Spatial Profiling of Nuclear Receptor Transcription Patterns over the Course ofDrosophilaDevelopment

Abstract

Previous work has shown that many of the 18 family members of Drosophila nuclear receptor transcription factors function in a temporal hierarchy to coordinate developmental progression and growth with the rate limiting process of metabolism. To gain further insight into these interactions and processes, we have undertaken a whole-family analysis of nuclear receptor mRNA spatial expression patterns over the entire process of embryogenesis, as well as the 3rd instar wandering larva stage, by using high-resolution fluorescence in situ hybridization. Overall, the patterns of expression are remarkably consistent with previously mapped spatial activity profiles documented during the same time points, with similar hot spots and temporal profiles in endocrine and metabolically important tissues. Among the more remarkable of the findings is that the majority of mRNA expression patterns observed show striking subcellular distributions, indicating potentially critical roles in the control of protein synthesis and subsequent subcellular distributions. These patterns will serve as a useful reference for future studies on the tissue-specific roles and interactions of nuclear receptor proteins, partners, cofactors and ligands.

Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs

“…In another example, the polarized distribution of Hsp83 transcripts on one side of the nucleus (Fig. 3E) looks remarkably similar to a number of nuclear receptor transcripts, which we previously colocalized together with subregions of ER (Wilk et al 2013). This colocalization could be associated with the known roles of Hsp83 as a chaperone and regulator of nuclear receptor folding and function, with perhaps an additional level of regulation at the level of translation.…”

“…Indeed, these biochemical approaches have suggested that as much as 50% of cytosolic protein-encoding transcripts are translated on ER (Reid and Nicchitta 2012;Jagannathan et al 2014). We also showed previously, using double labeling, that transcripts encoding transcription factors can also be enriched and translated on ER (Wilk et al 2013), suggesting that ER localization may be a common means of localizing transcripts that encode numerous types of proteins. In the case of the nuclear receptor transcripts that we found to be associated with ER, this may provide a number of advantages, including proximity to the nucleus, proximity to translational regulators and chaperone cofactors (e.g., Hsp83 RNA also appears to be similarly localized), or proximity to ER-derived ligands (e.g., the E75 ligand heme is enriched in ER) (Reinking et al 2005).…”

“…It is during this stage that many interesting hormonal and metabolic changes occur, which has made it a particularly important and prevalent developmental stage for Drosophila research (Ashburner 1973;Muskavitch and Hogness 1980;Basler and Struhl 1994;Mirth et al 2005;Baker and Thummel 2007;Gerber and Stocker 2007;Gomez-Marin and Louis 2012;Makhijani and Bruckner 2012;Tennessen et al 2014). To gain some insight into transcript expression and localization during this stage, we developed a method for semi-high-throughput TSA-FISH that includes the majority of larval tissues (Materials and Methods; Wilk et al 2010Wilk et al , 2013. To date, we have analyzed ∼800 genes and defined >150 new terms to describe these new expression and localization patterns.…”

“…Dissection, fixation, and in situ hybridization of third instar larval tissues Climbing third instar larval tissue preparation and hybridization were performed as described (Wilk et al 2010(Wilk et al , 2013 with the following modifications: 40% paraformaldehyde (PFA) stock that was made fresh prior to use (3.68 g of PFA, 10 mL of DEPC H 2 O, 70 µL of 2N KOH), 1× PBT (PBS with 0.1% Tween-20), 4% PFA (1 mL of 40% PFA stock diluted in 9 mL of 1× PBT and then filtered through a 0.45-µm filter unit), PBTT fix solution (1 mL of 40% PFA stock diluted in 9 mL of 1× PBT, 30 µL of 0.3% Triton-X100, and 10 µL of 0.1% picric acid solution and filtered through a 0.45-µm filter unit), 0.3% H 2 O 2 in 1× PBS (100 µL of 30% H 2 O 2 stock [Sigma, catalog no. 216763] in 9.9 mL of 1× PBS), and 80% acetone (40 mL of acetone with 10 mL of DEPCddH 2 O chilled at −20°C).…”

Nuclear receptor ecdysone‐induced protein 75 is required for larval–pupal metamorphosis in the Colorado potato beetle Leptinotarsa decemlineata (Say)

“…The three LdE75 s were detectable in all tested larval tissues. Similar expression profiles of Nuclear receptors (NRs) have been found in the same 20E‐response tissues in other insect species (Fahrbach et al ., ; Wilk et al ., ). Interestingly, our results revealed that the expression levels of LdE75A and LdE75C were lower than that of LdE75B in the L. decemlineata PGs.…”