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Contrasting evolutionary dynamics of the developmental regulator PAX9, among bats, with evidence for a novel post-transcriptional regulatory mechanism.

Phillips CD, Butler B, Fondon JW, Mantilla-Meluk H, Baker RJ - PLoS ONE (2013)

Bottom Line: Morphological evolution can be the result of natural selection favoring modification of developmental signaling pathways.Although a connection between morphology and binding element frequency was not apparent, results indicate this regulation would vary among craniofacially divergent bat species, but be static among conserved species.The presence of Musashi-binding elements within PAX9 of all mammals examined, chicken, zebrafish, and the fly homolog of PAX9, indicates this regulatory mechanism is ancient, originating basal to much of the animal phylogeny.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, United States of America. caleb.phillips@ttu.edu

ABSTRACT
Morphological evolution can be the result of natural selection favoring modification of developmental signaling pathways. However, little is known about the genetic basis of such phenotypic diversity. Understanding these mechanisms is difficult for numerous reasons, yet studies in model organisms often provide clues about the major developmental pathways involved. The paired-domain gene, PAX9, is known to be a key regulator of development, particularly of the face and teeth. In this study, using a comparative genetics approach, we investigate PAX9 molecular evolution among mammals, focusing on craniofacially diversified (Phyllostomidae) and conserved (Vespertilionidae) bat families, and extend our comparison to other orders of mammal. Open-reading frame analysis disclosed signatures of selection, in which a small percentage of residues vary, and lineages acquire different combinations of variation through recurrent substitution and lineage specific changes. A few instances of convergence for specific residues were observed between morphologically convergent bat lineages. Bioinformatic analysis for unknown PAX9 regulatory motifs indicated a novel post-transcriptional regulatory mechanism involving a Musashi protein. This regulation was assessed through fluorescent reporter assays and gene knockdowns. Results are compatible with the hypothesis that the number of Musashi binding-elements in PAX9 mRNA proportionally regulates protein translation rate. Although a connection between morphology and binding element frequency was not apparent, results indicate this regulation would vary among craniofacially divergent bat species, but be static among conserved species. Under this model, Musashi's regulatory control of alternative human PAX9 isoforms would also vary. The presence of Musashi-binding elements within PAX9 of all mammals examined, chicken, zebrafish, and the fly homolog of PAX9, indicates this regulatory mechanism is ancient, originating basal to much of the animal phylogeny.

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Comparative genetic and post-transcriptional reporter analysis.A) Patterns of nucleotide conservation across the 3′ UTR of PAX9 based on an alignment of Homo, Canis, Mus, 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). Homo long and Homo 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.
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pone-0057649-g004: Comparative genetic and post-transcriptional reporter analysis.A) Patterns of nucleotide conservation across the 3′ UTR of PAX9 based on an alignment of Homo, Canis, Mus, 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). Homo long and Homo 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.

Mentions: Outlier areas of nucleotide conservation within non-coding regions indicate potential regions of regulatory function. To identify any such motifs UTRs were analyzed for the presence of conserved islands. Both UTRs were found to contain conserved islands when comparisons included all non-bat mammalian taxa and phyllostomids or vespertilionids. When all samples were included the same conserved nucleotide domains were retained, although not statistically classifiable as conserved islands. Within the 5′ UTR a 146 bp conserved island 113 bp from the start codon was identified, and further analysis determined that this island is directly upstream from a putative internal ribosomal entry site. Within the 3′ UTR a 204 bp conserved island was identified 196 bp downstream of the stop codon. Next, nucleotide sequences of UTRs were surveyed for the presence of known regulatory elements identified in other studies using UTRscan [41]. This analysis revealed the presence of four Musashi-binding elements (MBEs; RTnAGT (n = 1 to 3)) within conserved regions of the 3′ UTR. However, among bats there was variation in the number of MBEs present in a given species 3′ UTR. Two MBEs were found to occur within the upstream conserved island, while another two were identified within a 64 bp region 588 bp downstream of the stop codon that is also highly conserved (Figure 4a). Among bats, there was variation in the number and combination of MBEs present in given species 3′ UTR; however this variation was almost exclusively observed in phyllostomids (Figure 4b). Within phyllostomids the four identified MBEs were observed in 92%, 0%, 25%, and 75% of species, respectively. Vespertilionids were fixed for presence of the first and fourth MBE, with the exception of Murina, which only had the first MBE. The first, second, and fourth MBEs were observed in both pteropodids examined. Analysis of the 3′ UTRs of human, dog, mouse, chicken, and zebrafish confirmed the presence of a single MBE within each of these species 3′ UTRs, with the exception of a proposed alternative transcript in human which contained seven MBEs. The discovery of MBEs within UTR regions led to an additional survey of open-reading frames for regulatory motifs. This analysis disclosed the presence of an additional MBE 32 bp from the stop codon in all chiropteran lineages except Murina.


