Regulation of Halloween genes and ecdysteroid responsive genes in molting gland of Blackback land crab Gecarcinus lateralis by molt-inhibiting hormone, mTOR and TGFβ signaling pathways pathways
Benrabaa, Samiha Abd Al Salem, author
Mykles, Donald, advisor
Garrity, Deborah, committee member
Kanatous, Shane, committee member
Stargell, Laurie, committee member
Molting is necessary for growth and development in all arthropods. Halloween genes are expressed in the Y-organs (YO) and encodes cytochrome P450 enzymes. These enzymes catalyze the synthesis of ecdysteroid hormones that regulate the molt cycle. This hormone binds to ecdysteroid receptor to activate a cascade of ecdysteroid-responsive genes that effect tissue responses to hormone. We used P. leptodactylus Halloween gene and insect ecdysteroid-responsive gene sequences to extract and characterize the land crab orthologs in the YO transcriptome. This resulted in identification and characterization of eight ecdysteroidogenic genes that encode phantom, disembodied, spook, shadow, Cyp18a1, neverland, NADK and ALAS and nine ecdysteroid-responsive genes that encoded EcR, RXR, broad complex, E75, E74, Hormone receptor 4, Hormone receptor 3, forkhead box transcription factor (FOXO) and Fushi tarazu factor-1. Sequences were validated by end-point PCR and Sanger sequencing. We used phylogenetic analysis to infer evolutionary relationships among contig sequences and ortholog of Halloween genes and ecdysteroid-responsive genes in other species. The results showed the contig sequences clustered with their corresponding orthologous genes. Tissue distributions for spook and phantom showed significantly higher mRNA levels in the YO compared to other tissues. By contrast, the mRNA levels of NADK, ALAS, and all ecdysteroid-responsive genes were not higher in the YO than those in other tissues. These data show that the YO is the primary source of ecdysteroid production and that the YO can respond to ecdysteroid, suggesting a feedback regulation on ecdysteroid synthesis and secretion. qPCR was used to quantify gene expression of Halloween and ecdysteroid-responsive gene expression in the YO of Gecarcinus lateralis induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). ESA decreased mRNA levels of Gl-Phm, Gl-E75 and Gl-RXR at 3 days post-ESA. Gl-Dib and shadow increased at 3 days post ESA and decreased at 7 and 14 days post-ESA. Gl-Cyp18a1, Gl-BR-C, Gl-NADK and Gl-ALAS mRNA were higher at Day 0 and 1 post-ESA and lower at Day14 post ESA. Gl-HR3, Gl-HR4, and Gl-E74 were expressed at low levels. MLA lowered mRNA levels of Halloween genes, Gl-Nev, and Gl-E75, except Gl-dib, at premolt and postmolt stages. Gl-Dib, Gl-NADK, Gl-ALAS, and Gl-BR-C mRNA levels were not affected by molt stage. Gl-EcR, Gl-HR4 and Gl-HR3 mRNA levels were highest in premolt and lowest in postmolt. Gl- FOXO mRNA levels were highest in premolt and lowest in intermolt. These data suggest that molting has an indirect effect on the regulation of Halloween genes and that molting directly regulates HR3, HR4, RXR and FOXO to increase ecdysteroid synthetic capacity of the YO in premolt animals. The presence of EcR/RXR and ecdysteroid-responsive genes suggest that elevated ecdysteroid represses the YO at the end of premolt. TGFβ/activin signaling mediates the transition of the YO from the activated to the committed state, as SB431542 blocks this transition. G. lateralis were eyestalk-ablated to induce molting and injected with vehicle (DMSO) or SB431542 at Day 0. In controls, ESA increased hemolymph ecdysteroid titers at 3, 7 and 14 days post-ESA. There were significant increases in the mRNA levels of Gl-Nvd at 7 and 14 days post-ESA and other Halloween genes (Gl-Spo, Gl-Phm, Gl-Dib, and Gl-Sad), as well as Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-BR-C,Gl- FOXO, Gl-EcR, and Gl-RXR, at 14 days post-ESA. SB431542 reduced hemolymph ecdysteroid titers at 7 and 14 days post-ESA compared to control animals, but titers were no different from controls at 1, 3, and 5 days post-ESA, indicating that SB431542 had no effect on YO activation. SB431542 blocked the increases in mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-Dib, Gl-Sad, Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-BR-C, Gl-EcR, and Gl-RXR by ESA. SB431542 had no effect on mRNA levels of the ecdysteroid-responsive genes Gl-HR3 Gl-HR4, Gl-E74, Gl-E75 and Gl-FTZ-F1. These data suggest that an Activin-like TGFβ factor stimulates YO ecdysteroidogenesis in the committed YO by up-regulating Halloween, Gl-BR-C, and Gl-FOXO genes.
Includes bibliographical references.
Includes bibliographical references.