Dynamic analysis of stochastic transcription cycles.

In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly increased the cyclicity. Stochastically timed bursts of transcription in an apparently random subset of cells in a tissue may thus produce an overall coordinated but heterogeneous phenotype capable of acute responses to stimuli.

Wnt signaling in estrogen-induced lactotroph proliferation.

Prolactinomas are the most common type of functioning pituitary adenoma in humans, but the control of lactotroph proliferation remains unclear. Here, using microarray analysis, we show that estrogen treatment increased expression of Wnt4 mRNA in adult Fischer rat pituitary tissue. Dual immunofluorescence analysis revealed that Wnt4 expression was not confined to lactotrophs, but that it was expressed in all anterior pituitary cell types. Estradiol induced proliferation in the somatolactotroph GH3 cell line, in parallel with Wnt4 mRNA and protein induction. A reporter gene assay for TCF- and LEF-dependent transcription revealed that there was no activation of the canonical Wnt pathway in GH3 cells upon stimulation with Wnt-conditioned culture medium or coexpression of constitutively active mutant ß-catenin. Expression of ß-catenin in both GH3 cells and normal rat anterior pituitary cells was restricted to the cell membrane and was unaltered by treatment with estradiol, with no nuclear ß-catenin being detected under any of the conditions tested. We show for the first time that Wnt4 affects non-canonical signaling in the pituitary by inhibiting Ca(2+) oscillations in GH3 cells, although the downstream effects are as yet unknown. In summary, Wnt4 is expressed in the adult pituitary gland, and its expression is increased by estrogen exposure, suggesting that its involvement in adult tissue plasticity is likely to involve ß-catenin-independent signaling pathways.

Dynamic organisation of prolactin gene expression in living pituitary tissue.

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.

Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model.

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states. Using the dual reporters in combination, dramatic and unexpected changes in PRL expression were observed after inflammatory challenge. Expression of PRL was shown by RT-PCR to be driven by activation of the alternative upstream extrapituitary promoter and flow cytometry analysis pointed at diverse immune cells expressing the reporter gene. These studies demonstrate the effective use of this type of model for molecular physiology and illustrate the potential for providing novel insight into human gene expression using a heterologous system.

Endogenous transforming growth factor beta 1 suppresses inflammation and promotes survival in adult CNS.

Transforming growth factor beta1 (TGFbeta1) is a pleiotropic cytokine with potent neurotrophic and immunosuppressive properties that is upregulated after injury, but also expressed in the normal nervous system. In the current study, we examined the regulation of TGFbeta1 and the effects of TGFbeta1 deletion on cellular response in the uninjured adult brain and in the injured and regenerating facial motor nucleus. To avoid lethal autoimmune inflammation within 3 weeks after birth in TGFbeta1-deficient mice, this study was performed on a T- and B-cell-deficient RAG2-/- background. Compared with wild-type siblings, homozygous deletion of TGFbeta1 resulted in an extensive inflammatory response in otherwise uninjured brain parenchyma. Astrocytes increased in GFAP and CD44 immunoreactivity; microglia showed proliferative activity, expression of phagocytosis-associated markers [alphaXbeta2, B7.2, and MHC1 (major histocompatibility complex type 1)], and reduced branching. Ultrastructural analysis revealed focal blockade of axonal transport, perinodal damming of axonal organelles, focal demyelination, and myelin debris in granule-rich, phagocytic microglia. After facial axotomy, absence of TGFbeta1 led to a fourfold increase in neuronal cell death (52 vs 13%), decreased central axonal sprouting, and significant delay in functional recovery. It also interfered with the microglial response, resulting in a diminished expression of early activation markers [ICAM1 (intercellular adhesion molecule 1), alpha6beta1, and alphaMbeta2] and reduced proliferation. In line with axonal and glial findings in the otherwise uninjured CNS, absence of endogenous TGFbeta1 also caused an approximately 10% reduction in the number of normal motoneurons, pointing to an ongoing and potent trophic role of this anti-inflammatory cytokine in the normal as well as in the injured brain.

Tumor necrosis factor-alpha activates the human prolactin gene promoter via nuclear factor-kappaB signaling.

Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-alpha activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-alpha in GH3 cells was confirmed using real-time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-alpha, Western blot analysis showed rapid inhibitory protein kappaB (IkappaBalpha) degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence-labeled p65 and IkappaBalpha fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IkappaBalpha degradation. This was associated with increased nuclear factor kappaB (NF-kappaB)-mediated transcription from an NF-kappaB-responsive luciferase reporter construct. Disruption of NF-kappaB signaling by expression of dominant-negative variants of IkappaB kinases or truncated IkappaBalpha abolished TNF-alpha activation of the prolactin promoter, suggesting that this effect was mediated by NF-kappaB. TNF-alpha signaling was found to interact with other endocrine signals to regulate prolactin gene expression and is likely to be a major paracrine modulator of lactotroph function.

