Investigating the roles of E-T coupling and social environment in the stimulus-dependent expression of parathyroid hormone 2.

Abstract

In larval zebrafish, parathyroid hormone 2 (pth2) codes for a peptide hormone that is expressed exclusively in cells near the ventral part of the posterior tuberculum, an area of the forebrain involved in sensori-motor control and social behavior. We identified pth2 in an RNA-seq experiment looking for genes regulated by voltage-gated calcium channel activity. Relative to wild type larvae, pth2 was down-regulated in larvae where the L-type Cav1.3 channel was inactivated either genetically (cav1.3a mutants) or pharmacologically (the Ca2+ channel blocker isradipine). Using mRNA in situ hybridization on cav1.3a mutant larval zebrafish and larvae treated with isradipine, we confirmed that pth2 expression in zebrafish requires Cav1.3a channel activity. Next, to characterize the regulatory region of the pth2 gene, we performed a transgenesis experiment using a 2 kbp fragment of the zebrafish genome immediately upstream of the pth2 coding sequence. This resulted in expression of GFP in cells of the posterior tuberculum and their axons.

Recently, a study investigating gene expression in zebrafish showed that social isolation led to decreased levels of pth2 expression. We confirmed these results using our stable line of transgenics. Using neomycin and CuSO4, the study also showed that expression of pth2 was dependent on mechanical stimulation due to the movements of other zebrafish. These findings provided evidence that pth2 is not expressed when hair cells of the lateral line are killed using ototoxic compounds, but the study did not explore the effects on pth2 expression when lateral line function is lost genetically. To test this, we performed mRNA in-situ hybridization on lateral line mutant zebrafish raised in social or isolated environments. Interestingly, mutants raised socially expressed more pth2 than wild type fish that were isolated, despite lacking a functional lateral line. Overall, this research provides further information about the regulation of pth2 and could help elucidate its role in the social behavior of zebrafish.