of the same hindlimb structures altered during the human transition to bipedalism. (B) Our results add a distal regulatory region to a collection of tissue and region-specific enhancers surrounding Gdf6 (Mortlock et al., 2003). Gain and loss of modular enhancers in BMP genes provides a flexible genomic mechanism for altering skeletal morphology in particular regions of the body. See also Figure S6.