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Differential expression of the aryl hydrocarbon receptor pathway associates with craniofacial polymorphism in sympatric Arctic charr.

Ahi EP, Steinhäuser SS, Pálsson A, Franzdóttir SR, Snorrason SS, Maier VH, Jónsson ZO - Evodevo (2015)

Bottom Line: The developmental basis of craniofacial morphology hinges on interactions of numerous signalling systems.The present study adds a second set of genes constituting an expanded gene network with strong, benthic-limnetic differential expression.To identify putative upstream regulators, we performed knowledge-based motif enrichment analyses on the regulatory sequences of the identified genes which yielded potential binding sites for a set of known transcription factors (TFs).

View Article: PubMed Central - PubMed

Affiliation: Institute of Life and Environmental Sciences, University of Iceland, Sturlugata 7, 101 Reykjavik, Iceland.

ABSTRACT

Background: The developmental basis of craniofacial morphology hinges on interactions of numerous signalling systems. Extensive craniofacial variation in the polymorphic Arctic charr, a member of the salmonid family, from Lake Thingvallavatn (Iceland), offers opportunities to find and study such signalling pathways and their key regulators, thereby shedding light on the developmental pathways, and the genetics of trophic divergence.

Results: To identify genes involved in the craniofacial differences between benthic and limnetic Arctic charr, we used transcriptome data from different morphs, spanning early development, together with data on craniofacial expression patterns and skeletogenesis in model vertebrate species. Out of 20 genes identified, 7 showed lower gene expression in benthic than in limnetic charr morphs. We had previously identified a conserved gene network involved in extracellular matrix (ECM) organization and skeletogenesis, showing higher expression in developing craniofacial elements of benthic than in limnetic Arctic charr morphs. The present study adds a second set of genes constituting an expanded gene network with strong, benthic-limnetic differential expression. To identify putative upstream regulators, we performed knowledge-based motif enrichment analyses on the regulatory sequences of the identified genes which yielded potential binding sites for a set of known transcription factors (TFs). Of the 8 TFs that we examined using qPCR, two (Ahr2b and Ap2) were found to be differentially expressed between benthic and limnetic charr. Expression analysis of several known AhR targets indicated higher activity of the AhR pathway during craniofacial development in benthic charr morphotypes.

Conclusion: These results suggest a key role of the aryl hydrocarbon receptor (AhR) pathway in the observed craniofacial differences between distinct charr morphotypes.

No MeSH data available.


Related in: MedlinePlus

Differential expression of Ahr2b and Ap2 in developing heads of Arctic charr morphs. a Relative expression of eight selected TF genes in developing heads of SB, LB, PI, PL and AC at six stages. Error bars represent standard deviation calculated from two biological replicates where each biological replicate contains a homogenate of six heads. b Relative expression ratios were subjected to an analysis of variance (ANOVA) to test for expression differences among five Arctic charr morphs and six stages (M morph; T stage (time); MxT morphs by time effects; P values of <0.05, 0.01 and 0.001 are indicated by one, two and three asterisks, respectively). Subsequently, a post hoc Tukey’s HSD test was performed to analyse the expression of candidates among the morphs. Green to red colour gradient of morphs represents low to high expression levels and morphs with no connecting line have significantly different expression (α = 0.05). The bold and underlined gene(s) displayed distinct benthic–limnetic expression dynamics
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Fig4: Differential expression of Ahr2b and Ap2 in developing heads of Arctic charr morphs. a Relative expression of eight selected TF genes in developing heads of SB, LB, PI, PL and AC at six stages. Error bars represent standard deviation calculated from two biological replicates where each biological replicate contains a homogenate of six heads. b Relative expression ratios were subjected to an analysis of variance (ANOVA) to test for expression differences among five Arctic charr morphs and six stages (M morph; T stage (time); MxT morphs by time effects; P values of <0.05, 0.01 and 0.001 are indicated by one, two and three asterisks, respectively). Subsequently, a post hoc Tukey’s HSD test was performed to analyse the expression of candidates among the morphs. Green to red colour gradient of morphs represents low to high expression levels and morphs with no connecting line have significantly different expression (α = 0.05). The bold and underlined gene(s) displayed distinct benthic–limnetic expression dynamics

Mentions: With the objective of finding potential upstream regulatory pathway(s) and/or TFs influencing the co-expressed genes, we selected the three genes, Chd4, Cspp1 and Dlg1, with persistent and strong reduction in expression in developing heads of benthic morphs and with positive co-expression in mammals and Arctic charr. We retrieved a list of over 200 genes showing co-expression in mammals with these three genes (Additional file 3: Table S3). This list was used as input for a knowledge-based TF enrichment analysis in human and mouse using WebGestalt v2 [34]. In both human and mouse, binding sites belonging to 17 TFs were significantly overrepresented on promoters of the input genes (Additional file 4: Table S4). We chose the eight most significantly enriched TFs for further gene expression analysis (Fig. 4a). The results revealed differential expression of all the eight TFs over time and seven TFs between the morphs (Fig. 4b). However, only two TFs (Ahr2b and Ap2) were differentially expressed between benthic and limnetic charr, with higher levels of expression for Ahr2b in benthic morphs and higher expression of Ap2 in limnetic morphs. This implicates Ahr2b and Ap2 as potential transcriptional regulators (i.e. transcriptional repressor and activator, respectively) of the co-expressed genes identified above.Fig. 4


Differential expression of the aryl hydrocarbon receptor pathway associates with craniofacial polymorphism in sympatric Arctic charr.

