Evolution of ligand specificity in vertebrate corticosteroid receptors

<p>Abstract</p> <p>Background</p> <p>Corticosteroid receptors include mineralocorticoid (MR) and glucocorticoid (GR) receptors. Teleost fishes have a single MR and duplicate GRs that show variable sensitivities to mineralocorticoids and glucocorticoids. How these receptors compare functionally to tetrapod MR and GR, and the evolutionary significance of maintaining two GRs, remains unclear.</p> <p>Results</p> <p>We used up to seven steroids (including aldosterone, cortisol and 11-deoxycorticosterone [DOC]) to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal (<it>Mus musculus</it>) and the midshipman fish (<it>Porichthys notatus</it>), a teleost model for steroid regulation of neural and behavioral plasticity. Variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of MR and GRs from the distantly related daffodil cichlid (<it>Neolamprologus pulcher</it>), another teleost model for neurobehavioral plasticity. Both teleost species had a single MR and duplicate GRs. All MRs were sensitive to DOC, consistent with the hypothesis that DOC was the initial ligand of the ancestral MR. Variation in GR steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences.</p> <p>Conclusion</p> <p>The mineralocorticoid sensitivity of duplicate GRs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two GRs.</p

Differentiation of VEGFR2+ Hepatic Endoderm.

<p><i>Hhex^-/-</i> DE cells did not differentiate into VEGFR2⁺ early hepatic progenitor cells. <b>A)</b> Analysis of FACS for ALB and VEGFR2 revealed that only <i>Hhex</i>^<i>+/+</i> (blue) cultures produced significant populations of HE progenitor cells and the majority of ALB⁺ cells were also VEGFR2⁺ in <i>Hhex</i>^<i>+/+</i> cultures. IGG plot confirms antigen specificity. <b>B and C)</b> Single channel and merged immunofluorescence staining of <i>Hhex</i>^<i>+/+</i> ALB⁺/VEGFR2⁺ sorted cells that were replated for 24 hours in HE media. Sorted cells showed co-expression of ALB and VEGFR2 (B), and were absent for the expression of the hematopoietic/endothelial marker CD34 (C). (Scale bars = 10μM.) <b>D and E)</b> Single channel and merged immunofluorescence staining of <i>Hhex</i>^<i>+/+</i> and <i>Hhex</i>^<i>-/-</i> ALB⁺/VEGFR2⁺ sorted cells that were replated for 7 days in HE media. Despite rapid expansion of the <i>Hhex</i>^<i>-/-</i> sorted cells, only <i>Hhex</i>^<i>+/+</i> sorted cells showed a co-expression of ALB and AAT that is indicative of further/continued hepatic differentiation. (Scale bars = 50μM.) <b>F)</b> Normalized mRNA expression of hepatic and <i>Vegf</i> signaling gene markers in ALB⁺/VEGFR2⁺ sorted cells from both <i>Hhex</i>^<i>+/+</i> and <i>Hhex</i>^<i>-/-</i> cultures. <i>Hhex</i>^<i>-/-</i> sorted cells show heavily attenuated mRNA expression for hepatic genes and no significant reduction in <i>Vegfa</i> expression when compared to cells from the previous DE differentiation stage. (*p<0.05, **p<0.01, ***p<0.001).</p

Differentiation of Definitive Endoderm.

<p><b>Fig 1:</b><i>Hhex</i>^<i>-/-</i> ESCs show no defects when differentiated toward definitive endoderm. <b>A)</b> Analysis of FACS for SOX17 and GSC revealed that both <i>Hhex</i>^<i>+/+</i> (blue) and <i>Hhex</i>^<i>-/-</i> (red) cultures produced similar percentages of differentiated DE cells. Light Scatter plot indicates the cells gated for sorting (green) and IGG plot (grey) confirms antigen specificity. <b>B)</b> Single channel and merged immunofluorescence staining of DE cultures after the 5-day culture. Both <i>Hhex</i>^<i>+/+</i> and <i>Hhex</i>^<i>-/-</i> cultures showed significantly more cells double positive for the nuclear expression of SOX17 and FOXA2/HNF3β compared to IGG controls. (Scale bars = 100μM, *p<0.05, **p<0.01)</p

<i>Hhex</i> Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via <i>Vegf</i> Signaling

