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. 2016 Mar 31;4:e1878.
doi: 10.7717/peerj.1878. eCollection 2016.

Investigation of the Effects of Estrogen on Skeletal Gene Expression During Zebrafish Larval Head Development

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Free PMC article

Investigation of the Effects of Estrogen on Skeletal Gene Expression During Zebrafish Larval Head Development

Ehsan Pashay Ahi et al. PeerJ. .
Free PMC article

Abstract

The development of craniofacial skeletal structures requires well-orchestrated tissue interactions controlled by distinct molecular signals. Disruptions in normal function of these molecular signals have been associated with a wide range of craniofacial malformations. A pathway mediated by estrogens is one of those molecular signals that plays role in formation of bone and cartilage including craniofacial skeletogenesis. Studies in zebrafish have shown that while higher concentrations of 17-β estradiol (E 2) cause severe craniofacial defects, treatment with lower concentrations result in subtle changes in head morphology characterized with shorter snouts and flatter faces. The molecular basis for these morphological changes, particularly the subtle skeletal effects mediated by lower E 2 concentrations, remains unexplored. In the present study we address these effects at a molecular level by quantitative expression analysis of sets of candidate genes in developing heads of zebrafish larvae treated with two different E 2 concentrations. To this end, we first validated three suitable reference genes, ppia2, rpl8 and tbp, to permit sensitive quantitative real-time PCR analysis. Next, we profiled the expression of 28 skeletogenesis-associated genes that potentially respond to estrogen signals and play role in craniofacial development. We found E 2 mediated differential expression of genes involved in extracellular matrix (ECM) remodelling, mmp2/9/13, sparc and timp2a, as well as components of skeletogenic pathways, bmp2a, erf, ptch1/2, rankl, rarab and sfrp1a. Furthermore, we identified a co-expressed network of genes, including cpn1, dnajc3, esr1, lman1, rrbp1a, ssr1 and tram1 with a stronger inductive response to a lower dose of E 2 during larval head development.

Keywords: 17-β estradiol; Craniofacial skeleton; Development; Estrogen; Gene expression; Reference genes; Zebrafish larvae; qPCR.

Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Expression analysis of candidate reference genes in developing heads of zebrafish larvae across control and E2 treated groups.
(A) Expression profiles of candidate reference genes in raw Cq values for all samples (3 treatments for each of 5 larval stages and with 3 biological replicates). The middle line denotes the median and boxes indicate the 25/75 percentiles. (B) Expression differences of candidate reference genes in the head of zebrafish during the larval development and three E2 treatment groups. Fold changes in expression calculated from the qPCR data, were subjected to ANOVA and Tukey’s HSD analysis to test the expression differences amongst three treatment groups (control, 2 µM and 5 µM) and across five larval stages (3 to 7dpf). White boxes represent low expression, while black boxes represent high expression. Two or more steps of shade differences in the boxes represent significantly different expression between the samples (alpha = 0.05). NS, not significant.
Figure 2
Figure 2. Expression differences of two estrogen receptors and components of hedgehog signaling pathway in developing heads of zebrafish larvae across control and E2 treated groups.
Expression of esrra, esr1, ptch1, ptch2, shha and shhb was examined with qPCR and normalised using three highest ranked reference genes (ppia2, rpl8 and tbp). For analysis of relative expression levels for each target gene a replicate of the control group at 3dpf was set to one. The white, grey, and black bars in each graph represent expression levels for control, 2 µM E2 treated and 5 µM E2 treated groups respectively. Statistical differences of each treatment group versus the others are shown in white, grey, and black circles representing higher expressed than control, 2 µM E2 treated and 5 µM E2 treated groups respectively (P < 0.05). Error bars represent standard deviation calculated from three biological replicates. Each biological replicate is from a homogenate of 30 heads.
Figure 3
Figure 3. Expression differences of six potential skeletogenic targets of estrogen pathway in developing heads of zebrafish larvae across control and E2 treated groups.
Expression of bmp2a, bmp2b, opg, rankl, runx2b and sox9b was examined with qPCR and normalised using three highest ranked reference genes (ppia2, rpl8 and tbp). For analysis of relative expression levels for each target gene a replicate of the control group at 3dpf was set to one. The white, grey, and black bars in each graph represent expression levels for control, 2 µM E2 treated and 5 µM E2 treated groups respectively. Statistical differences of each treatment group versus the others are shown in white, grey, and black circles representing higher expressed than control , 2 µM E2 treated and 5 µM E2 treated groups respectively (P < 0.05). Error bars represent standard deviation calculated from three biological replicates. Each biological replicate was made from a homogenate of 30 heads.
Figure 4
Figure 4. Expression differences of eight potential targets of estrogen pathway involved in skeletal ECM formation examined during zebrafish larval head development across control and E2 treated groups.
Expression of col2a1a, ctsk, mmp2, mmp9, mmp13, sparc, spp1 and timp2 was examined with qPCR and normalised using three highest ranked reference genes (ppia2, rpl8 and tbp). For analysis of relative expression levels for each target gene a replicate of the control group at 3dpf was set to one. The white, grey, and black bars in each graph represent expression levels for control, 2 µM E2 treated and 5 µM E2 treated groups respectively. Statistical differences of each treatment group versus the others are shown in white, grey, and black circles representing higher expressed than control, 2 µM E2 treated and 5 µM E2 treated groups respectively (P < 0.05). Error bars represent standard deviation calculated from three biological replicates. Each biological replicate was made from a homogenate of 30 heads.
Figure 5
Figure 5. Expression differences of eight other potential targets of estrogen pathway involved in jaw skeletal elongation examined during zebrafish larval head development across control and E2 treated groups.
Expression of alx4, dlx1, erf, ets2, pbx1a, pbx1b, rarab and sfrp1a was examined with qPCR and normalised using three best ranked reference genes (ppia2, rpl8 and tbp). For analysis of relative expression levels for each target gene a replicate of the control group at 3dpf was set to one. The white, grey, and black bars in each graph represent expression levels for control, 2 µM E2 treated and 5 µM E2 treated groups respectively. Statistical differences of each treatment group versus the others are shown in white, grey, and black circles representing higher expressed than control, 2 µM E2 treated and 5 µM E2 treated groups respectively (P < 0.05). Error bars represent standard deviation calculated from three biological replicates. Each biological replicate was made from a homogenate of 30 heads.
Figure 6
Figure 6. Expression differences of esr1 coexpressed genes in developing heads of zebrafish larvae across control and E2 treated groups.
Expression levels of eleven candidate genes coexpresed with esr1, based on data from COXPRESdb in zebrafish, were examined with qPCR and normalised using three best ranked reference genes (ppia2, rpl8 and tbp). For analysis of relative expression levels for each target gene a replicate of the control group at 3dpf was set to one. The white, grey, and black bars in each graph represent expression levels for control, 2 µM E2 treated and 5 µM E2 treated groups respectively. Statistical differences of each treatment group versus the others are shown in white, grey, and black circles representing higher expressed than in control, 2 µM E2 treated and 5 µM E2 treated groups respectively (P < 0.05). Error bars represent standard deviation calculated from three biological replicates. Each biological replicate is based on a homogenate of 30 heads.

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Grant support

This project was supported by The University of Iceland Research Fund, the Eimskip University Fund and Roanoke College, USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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