Athyroid Pax8-/- mice cannot be rescued by the inactivation of thyroid hormone receptor alpha1.

The Pax8(-/-) mouse provides an ideal animal model to study the consequences of congenital hypothyroidism, because its only known defect is the absence of thyroid follicular cells. Pax8(-/-) mice are, therefore, completely athyroid in postnatal life and die around weaning unless they are substituted with thyroid hormones. As reported recently, Pax8(-/-) mice can also be rescued and survive to adulthood by the additional elimination of the entire thyroid hormone receptor alpha (TRalpha) gene, yielding Pax8(-/-)TRalpha(o/o) double-knockout animals. This observation has led to the hypothesis that unliganded TRalpha1 might be responsible for the lethal phenotype observed in Pax8(-/-) animals. In this study we report the generation of Pax8(-/-)TRalpha1(-/-) double-knockout mice that still express the non-T(3)-binding TR isoforms alpha2 and Deltaalpha2. These animals closely resemble the phenotype of Pax8(-/-) mice, including growth retardation and a completely distorted appearance of the pituitary with thyrotroph hyperplasia and hypertrophy, extremely high TSH mRNA levels, reduced GH mRNA expression, and the almost complete absence of lactotrophs. Like Pax8(-/-) mice, Pax8(-/-)TRalpha1(-/-) compound mutants die around weaning unless they are substituted with thyroid hormones. These findings do not support the previous interpretation that the short life span of Pax8(-/-) mice is due to the negative effects of the TRalpha1 aporeceptor, but, rather, suggest a more complex mechanism involving TRalpha2 and an unliganded TR isoform.

Regulation of iodothyronine deiodinases in the Pax8-/- mouse model of congenital hypothyroidism.

Thyroid hormones are essential for a variety of developmental and metabolic processes. Congenital hypothyroidism (CHT) results in severe defects in the development of different tissues, in particular brain. As an animal model for CHT, we studied Pax8(-/-) mice, which are born without a thyroid gland. We determined the expression of iodothyronine deiodinase D1 in liver and kidney, D2 in brain and pituitary, and D3 in brain, as well as serum T(4), T(3), and rT(3) levels in Pax8(-/-) vs. control mice during the first 3 wk of life. In control mice, serum T(4) and T(3) were undetectable on the day of birth (d 0) and increased to maximum levels on d 15. In Pax8(-/-) mice, serum T(4) and T(3) remained below detection limits. Serum rT(3) was high on d 0 in both groups and rapidly decreased in Pax8(-/-), but not in control mice. Hepatic and renal D1 activities and mRNA levels were low on d 0 and increased in control mice roughly parallel to serum T(4) and T(3) levels. In Pax8(-/-) mice, tissue D1 activities and mRNA levels remained low. Cerebral D2 activities were low on d 0 and increased to maximum levels on d 15, which were approximately 10-fold higher in Pax8(-/-) than in control mice. D2 mRNA levels were higher in Pax8(-/-) than in control mice only on d 21. Cerebral D3 activities and mRNA levels were high on d 0 and showed a moderate decrease between d 3 and 15, with values slightly lower in Pax8(-/-) than in control mice. One day after the injection of 200 ng T(4) or 20 ng T(3)/g body weight, tissue deiodinase activities and mRNA levels were at least partially restored toward control levels, with the exception of cerebral D3 activity. In conclusion, these findings show dramatic age and thyroid state-dependent changes in the expression of deiodinases in central and peripheral tissues of mice during the first 3 wk of life.

Expression of pituitary hormones in the Pax8-/- mouse model of congenital hypothyroidism.

Signaling mechanisms in pituitary morphogenesis as well as pituitary cell fate determination during early embryonic development are relatively well characterized. In contrast, the cues that determine the progression of the various anterior pituitary cell types during postnatal periods are poorly defined. Pax8-/- mice, which are born without a thyroid gland, were used to study the influence of thyroid hormones on the expression of pituitary hormones during early postnatal life. Serum pituitary hormones were determined by RIAs, and the pituitaries were analyzed by Northern blotting, in situ hybridization histochemistry, and immunocytochemistry. In 21-d-old Pax8-/- mice, the cellular composition of the anterior pituitary was dramatically distorted. Thyrotropes exhibited hypertrophy and hyperplasia, the number of detectable somatotropes was drastically reduced, and lactotropes were almost undetectable. Expression of LH and FSH was also reduced, but ACTH and proopiomelanocortin expression was not significantly different. Serum pituitary hormone levels were changed correspondingly. T(4) replacement therapy for variable time periods normalized TSH and GH mRNA expression within 3 d but not prolactin expression, not even when T(4) was administered for 6 d in combination with estradiol. These findings reveal the importance of thyroid hormones in developing the appropriate proportions of anterior pituitary cell types, especially with regard to lactotropes.

Analysis of hypertrophic thyrotrophs in pituitaries of athyroid Pax8-/- mice.