Ahi EP, Steinhäuser SS, Pálsson A, Franzdóttir SR, Snorrason SS, Maier VH, Jónsson ZO - Evodevo (2015)

Differential expression of Ahr2b and Ap2 in developing heads of Arctic charr morphs. a Relative expression of eight selected TF genes in developing heads of SB, LB, PI, PL and AC at six stages. Error bars represent standard deviation calculated from two biological replicates where each biological replicate contains a homogenate of six heads. b Relative expression ratios were subjected to an analysis of variance (ANOVA) to test for expression differences among five Arctic charr morphs and six stages (M morph; T stage (time); MxT morphs by time effects; P values of <0.05, 0.01 and 0.001 are indicated by one, two and three asterisks, respectively). Subsequently, a post hoc Tukey’s HSD test was performed to analyse the expression of candidates among the morphs. Green to red colour gradient of morphs represents low to high expression levels and morphs with no connecting line have significantly different expression (α = 0.05). The bold and underlined gene(s) displayed distinct benthic–limnetic expression dynamics
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4574265&req=5

Fig4: Differential expression of Ahr2b and Ap2 in developing heads of Arctic charr morphs. a Relative expression of eight selected TF genes in developing heads of SB, LB, PI, PL and AC at six stages. Error bars represent standard deviation calculated from two biological replicates where each biological replicate contains a homogenate of six heads. b Relative expression ratios were subjected to an analysis of variance (ANOVA) to test for expression differences among five Arctic charr morphs and six stages (M morph; T stage (time); MxT morphs by time effects; P values of <0.05, 0.01 and 0.001 are indicated by one, two and three asterisks, respectively). Subsequently, a post hoc Tukey’s HSD test was performed to analyse the expression of candidates among the morphs. Green to red colour gradient of morphs represents low to high expression levels and morphs with no connecting line have significantly different expression (α = 0.05). The bold and underlined gene(s) displayed distinct benthic–limnetic expression dynamics
Mentions: With the objective of finding potential upstream regulatory pathway(s) and/or TFs influencing the co-expressed genes, we selected the three genes, Chd4, Cspp1 and Dlg1, with persistent and strong reduction in expression in developing heads of benthic morphs and with positive co-expression in mammals and Arctic charr. We retrieved a list of over 200 genes showing co-expression in mammals with these three genes (Additional file 3: Table S3). This list was used as input for a knowledge-based TF enrichment analysis in human and mouse using WebGestalt v2 [34]. In both human and mouse, binding sites belonging to 17 TFs were significantly overrepresented on promoters of the input genes (Additional file 4: Table S4). We chose the eight most significantly enriched TFs for further gene expression analysis (Fig. 4a). The results revealed differential expression of all the eight TFs over time and seven TFs between the morphs (Fig. 4b). However, only two TFs (Ahr2b and Ap2) were differentially expressed between benthic and limnetic charr, with higher levels of expression for Ahr2b in benthic morphs and higher expression of Ap2 in limnetic morphs. This implicates Ahr2b and Ap2 as potential transcriptional regulators (i.e. transcriptional repressor and activator, respectively) of the co-expressed genes identified above.Fig. 4

Bottom Line: The developmental basis of craniofacial morphology hinges on interactions of numerous signalling systems.The present study adds a second set of genes constituting an expanded gene network with strong, benthic-limnetic differential expression.To identify putative upstream regulators, we performed knowledge-based motif enrichment analyses on the regulatory sequences of the identified genes which yielded potential binding sites for a set of known transcription factors (TFs).

View Article: PubMed Central - PubMed

Affiliation: Institute of Life and Environmental Sciences, University of Iceland, Sturlugata 7, 101 Reykjavik, Iceland.

ABSTRACT

Background: The developmental basis of craniofacial morphology hinges on interactions of numerous signalling systems. Extensive craniofacial variation in the polymorphic Arctic charr, a member of the salmonid family, from Lake Thingvallavatn (Iceland), offers opportunities to find and study such signalling pathways and their key regulators, thereby shedding light on the developmental pathways, and the genetics of trophic divergence.

Results: To identify genes involved in the craniofacial differences between benthic and limnetic Arctic charr, we used transcriptome data from different morphs, spanning early development, together with data on craniofacial expression patterns and skeletogenesis in model vertebrate species. Out of 20 genes identified, 7 showed lower gene expression in benthic than in limnetic charr morphs. We had previously identified a conserved gene network involved in extracellular matrix (ECM) organization and skeletogenesis, showing higher expression in developing craniofacial elements of benthic than in limnetic Arctic charr morphs. The present study adds a second set of genes constituting an expanded gene network with strong, benthic-limnetic differential expression. To identify putative upstream regulators, we performed knowledge-based motif enrichment analyses on the regulatory sequences of the identified genes which yielded potential binding sites for a set of known transcription factors (TFs). Of the 8 TFs that we examined using qPCR, two (Ahr2b and Ap2) were found to be differentially expressed between benthic and limnetic charr. Expression analysis of several known AhR targets indicated higher activity of the AhR pathway during craniofacial development in benthic charr morphotypes.

Conclusion: These results suggest a key role of the aryl hydrocarbon receptor (AhR) pathway in the observed craniofacial differences between distinct charr morphotypes.

No MeSH data available.


Related in: MedlinePlus