Smad-interacting protein 1 affects acute and tonic, but not chronic pain.

BACKGROUND: Smad-interacting protein 1 (also named Zeb2 and Zfhx1b) is a transcription factor that plays an important role in neuronal development and, when mutated, causes Mowat-Wilson syndrome (MWS). A corresponding mouse model carrying a heterozygous Zeb2 deletion was comprehensively analysed in the German Mouse Clinic. The most prominent phenotype was the reduced pain sensitivity. In this study, we investigated the role of Zeb2 in inflammatory and neuropathic pain. METHODS: For this, we tested mutant Zeb2 animals in different models of inflammatory pain like abdominal constriction, formalin and carrageenan test. Furthermore, we studied the pain reactivity of the mice after peripheral nerve ligation. To examine the nociceptive transmission of primary sensory dorsal root ganglia (DRG) neurons, we determined the neuronal activity in the spinal dorsal horn after the formalin test using staining of c-Fos. Next, we characterized the neuronal cell population in the DRGs and in the sciatic nerve to study the effect of the Zeb2 mutation on peripheral nerve morphology. RESULTS: The present data show that Zeb2 is involved in the development of primary sensory DRG neurons, especially of C- and Aδ fibres. These alterations contribute to a hypoalgesic phenotype in inflammatory but not in neuropathic pain in these Zeb2(+/-) mice. CONCLUSION: Our data suggest that the under-reaction to pain observed in MWS patients results from a reduced responsivity to nociceptive stimulation rather than an inability to communicate discomfort.

A balanced translocation t(6;14)(q25.3;q13.2) leading to reciprocal fusion transcripts in a patient with intellectual disability and agenesis of corpus callosum.

We identified a male patient presenting with intellectual disability and agenesis of the corpus callosum, carrying an apparently balanced, reciprocal, de novo translocation t(6;14)(q25.3;q13.2). Breakpoint mapping, using array painting, identified 2 interesting candidate genes, ARID1B and MRPP3, disrupted in the patient. Unexpectedly, the rearrangement produced 3 in-frame reciprocal fusion transcripts that were further characterized. Formation of fusion transcripts is mainly reported in acquired malignancies and is very rarely observed in patients with intellectual disability (ID) and/or multiple congenital malformations (MCA). Additional experimental results suggest that ARID1B, a gene involved in chromatin remodeling, constitutes a good candidate for the central nervous system phenotype present in the patient.

Combinatorial expression of phenotypes of different cell lineages in the rat and mouse pituitary.

As studied by single cell RT-PCR of pituitary hormones, we demonstrated that the pituitaries of rats and mice contain a subpopulation of cells that express two or more hormone phenotypes typically belonging to lineages that are branched separately early during embryonic development, such as glycoprotein hormone alpha-subunit (alphaGSU) mRNA + PRL mRNA, alphaGSU mRNA + POMC mRNA, and POMC mRNA + GH or PRL mRNA. GnRH in vitro selectively expands the population of cells coexpressing alphaGSU mRNA + PRL mRNA, and CRH selectively increases the proportion of cells coexpressing alphaGSU mRNA + POMC mRNA. Colocalization of alphaGSU + PRL or alphaGSU + POMC could not be detected by double immunofluorescence. This lineage promiscuity was also observed in the pituitary in vivo.

Stimulation of combinatorial expression of prolactin and glycoprotein hormone alpha-subunit genes by gonadotropin-releasing hormone and estradiol-17beta in single rat pituitary cells during aggregate cell culture.

Previously we showed the existence of rat and mouse anterior pituitary cells coexpressing mRNA from two or more hormone genes in which production and/or storage of the corresponding hormones were not detectable. To substantiate a putative function for these cells, we investigated whether these phenotypes were retained during long-term reaggregate cell culture and whether protagonist regulatory factors could expand cell populations expressing particular hormone mRNA combinations. After 4-wk culture and treatments, aggregates were trypsinized and single cells collected by means of a fluo-rescence-activated cell sorter. Hormone mRNAs were detected by single-cell RT-PCR. Combinatorial hormone mRNA expression was retained in culture. Both estradiol (E2) and GnRH (1 nM) markedly augmented the proportion of cells expressing prolactin (PRL) mRNA together with other hormone mRNAs and cells expressing glycoprotein subunit (GSU)-alpha mRNA together with other hormone mRNAs. GnRH strongly increased the proportion of cells containing alphaGSU mRNA alone, but E2 did not. GnRH and (E2) affected the expansion of a population (approximately 20% of all cells) coexpressing PRL and alphaGSU mRNA without betaGSUs. Immunostaining of stored hormone on tissue sections revealed colocalization of PRL and alphaGSU in the E2- but not in the GnRH-treated cells. The present findings suggest that cells coexpressing different pituitary hormone mRNAs form a distinct population that survives without extrapituitary factors. Their occurrence can be markedly modified by regulatory factors. Certain hormone regimens favor unique coexpressions distinctly at mRNA and protein level. These peculiar characteristics support the notion that combinatorial expression of hormone genes in the pituitary serves a biological role.

