Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis

Abstract

BackgroundBiliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics.ResultsWe hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection.ConclusionsHSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0097-2) contains supplementary material, which is available to authorized users.

TGF‐β Signaling Plays a Pivotal Role During Developmental Biliary Atresia in Sea Lamprey (Petromyzon marinus)

“…To identify candidate pathways underlying the developmental processes during BA, we analyzed sea lamprey hepatobiliary transcriptomes at four landmark stages: M0 (National Center for Biotechnology Information Sequence Read Archive database accession number SRX2721810), M2 (SRX2721809), M5 (SRX2721808), and JV (SRX2721807) . We annotated 22,297 genes from the hepatobiliary transcriptome assembly (gene‐expression data listed in Supporting Table ).…”

“…Total RNAs of pooled liver samples from different developmental stages (M0, M2, M5, and JV; five per stage) were extracted using TRIzol Reagent (Invitrogen/Thermo Fisher Scientific, Madison, WI) and treated with TURBO DNA‐free kit (Invitrogen) as described . RNA samples (2 µg/sample) were enriched by the mRNA‐seq 8 sample Prep Kit, and the transcriptomes were obtained using an Illumina Genome Analyzer II (San Diego, CA) and mRNA‐seq protocol (75‐mers; Illumina) at the Genomics Technology Support Facility at MSU.…”

“…RNA samples (2 µg/sample) were enriched by the mRNA‐seq 8 sample Prep Kit, and the transcriptomes were obtained using an Illumina Genome Analyzer II (San Diego, CA) and mRNA‐seq protocol (75‐mers; Illumina) at the Genomics Technology Support Facility at MSU. These sequences were trimmed with Trimmomatic‐0.30 software, filtered for polynucleotides (more than 36 nt) with in‐house Perl script, and aligned to the sea lamprey reference genome using TopHat software . Gene‐expression analysis of mRNA‐seq was based on the gene model of Petromyzon marinus (Pmarinus_7.0.66) .…”

“…TaqMan minor groove binder (MGB) or SYBR Green system (Invitrogen) was used as described . Briefly, total RNAs were reverse‐transcribed into complementary DNA (cDNA) using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random hexamers (Promega Corp., Madison, WI) according to the manufacturer’s protocols.…”

“…An additional 30 M2 larvae were injected with vehicle (3.3% lipofectamine), 0.3 mM TGF‐β1 siRNA, or TGF‐βRII siRNA intraperitoneally (n = 10 for each group). Liver samples were collected 2 weeks after treatment and fixed in hydrogel for CLARITY and immunofluorescent confocal microscopy imaging . Two‐week treatment was chosen because it took 1 to 2 weeks for M2 larvae to progress to M3 .…”