Thyroid hormone is important for pituitary development and maintenance. We previously reported that in the Pax8(-/-) mouse model of congenital hypothyroidism, lactotrophs are almost undetectable, whereas the thyrotrophs exhibit hyperplasia and hypertrophy. Because the latter might be caused by an overstimulation of thyrotrophs with TRH, we analyzed TRH-R1(-/-)Pax8(-/-) double-knockout mice, which miss a functional thyroid gland and the TRH transducing receptor-1 at pituitary target sites. Interestingly, in these double mutants, the hypertrophy and hyperplasia of the thyrotrophs still persist, suggesting that the phenotype is rather a direct consequence of the athyroidism of the animals. The increased expression of TSH in the Pax8(-/-) mice was paralleled by a strongly up-regulated expression of deiodinase type 2 (Dio2) in thyrotrophic cells. Moreover, coexpression of TSH and Dio2 could also be demonstrated in the pituitary of wild-type mice, underlining the important role of this enzyme in the negative feedback regulation of TSH by thyroid hormone. As another consequence of the athyroidism in the mutant mice, tyrosine hydroxylase mRNA expression was found to be also highly up-regulated in thyrotrophic cells of the pituitaries from Pax8(-/-) mice, whereas the transcript levels in the hypothalamus were not affected. Accordingly, tyrosine hydroxylase protein levels, enzyme activities, and ultimately dopamine concentrations were found to be strongly increased in the pituitaries of Pax8(-/-) mice compared with wild-type animals. These findings may explain in part the reduced number of lactotrophs found in the pituitary of athyroid Pax8(-/-) mice and suggest a novel paracrine regulatory mechanism of lactotroph activity.

Gene expression of connective tissue growth factor in adult mouse.

The connective tissue growth factor (CTGF) is a well-known fibroblast mitogen and angiogenic factor that plays an important role in bone formation during embryogenesis. In the adult, CTGF is involved in wound healing as well as fibrotic and vascular disease. However, little is known about its physiological functions under non-pathological conditions in the adult organism. Here, we describe the cellular site of the CTGF mRNA expression in adult male and female mice as revealed by in situ hybridization histochemistry. Strong and persistent CTGF gene expression was particularly prominent in the mesenchyme of the cardiovascular system (aorta, auricular tissue, renal glomeruli), the mesenchyme surrounding the ovarian follicles or the testicular tubes in the gonadal tissue, and the subcapsular mesenchyme bordering densely innervated parts of whisker hair vibrissae. CTGF hybridization signals were not observed in the mesenchyme of many other organs including gut, muscle, liver or most parts of the lymphatic tissue. Strong expression was also present in the primary (early) ovarian follicles, the epithelium of the deep uterine glands and on myenteric ganglia neurons. These data suggest a selective and continuous mesenchymal function in the gonads and those tissues attracting very strong vascular supply or peripheral innervation. CTGF may also be involved in the cyclical proliferation of the uterine gland epithelium and in the early stages of follicular maturation, as well as in the neuropeptide regulation in the gut, cardiovascular and renal systems.

Hearing loss in athyroid pax8 knockout mice and effects of thyroxine substitution.

Pax8-/- mice do not develop thyroid follicular structures and thus provide an ideal animal model to study the consequences of congenital hypothyroidism. Despite their athyroidism, Pax8-/- mice survive up to postnatal day 21 (P21). No auditory brain stem responses (ABR) to sound could be recorded in these animals at 130 dB SPL, even at P21, when hearing reaches adult sensitivity in control mice. Abnormalities in the outer and middle ear structures were found in a considerable percentage of Pax8-/- animals. Maturation of the inner ear appeared delayed by about 1 week with respect to euthyroid controls. Hearing of adult Pax8-/- mice could be nearly normalized by early postnatal substitution with thyroxine (T(4)), but structural and functional restoration of hearing was incomplete. Even when T(4) substitution was initiated at P1, ABR thresholds, measured at 6 weeks of age or more, were increased by about 20 dB, and each day of delay in the start of T(4) substitution resulted in an additional threshold loss of about 4 dB. The most prominent structural deficit in Pax8-/- animals in which T(4) substitution was started at P8 or later was an abnormally thick tectorial membrane. In these late-substituted animals, disarray of stereovilli from inner and outer hair cells was observed and also outer hair cell loss was found, predominantly in the basal part of the cochlea. The degree of structural disorder increased the later T(4) substitution was initiated. The structural and functional consequences of postnatal athyroidism observed in Pax8-/- mice are largely in agreement with and extend those data obtained from hypothyroid animal models in which hypothyroidism was induced by goitrogenic agents (methimazole, propylthiouracil) or animal models with disrupted genes for the TSH receptor or the thyroid hormone receptors. The hearing loss and also the recovery effect by T(4) substitution in Pax8-/- mice is larger than that in the other models. Although Pax8-/- mice are born by euthyroid Pax8+/- dams, the Pax8-/- phenotype could not be completely restored by immediate postnatal T(4) substitution, indicating that some deficits are the consequence of prenatal T(4) deficiency of the offspring.