Contrasting evolutionary dynamics of the developmental regulator PAX9, among bats, with evidence for a novel post-transcriptional regulatory mechanism.

Phillips CD, Butler B, Fondon JW, Mantilla-Meluk H, Baker RJ - PLoS ONE (2013)

Comparative genetic and post-transcriptional reporter analysis.A) Patterns of nucleotide conservation across the 3′ UTR of PAX9 based on an alignment of Homo, Canis, Mus, 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). Homo long and Homo 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585407&req=5

pone-0057649-g004: Comparative genetic and post-transcriptional reporter analysis.A) Patterns of nucleotide conservation across the 3′ UTR of PAX9 based on an alignment of Homo, Canis, Mus, 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). Homo long and Homo 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.
Mentions: Outlier areas of nucleotide conservation within non-coding regions indicate potential regions of regulatory function. To identify any such motifs UTRs were analyzed for the presence of conserved islands. Both UTRs were found to contain conserved islands when comparisons included all non-bat mammalian taxa and phyllostomids or vespertilionids. When all samples were included the same conserved nucleotide domains were retained, although not statistically classifiable as conserved islands. Within the 5′ UTR a 146 bp conserved island 113 bp from the start codon was identified, and further analysis determined that this island is directly upstream from a putative internal ribosomal entry site. Within the 3′ UTR a 204 bp conserved island was identified 196 bp downstream of the stop codon. Next, nucleotide sequences of UTRs were surveyed for the presence of known regulatory elements identified in other studies using UTRscan [41]. This analysis revealed the presence of four Musashi-binding elements (MBEs; RTnAGT (n = 1 to 3)) within conserved regions of the 3′ UTR. However, among bats there was variation in the number of MBEs present in a given species 3′ UTR. Two MBEs were found to occur within the upstream conserved island, while another two were identified within a 64 bp region 588 bp downstream of the stop codon that is also highly conserved (Figure 4a). Among bats, there was variation in the number and combination of MBEs present in given species 3′ UTR; however this variation was almost exclusively observed in phyllostomids (Figure 4b). Within phyllostomids the four identified MBEs were observed in 92%, 0%, 25%, and 75% of species, respectively. Vespertilionids were fixed for presence of the first and fourth MBE, with the exception of Murina, which only had the first MBE. The first, second, and fourth MBEs were observed in both pteropodids examined. Analysis of the 3′ UTRs of human, dog, mouse, chicken, and zebrafish confirmed the presence of a single MBE within each of these species 3′ UTRs, with the exception of a proposed alternative transcript in human which contained seven MBEs. The discovery of MBEs within UTR regions led to an additional survey of open-reading frames for regulatory motifs. This analysis disclosed the presence of an additional MBE 32 bp from the stop codon in all chiropteran lineages except Murina.

Bottom Line: Morphological evolution can be the result of natural selection favoring modification of developmental signaling pathways.Although a connection between morphology and binding element frequency was not apparent, results indicate this regulation would vary among craniofacially divergent bat species, but be static among conserved species.The presence of Musashi-binding elements within PAX9 of all mammals examined, chicken, zebrafish, and the fly homolog of PAX9, indicates this regulatory mechanism is ancient, originating basal to much of the animal phylogeny.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, United States of America. caleb.phillips@ttu.edu

ABSTRACT
Morphological evolution can be the result of natural selection favoring modification of developmental signaling pathways. However, little is known about the genetic basis of such phenotypic diversity. Understanding these mechanisms is difficult for numerous reasons, yet studies in model organisms often provide clues about the major developmental pathways involved. The paired-domain gene, PAX9, is known to be a key regulator of development, particularly of the face and teeth. In this study, using a comparative genetics approach, we investigate PAX9 molecular evolution among mammals, focusing on craniofacially diversified (Phyllostomidae) and conserved (Vespertilionidae) bat families, and extend our comparison to other orders of mammal. Open-reading frame analysis disclosed signatures of selection, in which a small percentage of residues vary, and lineages acquire different combinations of variation through recurrent substitution and lineage specific changes. A few instances of convergence for specific residues were observed between morphologically convergent bat lineages. Bioinformatic analysis for unknown PAX9 regulatory motifs indicated a novel post-transcriptional regulatory mechanism involving a Musashi protein. This regulation was assessed through fluorescent reporter assays and gene knockdowns. Results are compatible with the hypothesis that the number of Musashi binding-elements in PAX9 mRNA proportionally regulates protein translation rate. Although a connection between morphology and binding element frequency was not apparent, results indicate this regulation would vary among craniofacially divergent bat species, but be static among conserved species. Under this model, Musashi's regulatory control of alternative human PAX9 isoforms would also vary. The presence of Musashi-binding elements within PAX9 of all mammals examined, chicken, zebrafish, and the fly homolog of PAX9, indicates this regulatory mechanism is ancient, originating basal to much of the animal phylogeny.

Show MeSH
Related in: MedlinePlus