Dynamic organisation of prolactin gene expression in living pituitary tissue

Abstract

SummaryGene expression in living cells is highly dynamic, but temporal patterns of gene expression in intact tissues are largely unknown. The mammalian pituitary gland comprises several intermingled cell types, organised as interdigitated networks that interact functionally to generate co-ordinated hormone secretion. Live-cell imaging was used to quantify patterns of reporter gene expression in dispersed lactotrophic cells or intact pituitary tissue from bacterial artificial chromosome (BAC) transgenic rats in which a large prolactin genomic fragment directed expression of luciferase or destabilised enhanced green fluorescent protein (d2EGFP). Prolactin promoter activity in transgenic pituitaries varied with time across different regions of the gland. Although amplitude of transcriptional responses differed, all regions of the gland displayed similar overall patterns of reporter gene expression over a 50-hour period, implying overall coordination of cellular behaviour. By contrast, enzymatically dispersed pituitary cell cultures showed unsynchronised fluctuations of promoter activity amongst different cells, suggesting that transcriptional patterns were constrained by tissue architecture. Short-term, high resolution, single cell analyses in prolactin-d2EGFP transgenic pituitary slice preparations showed varying transcriptional patterns with little correlation between adjacent cells. Together, these data suggest that pituitary tissue comprises a series of cell ensembles, which individually display a variety of patterns of short-term stochastic behaviour, but together yield long-range and long-term coordinated behaviour.

Pulsatile patterns of pituitary hormone gene expression change during development

“…4E). These patterns also differed from that of adult pituitary tissue (Harper et al, 2010). Differences could also be detected in the transcription patterns of the tissue ( …”

“…To assess the real-time transcription activity of the PRL gene, transgenic rats were previously generated that express the luciferase reporter gene under the regulation of the human PRL locus (hPRL-Luc49), followed by bioluminescence imaging of cells or tissue in culture in a temperature-, CO -and humiditycontrolled imaging chamber (Semprini et al, 2009;Harper et al, 2010). Characterisation of the reporter transgene showed that it faithfully reflects endogenous Prl gene transcription activity as it is expressed in the correct cell type (lactotroph cells) and responds appropriately to various regulators of Prl expression (Semprini et al, 2009;Harper et al, 2010).…”

“…Characterisation of the reporter transgene showed that it faithfully reflects endogenous Prl gene transcription activity as it is expressed in the correct cell type (lactotroph cells) and responds appropriately to various regulators of Prl expression (Semprini et al, 2009;Harper et al, 2010). The location of transcriptionally active lactotroph cells in the rat pituitary has been assessed by bioluminescence imaging of adult pituitary 2 tissue slices 7. and, in this study, in whole neonatal and embryonic pituitaries from transgenic hPRLLuc49 rats.…”

Noise-Driven Heterogeneity in the Rate of Genetic-Variant Generation as a Basis for Evolvability

“…An important study has recently used transgenic rat models with fluorescent reporter and real-time confocal imaging of fluorescent pituitary tissue to analyze the spatiotemporal patterns of gene expression in relation to tissue structure (Harper et al 2010). This study showed a long-term coordination of pituitary cell behavior when the tissue structure appears whereas dispersion of cells in a primary culture provides high variability and independence of promoter activity.…”

Novel approaches to in vitro transgenesis

“…The library of reporter genes available has significantly expanded over the years, and includes enzymes such as b-galactosidase, which generate a colourimetric product or chloramphenicol acetyl transferase, which catalyses the synthesis of radiolabelled (and latterly fluorescent) product, to enzymes such as luciferase that catalyses the production of light from the substrate luciferin to give a measure of gene activation (Frawley et al 1994, White et al 1995. Luciferase can be used to analyse the induction of gene expression across a population of cells by assaying the whole cell lysate, and also to dynamically analyse gene expression in a single cell through microscopy, measuring the photon output from a single cell (Harper et al 2010).…”