Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

How does proline-repeat motif length in the proteins of teeth and bones relate to the evolution of vertebrates? Counterintuitively, longer repeat stretches are associated with smaller aggregated subunits within a supramolecular matrix, resulting in enhanced crystal length in mammalian versus amphibian tooth enamel

Cladogram reconstructed from [22], [28]–[29] for phyllostomid bats included in this study.

<p>Node values are estimated divergence times taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057649#pone.0057649-Baker1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057649#pone.0057649-Datzmann1" target="_blank">[28]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057649#pone.0057649-Rojas1" target="_blank">[29]</a>. Each leaf of the cladogram includes genus, lateral image of skulls, and symbols of insect, blood, flower, or fruit to indicate dietary strategy of that genus.</p

Diagram of reporter construct design.

<p>SV 40 = Simian virus 40 polyadenylation signal, AcGFP1 = green fluorescent protein, Bi-Cis = bicistronic promoter, mOrange = orange fluorescent protein, Pax9 3′ UTR = 3′ sequences of PAX9 from bat species described in the text. Experimental constructs differed only in the species from which PAX9 sequence was amplified. The control construct did not include a PAX9 3′ sequence.</p

Comparative genetic and post-transcriptional reporter analysis.

<p>A) Patterns of nucleotide conservation across the 3′ UTR of PAX9 based on an alignment of <i>Homo</i>, <i>Canis</i>, <i>Mus</i>, and all bat species of this study, in which the vertical axis represents sequence divergence and the horizontal axis represents base pairs from the stop codon. Conserved domains are represented by grey areas and the locations of MBEs are demarked with arrows. The location of all seven MBEs occurring in a human alternative transcript are not show to conserve space (length >3Kb). B) Histogram summarizing the frequency of MBEs across taxa surveyed (checkered = pteropodids, black = phyllostomids, grey = vespertilionids, and white = non-bat taxa). <i>Homo</i> long and <i>Homo</i> short refer to the two alternative transcripts of PAX9 observed in humans. C) Results of the fluorescent reporter assay, with error bars representing standard mean error. Cell lines labeled as ‘3 MBEs’ and ‘3b MBEs’ are alternative spatial combination of this motif frequency and, SV40 represents the construct with only SV40 polyadenylation signal containing no MBE. D) Result of the knockdown assay. Cell lines denoted with KD indicate knockdown lines. Error bars represent standard mean error, and are not visible in some instances due to the error being within histogram bar thickness.</p

Patterns of PAX9 open-reading frame evolution.

<p>Although under tight purifying selection, PAX9 exhibits patterns of saturation and recurrent substitution contingent on the level of comparison (amino acid versus nucleotide) as well as the amount of evolutionary time considered in the data. A) and B) display the pairwise nucleotide and predicted amino acid differences among orders regressed against <i>t_mrca</i>, respectively. C) and D) show similar plots, but among species from the families Phyllostomidae, Vespertilionidae and Miniopteridae, and the order Primates. E) Confidence in PSIPRED secondary structure prediction (greater confidence represented by larger bars) for each of the 341 residues of Pax9, and shading demarks exons. Directly below this histogram is the structural prediction in which white bars represent coiled structures and black represent helical regions. The solid grey bar at the bottom defines the limits of the paired-binding domain described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057649#pone.0057649-Wang2" target="_blank">[7]</a>. Vertical lines pointing to each codon position in the histogram indicate positions that vary across the mammalian taxa examined, and taller lines demark sights inferred to have accumulated recurrent substitutions. The number of inferred substitutions at these sights from left to right are as follows: 3, 2, 3, 2, 7, 4, 3, 4, 8, 6, 3, 2, 4, 4, 2, 3, 2, and 5. F) Reticulation network based on predicted amino acid translations. Closed loops in the network indicate homoplasies, and edge thickness is in proportion to bootstrap support.</p

Major analyses implemented in this study with short descriptions for the information obtained from each analysis.

<p>Analyses are grouped into open-reading frame or regulatory subheadings, indicating to which major hypothesis each analysis was relevant.</p

Quantitative and qualitative assessments of PC1 on canine cranioskeletal shape.

<p>(A) Gray wolf (mesocephalic, ancestor to dogs) (B) Afghan hound (dolichocephalic), (C) Leonberger (mesocephalic), (D) Pug (brachycephalic). (E) Boxplots of PC1 (corresponding breed names are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002849#pgen.1002849.s008" target="_blank">Table S2</a>). (F) Surface scans of a gray wolf skull illustrate morphological changes associated with PC1. Columns (left to right) are dorsal, lateral, and rostral views. Top row: a gray wolf skull morphed by positive PC1. Middle row: a gray wolf skull (no morphing). Bottom row: a gray wolf skull morphed by negative PC1. Pseudocoloring of the gray wolf skull indicates rostrum (ros) and neurocranium (nc). Line indicates width of the zygomatic arches (za).</p

PC1 GWAS and fine mapping at CFA32.

<p>All GWAS used the mixed-model GEMMA. Chromosomes listed on the <i>x</i>-axis, −log₁₀(<i>P</i>) on the <i>y</i>-axis. SNPs remaining significant following Bonferroni correction are colored blue. Q-Q plots of observed versus expected −log₁₀(<i>P</i>) are depicted on right, with full SNP dataset (black circles), pruned dataset (grey circles), expected values (red lines), and 95% confidence intervals (black lines). Scan results using breed-sex averages of PC1 without (A) and with a breed-sex average size covariate (B). Including a size covariate in the mixed-model overcorrects, leading to loss of associations on CFA 30 and 32.(C) Scan results using PC1 breed-sex averages and breed-sex size covariates. In this scan, only breeds whose neurocranium size ranked within the smallest 50% of our dataset where analyzed. By reducing relatedness disparity in our study population, the association on CFA32 remains significant despite size correction. (D) Scan results using all breed-sex averages of PC1, but excluding extreme brachycephalic breeds (Pug, Pekingese, Boston Terrier, Shih Tzu, Brussels Griffon, French Bulldog, Bulldog, Boxer, Cavalier King Charles Spaniel, Chihuahua). (E) Average log(H_O ratios) or F_ST from ten-SNP sliding windows. (F) Regional H_O or F_ST values, and their respective Lowess best fit curves.</p

Genetic variation at the CFA32 QTL includes a brachycephaly-associated missense mutation within <i>BMP3</i>.

<p>For display purposes, we set the reference sequence to be the allele most common to Pekingese and Bulldog. Variants located within 8.15–8.27 Mb (A) or the 85 kb critical interval (B) are illustrated (homozygous reference = yellow, heterozygous = orange, homozygous variant = red). (A) Pekingese and Bulldog agree across an 85 kb interval (black bar) including <i>BMP3</i> (red) and a portion of <i>PRKG2</i> (aqua). Line graphs below genes plot conservation (phastCons4way) and association (−log₁₀(<i>P</i>)) with respect to variant position (<i>28</i>). (B) Variants of interest met one or more of the following criteria: conserved (phastCons4way score ≥0.7), associated (an association <i>P</i>-value among the smallest 5% of <i>P</i>-values, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002849#s4" target="_blank">Materials and Methods</a>), exonic (untranslated regions and coding), or splice (located within 20 bp of an exon boundary). Forty-eight variants of interest remained after applying filtering criteria, including a F452L mutation in <i>BMP3</i> at position 8,196,098.</p