Ontogeny of plurihormonal cells in the anterior pituitary of the mouse, as studied by means of hormone mRNA detection in single cells.

The expression of mRNA of growth hormone (GH), prolactin (PRL), pro-opiomelanocortin (POMC) and the common glycoprotein hormone alpha-subunit (alphaGSU) was studied by means of single cell reverse transcriptase-polymerase chain reaction in male mouse pituitary cells at key time points of fetal and postnatal development: embryonic day 16 (E16); postnatal day 1 (P1) and young-adult age (P38). At E16, the hormone mRNAs examined were detectable, although only in 44% of total cells. Most of the hormone-positive cells expressed only one of the tested hormone mRNAs (monohormonal) but 14% of them contained more than one hormone mRNA (plurihormonal cells). Combinations of GH mRNA with PRL mRNA, of alphaGSU mRNA with GH and/or PRL mRNA and of POMC mRNA with GH and/or PRL mRNA or alphaGSU mRNA were found. As expected, the proportion of hormone-positive cells rose as the mouse aged. The proportions of plurihormonal cells followed a developmental pattern independent of that of monohormonal cells and characteristic for each hormone mRNA examined. Cells coexpressing POMC mRNA with GH or PRL mRNA significantly rose in proportion between E16 and P1, while the proportion of cells coexpressing GH and PRL mRNA markedly increased between P1 and P38. The occurrence of cells displaying combined expression of alphaGSU mRNA with GH and/or PRL mRNA did not significantly change during development. Remarkably, the population of cells expressing PRL mRNA only, was larger at E16 than at P1 and expanded again thereafter. In conclusion, the normal mouse pituitary develops a cell population that is capable of expressing multiple hormone mRNAs, thereby combining typical phenotypes of different cell lineages. These plurihormonal cells are already present during embryonic life. This population is of potential physiological relevance because development-related factors appear to determine which hormone mRNAs are preferentially coexpressed. Coexpression of multiple hormone mRNAs may represent a mechanism to respond to temporally increased endocrine demands. The data also suggest that the control of combined hormone expression is different from that of single hormone expression, raising questions about the current view on pituitary cell lineage specifications.

Combined expression of different hormone genes in single cells of normal rat and mouse pituitary.

Cells displaying combined expression of different pituitary hormone genes (further referred to as 'multi-hormone mRNA cells') were identified in normal rat and mouse pituitary by single cell RT-PCR. These cells do not seem to produce or store all the respective hormones the mRNAs encode for. The cells are already developed at day 16 of embryonic life (E16) in the mouse. Different peptides, such as gamma3-melanocyte-stimulating hormone (gamma3-MSH) and gonadotropin-releasing hormone (GnRH), affect different subsets of these cells. In culture, estrogen and GnRH increase the number of 'multi-hormone mRNA cells' that contain prolactin (PRL) mRNA or mRNA of the alpha-subunit of the glycoprotein hormones (alpha-GSU) but not the number of 'multi-hormone mRNA cells' not containing PRL or alpha-GSU mRNA. 'Multi-hormone mRNA cells' may function as 'reserve cells' in which a particular hormone mRNA may be translated under a particular physiological condition demanding a rapid increase of that hormone.

Progenitor cells in the embryonic anterior pituitary abruptly and concurrently depress mitotic rate before progressing to terminal differentiation.

