Dynamic organisation of prolactin gene expression in living pituitary tissue

Abstract

Gene 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 co-ordination 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.

Fig. 1.

Lactotroph organisation within the anterior pituitary gland. Localisation of luminescent signal (A) and fluorescent signal (B) from 400 μm thick pituitary slices, taken from transgenic rats expressing PRL-luciferase or PRL-d2EGFP, respectively. Left panels show transmitted-light image. Right panels show transmitted-light image merged with localisation of reporter gene signal. (C) Immunohistochemistry of PRL protein expression (brown) in a wild type rat pituitary. Scale bars: 500 μm.

Fig. 2.

Real-time luminescence imaging of PRL promoter-directed transcription in living transgenic pituitary tissue slices. (A,D,G) Luminescent images of 400 μm thick pituitary tissue slices, (B,E,H) graphs of luminescent photon flux from whole pituitary slices and (C,F,I) selected regions within the slices over approximately 3 days, with no treatment (control; A-C), 1 μM dopamine (D-F), or 5 μM forskolin and 0.5 μM BayK8644 (FBK; G,H,I). Arrow in B shows time of stimulation with 5 μM forskolin. Grey bars indicate 12-hour periods. Scale bars: 500 μm.

Fig. 3.

Real-time luminescence imaging of PRL transcription in living luminescent transgenic pituitary cells. (A-C) Representative luminescence images of individual cells dispersed from whole pituitary glands taken from luminescent transgenic rats with no treatment (control; A), 1 μM dopamine (B) or 5 μM forskolin and 0.5 μM BayK8644 (C). (D-F) Graphs of luminescent photon flux from individual cells with no treatment (control; D), 1 μM dopamine (E) or 5 μM forskolin and 0.5 μM BayK8644 (F). Each graph shows seven representative cell traces where luminescence signal per cell is normalised to the initial value per cell. Numbers above A show time in hours. Scale bar: 50 μm.

Fig. 4.

Regional expression of the time-course of PRL promoter-directed luciferase expression in unstimulated pituitary slices and dispersed cells from transgenic rats with differing expression levels. (A,B) Analysis of PRL expression in ‘cell areas’ (approximately 50 μm; represented by coloured lines on graphs) over a period of 50 hours. Comparison of responses in high copy (A) and low copy (B) 400 μm thick transgenic pituitary slices. The arrows on the images mark the orientation of the analysis. (C,D) Corresponding expression profiles from individual dispersed pituitary cells were analysed from the same high (C) and low (D) copy transgenic lines in unstimulated conditions. Each line represents an individual cell with the black lines representing the average response. Scale bars: 500 μm (A,B), 75 μm (C,D).

Fig. 5.

Visualisation of the dynamics of PRL promoter-directed d2EGFP expression in single lactotroph cells within living transgenic pituitary tissue by confocal microscopy. (A) Whole fluorescent transgenic pituitary slice (2.5× objective; scale bar: 500 μm). Fluorescent (green) signal indicates PRL promoter expression. (B) Higher magnification of boxed region in A reveals single cell resolution (10× objective; scale bar: 150 μm). (C) A small region within the pituitary slice (boxed region in B) was imaged sequentially over 15 hours (20× objective; scale bar: 75 μm). (D) Single cell transcriptional responses from cells within the section of pituitary in C clustered into five groups based on transcriptional profile (data taken from 239 cells with the groups containing 18, 61, 57, 64 and 39 cells, respectively). (E) Average transcriptional response for each of the five groups. (F) Location of the cells showing each profile within the pituitary slice (cells shown by green dots). (G) Three cells were selected within the slice and the correlation was compared between the transcriptional profile of the chosen cells and the cells surrounding it (red denotes a correlation coefficient of 0.75-1.0; orange, 0.5-0.75; yellow <0.5).