Investigation of the effects of estrogen on skeletal gene expression during zebrafish larval head development

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-βestradiol (E2) 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 lowerE2concentrations, 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 differentE2concentrations. To this end, we first validated three suitable reference genes,ppia2,rpl8andtbp, 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 foundE2mediated differential expression of genes involved in extracellular matrix (ECM) remodelling,mmp2/9/13,sparcandtimp2a, as well as components of skeletogenic pathways,bmp2a,erf,ptch1/2,rankl,rarabandsfrp1a. Furthermore, we identified a co-expressed network of genes, includingcpn1,dnajc3,esr1,lman1,rrbp1a,ssr1andtram1with a stronger inductive response to a lower dose ofE2during larval head development.