The control of progenitor cell proliferation in concert with terminal differentiation during embryonic development is poorly understood. The present paper examines this issue in the different cell lineages of the fetal mouse pituitary. Mouse fetuses were pulse-exposed to 3H-thymidine (3H-T) on a single day between embryonic day (E) 10 and E16 (prior to the onset of hormone phenotype expression) and the 3H-T labeling index of each cell type determined 3 or 4 days later (E13-19), when hormone phenotypes were detectable. In the pars tuberalis primordium, TSHbeta appeared from E13. Of these cells 75.5% were labeled when 3H-T had been administered on E10. Label decreased to 40.8% when it had been incorporated on E11 and was negligible (4.2%) when it had been taken up on E12. In the pars distalis, ACTH appeared on E13, TSHbeta, and PRL on E14, LHbeta/FSHbeta on E15 and GH on E16. When examined on E16, all these cell types were labeled for 50-60% if 3H-T had been injected on E12, but this number dropped to about 15% when 3H-T had been given on E13. Only 5-10% of the hormonal cells had taken up label when E14, 15, and 16 were the days of 3H-T administration. The decline in overall labeling index (LI) within both parts of the pituitary was significantly smaller than that in the hormone expressing cells. It is concluded that an outspoken decline in proliferation of the cells destined to become hormone-expressing cell types occurs one to several days before these hormones come to expression. In the pars distalis, this decline occurs at a common time point i.e. between E12 and E13 for each cell type. Pars tuberalis and pars distalis TSHbeta cells show distinct 3H-T labeling profiles, suggesting distinct cell lineage sources for each.

Targeted ablation of gonadotrophs in transgenic mice affects embryonic development of lactotrophs.

Ablation of pituitary gonadotrophs was obtained in transgenic mice expressing diphtheria toxin A (DTA) under control of the -313/+48 bovine glycoprotein hormone alpha-subunit (alphaSU) promoter, previously shown to be active in mouse gonadotrophs but not in thyrotrophs. Development of hormone-producing cell types was assessed on the day of birth by computer-assisted image analysis on paraffin-embedded, immunostained pituitary sections. Six out of 50 transgenic F0 ('founder') mice (3 males and 3 females) showed a nearly complete disappearance of gonadotrophs but not of thyrotrophs. The number of lactotrophs and the relative area occupied by PRL-immunoreactivity were significantly reduced in the gonadotroph-depleted mice. The size of lactotroph clusters was smaller in the absence of gonadotrophs. The number and immunoreactive area of corticotrophs and somatotrophs, on the other hand, were not significantly affected by gonadotroph ablation. Based on the reported evidence that fetal ovaries do not produce steroid hormones as a result of lack of expression of at least three of the steroidogenic enzymes, P450scc, P450c17, and P450arom, the present observations can hardly be explained by a decline in estrogen levels due to gonadotroph ablation. Rather, the present data indicate that gonadotrophs directly stimulate the development of lactotrophs during fetal and early postnatal life, consistent with previous in vitro observations, and/or that gonadotrophs may share a cell-lineage relationship with a subpopulation of lactotrophs.

Presence of gonadotropin-releasing hormone (GnRH) mRNA in Rathke's pouch and effect of the GnRH-antagonist ORG 30276 on lactotroph development in vitro.

Reverse transcription-polymerase chain reaction (RT-PCR) with specific GnRH cDNA primers performed on RNA from Rathke's pouches removed from pregnant rats at day 12 of gestation (e12) generated an amplified DNA fragment of the expected length (357 bp). The fragment hybridized with a labeled GnRH cDNA probe in Southern blotting. DNA sequencing demonstrated identity with the known nucleotide sequence of the corresponding segment of rat GnRH cDNA. To determine whether GnRH mRNA was located in the Rathke's pouch cells or in remnants of surrounding tissue not completely removed during preparation, the pouches were treated with collagenase. Based on light and electron microscopic examination, this treatment disconnected virtually all contaminating tissue, allowing the 'pure' Rathke's pouches to be picked-up separately. Again, RT-PCR generated a DNA fragment of the expected length, the fragment hybridized with the GnRH cDNA probe and showed the nucleotide sequence of the corresponding region of rat GnRH cDNA. In Rathke's pouches established in explant culture on e12, lactotrophs were well developed when examined 9 days later by immunostaining of prolactin in paraffin-embedded sections of the tissue. Computerized image analysis showed prolactin immunoreactivity in 8.0+/-1.1% of the section area. Addition of the potent and long-acting GnRH antagonist ORG 30276 to the crude preparation of Rathke's pouches caused a significant decrease in the relative area staining for prolactin. The latter effect was abolished by concomitant addition of GnRH. In preparations of pure Rathke's pouches (collagenase-treated), ORG 30276 failed to affect the relative area of prolactin immunoreactivity. GnRH mRNA remained expressed in explants of both crude and pure Rathke's pouches until the end of the culture period. It is concluded that the GnRH gene is expressed in Rathke's pouch as early as e12 and that GnRH may be a physiological paracrine/autocrine peptide stimulating the development of lactotrophs. Mesenchymal and/or neural factors may be essential for the latter system to function.