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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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Related in: MedlinePlus

Diagram of reporter construct design.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.
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pone-0057649-g002: Diagram of reporter construct design.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.

Mentions: The analyses of PAX9 disclosed the presence of Musashi binding-elements (MBEs) that varied in frequency among lineages (specific findings reported in Results). Musashi is involved in maintenance of stem cell state, cellular differentiation, and tumorigenesis and has been shown to accomplish this regulation through repression of target mRNAs [42]. To investigate the functionality of these identified regulatory motifs a fluorescent reporter assay was developed. A tetracycline responsive bicistronic expression vector (pTRE-Tight-Bi-AcGFP1; Clontech Laboratories, Mountain View, CA) was constructed to contain mOrange fluorescent protein followed by the 3′ 130 bp of the open-reading frame and the contiguous 3′ UTRs (Figure 2). The purpose of including the 3′ end of the open-reading frame as part of the heterologous 3′ UTR was to include the MBE identified in this region, as well as those found in 3′ UTRs. Multiple constructs were created that differed only in the specific bat lineage from which the PAX9 sequence was amplified. Bat lineages were selected to include the major phylogenetic and morphological divergence among nectarivorous taxa, the highly derived vampire bat lineage, and the observed frequency variation of Musashi binding elements among phyllostomids. Final vectors contained two MBEs (TK19556, Musonycteris harrisoni), two different spatial combinations of three MBEs (TK101009, Desmodus rotundus; TK104582, Lonchophylla concava), four MBEs (TK101008, Glossophaga soricina), or only the vector’s native simian virus 40 (SV40) polyadenylation signal containing no MBE, resulting in five unique expression constructs. In all constructs acGFP1 served as an internal control, being expressed in the opposite direction of the bicistronic promoter region from mOrange. Vectors were transfected (Nucleofector Reagent R; Lonza, Basel, CH) into Tet-Off Advanced HeLa cells (Clontech Laboratories, Mountain View, CA). HeLa was selected as the cell line to be used for these experiments because Western blotting confirmed the expression of both Musashi-2 and Musashi-1 (data not shown). Forty-eight hours post-transfection, relative fluorescence of both acGFP1 and mOrange was measured using a SpectraMax Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA). Transfection and measurement for each cell line was replicated four times and the ratio of acGFP1 to mOrange for each measurement was taken as the comparable measure of reporter protein expression across cell lines and transfections. Expression levels across cell lines were compared using a Kruskal-Wallis test after a Shapiro-Wilk test indicated the data were not normally distributed (W = 0.9, P<0.01).


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)

Diagram of reporter construct design.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057649-g002: Diagram of reporter construct design.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.
Mentions: The analyses of PAX9 disclosed the presence of Musashi binding-elements (MBEs) that varied in frequency among lineages (specific findings reported in Results). Musashi is involved in maintenance of stem cell state, cellular differentiation, and tumorigenesis and has been shown to accomplish this regulation through repression of target mRNAs [42]. To investigate the functionality of these identified regulatory motifs a fluorescent reporter assay was developed. A tetracycline responsive bicistronic expression vector (pTRE-Tight-Bi-AcGFP1; Clontech Laboratories, Mountain View, CA) was constructed to contain mOrange fluorescent protein followed by the 3′ 130 bp of the open-reading frame and the contiguous 3′ UTRs (Figure 2). The purpose of including the 3′ end of the open-reading frame as part of the heterologous 3′ UTR was to include the MBE identified in this region, as well as those found in 3′ UTRs. Multiple constructs were created that differed only in the specific bat lineage from which the PAX9 sequence was amplified. Bat lineages were selected to include the major phylogenetic and morphological divergence among nectarivorous taxa, the highly derived vampire bat lineage, and the observed frequency variation of Musashi binding elements among phyllostomids. Final vectors contained two MBEs (TK19556, Musonycteris harrisoni), two different spatial combinations of three MBEs (TK101009, Desmodus rotundus; TK104582, Lonchophylla concava), four MBEs (TK101008, Glossophaga soricina), or only the vector’s native simian virus 40 (SV40) polyadenylation signal containing no MBE, resulting in five unique expression constructs. In all constructs acGFP1 served as an internal control, being expressed in the opposite direction of the bicistronic promoter region from mOrange. Vectors were transfected (Nucleofector Reagent R; Lonza, Basel, CH) into Tet-Off Advanced HeLa cells (Clontech Laboratories, Mountain View, CA). HeLa was selected as the cell line to be used for these experiments because Western blotting confirmed the expression of both Musashi-2 and Musashi-1 (data not shown). Forty-eight hours post-transfection, relative fluorescence of both acGFP1 and mOrange was measured using a SpectraMax Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA). Transfection and measurement for each cell line was replicated four times and the ratio of acGFP1 to mOrange for each measurement was taken as the comparable measure of reporter protein expression across cell lines and transfections. Expression levels across cell lines were compared using a Kruskal-Wallis test after a Shapiro-Wilk test indicated the data were not normally distributed (W = 0.9, P<0.01).

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