<div><p>Elucidating the molecular mechanisms involved in the differentiation of stem cells to hepatic cells is critical for both understanding normal developmental processes as well as for optimizing the generation of functional hepatic cells for therapy. We performed <i>in vitro</i> differentiation of mouse embryonic stem cells (mESCs) with a null mutation in the homeobox gene <i>Hhex</i> and show that <i>Hhex</i>^<i>-/-</i> mESCs fail to differentiate from definitive endoderm (Sox17⁺/Foxa2⁺) to hepatic endoderm (Alb⁺/Dlk⁺). In addition, hepatic culture elicited a >7-fold increase in <i>Vegfa</i> mRNA expression in <i>Hhex</i>^<i>-/-</i> cells compared to <i>Hhex</i>^<i>+/+</i> cells. Furthermore, we identified VEGFR2⁺/ALB⁺/CD34^- in early <i>Hhex</i>^<i>+/+</i> hepatic cultures. These cells were absent in <i>Hhex</i>^<i>-/-</i> cultures. Finally, through manipulation of <i>Hhex</i> and <i>Vegfa</i> expression, gain and loss of expression experiments revealed that <i>Hhex</i> shares an inverse relationship with the activity of the <i>Vegf</i> signaling pathway in supporting hepatic differentiation. In summary, our results suggest that <i>Hhex</i> represses <i>Vegf</i> signaling during hepatic differentiation of mouse ESCs allowing for cell-type autonomous regulation of <i>Vegfr2</i> activity independent of endothelial cells.</p></div

Genotype Comparison using QPCR at each Differentiation Stage.

<p><i>Hhex</i>^<i>-/-</i> HE cells did not show mRNA expression consistent with hepatic differentiation. <b>A-E)</b> Comparison of fold-change in normalized mRNA gene expression using differentiation-stage specific markers. Comparison of pluripotency gene markers reveal <i>Hhex</i>^<i>-/-</i> (red) cells showed increased pluripotency relative to <i>Hhex</i>^<i>+/+</i> (blue) at each differentiation stage, particularly during HE differentiation <b>(A, B, and E)</b>. Comparison of definitive endodermal gene markers reveal <i>Hhex</i>^<i>-/-</i> cells fail to exhibit significant decreases in definitive endodermal gene expression characteristic of HE differentiation <b>(C)</b>, and as seen in <i>Hhex</i>^<i>+/+</i> HE cells. <i>Hhex</i>^<i>+/+</i> HE cells showed dramatic increase in hepatic gene expression <b>(D)</b>, while <i>Hhex</i>^<i>-/-</i> cells showed a heavily attenuated expression. Comparison of <i>Vegf</i> signaling gene markers showed that <i>Hhex</i>^<i>-/-</i> cells exhibit increased levels of ligands (<i>Vegf-a</i>) and receptor (<i>Vegfr1</i> and <i>Vegfr2</i>) gene expression at each differentiation stage, but particularly during HE differentiation <b>(A, B, and C)</b>. (Note raw data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146806#pone.0146806.s001" target="_blank">S1 Fig</a>) (*p<0.05, **p<0.01, and ***p<0.001).</p

Differentiation of Hepatic Endoderm.

<p><i>Hhex</i>^<i>-/-</i> definitive endodermal cells did not differentiate toward hepatic endoderm. <b>A)</b> Analysis of FACS for ALB and DLK revealed that only <i>Hhex</i>^<i>+/+</i> (blue) cultures produced significant populations of differentiated HE cells. IGG plot (grey) confirms antigen specificity. <b>B)</b> Single channel and merged immunofluorescence staining of HE cultures. Only <i>Hhex</i>^<i>+/+</i> cells showed a uniform and highly differentiated population of HE cells that were double positive for ALB and AAT compared to IGG controls. (Scale bars = 50μM, *p<0.05, **p<0.01).</p

Proposed <i>Hhex-Vegf</i> Hepatic Regulatory Pathway.

<p><i>Hhex</i> and <i>Vegf</i> signaling modulate the commitment of DE cells toward the HE lineage. <b>A)</b> The addition of VEGFA protein to <i>Hhex</i>^<i>+/+</i> HE cells facilitated HE differentiation as indicated by increased hepatic gene marker expression (black bars). Similar results were seen with the reduction of VEGF signaling in <i>Hhex</i>^<i>-/-</i> DE cells cultured in HE media (white bars). <b>B)</b> The reduction of <i>Hhex</i> using siRNA in <i>Hhex</i>^<i>+/+</i> HE cells resulted in decreased expression of hepatic gene markers and increased <i>Vegfa</i> expression. <b>C)</b> Comparative qPCR analysis for the expression of hepatic markers during the stages of mouse embryonic hepatic expansion, E11.5-E13.5. Hepatic markers change similarly <i>in vivo</i> when compared to the results obtained from the <i>in vivo</i> differentiation from ESC in the current report. <b>D)</b> We propose an <i>Hhex</i>-regulated model of HE specification whereby <i>Hhex</i> is necessary for the expression of hepatic genes, in part, via reducing/regulating <i>Vegf</i> signaling. (* = p<0.05, **p<0.01, ***p<0.001).</p