Cytokine gene expression during postnatal small intestinal development: regulation by glucocorticoids.

BACKGROUND: In the intestinal mucosa, numerous cytokines produced by the epithelium, fibroblasts, and immune cells were shown to affect epithelial differentiation and proliferation through epithelial-mesenchymal and epithelial-immune cell interactions. To date, the importance of cytokines in postnatal development of the rat small intestine has not been established. AIM: To investigate the developmental changes in expression of mucosal cytokines in the postnatal maturation of the rat small intestinal epithelium and their regulation by glucocorticoids (GC). METHODS: Mucosal maturation was assessed by the onset of sucrase-isomaltase (SI) mRNA, analysed by in situ hybridisation. The amount of transforming growth factor beta1 (TGF-beta1), beta2 (TGF-beta2), tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and TGF-alpha was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in mucosal extracts from weaning (14-21 days old) and adult rats, or one day after an injection of hydrocortisone (HC) in 11 day old rats. Similarly, expression of cytokines and the regulatory effect of GC were studied on cultured subepithelial myofibroblasts cloned from postnatal jejunum and ileum cultured in the absence or presence of dexamethasone (DX). RESULTS: TGF-beta1, TGF-beta2, and IL-1beta decreased during the third week of life while levels of TNF-alpha increased and TGF-alpha remained constant. In parallel, SI transcripts increased and showed a progressive accumulation in the apical part of the enterocytes first localised at the base of the villi from 18 days onwards. Interestingly, precocious induction of SI mRNA by HC paralleled the decrease in expression of TGF-beta isoforms and of IL-1beta. All cytokines were expressed in the myofibroblast cell lines. In addition, the results showed that TNF-alpha was differentially expressed in basal culture conditions and after DX stimulation in jejunal and ileal myofibroblasts. DX decreased IL-1beta but not the TGF-beta isoforms, similar to that in vivo. CONCLUSIONS: This study shows that mucosal cytokines are developmentally regulated and that GC are potentially involved in this regulation in parallel with maturation of the gut mucosa at weaning.

Expression of the c-ets1 gene in the hypothalamus and pituitary during rat development.

The Ets gene family codes for transcription factors containing a conserved DNA binding domain: the Ets-binding domain. The proto-oncogene c-ets1 is highly expressed in lymphoid organs and in developing mesodermal-originating structures. We studied c-ets1 gene expression in the developing rat hypothalamo-hypophyseal system, using in situ hybridization on paraformaldehyde-fixed frozen sections. At embryonic day 12 (E12) and E13, cells synthesizing c-ets1mRNA are found in the neural tube where they form small, heavily labeled strand-like and punctate structures; positive mesenchymatous cells, corresponding to the surface capillary network, surround the brain and hypophysis. C-ets1mRNA is synthesized from E14 in the neural pituitary and E15 in the adenohypophysis, during angiogenesis; no c-ets1mRNA is detected in the avascular intermediate pituitary at any stage. Strand-like c-ets1mRNA labeling is intense from E14 to E21 in the diencephalon. This labeling is also detected during perinatal stages in the hypothalamic magnocellular nuclei, one of the most richly vascularized brain areas. In the rat hypothalamo-hypophyseal system, c-ets1 gene expression is maximal during fetal and perinatal stages and progressively decreases thereafter until adulthood. The spatio-temporal correlation observed between c-ets1 gene expression and blood vessel formation in the rat hypothalamus and pituitary suggests a role for c-ets1 in angiogenesis in this system.

Expression of neural cell adhesion molecules, NCAMs, and their polysialylated forms, PSA-NCAMs, in the developing rat pituitary gland.

Neural cell adhesion molecules (NCAMs) can undergo post-translational modifications, such as the addition of polysialic acid chains, thus generating PSA-NCAMs, which are expressed mainly during development. Since polysialylation considerably modifies NCAM adhesivity, expression of NCAMs and PSA-NCAMs has been investigated in the developing hypophysis by immunohistochemistry. At embryonic day 13 (E13), an antibody against NCAM outlined all cellular profiles in the entire Rathke's pouch; this labelling persisted until adulthood. NCAM expression increased in all lobes during development and concerned all pituitary cell types. In contrast, at E13, PSA-NCAMs were only detected in the neural lobe, solely constituted of pituicytes at this stage, and the tuberal lobe, the only lobe expressing hormonal mRNA at the same stage. PSA-NCAMs expression increased in the neural lobe at E17 with the arrival of the neurosecretory fibres and persisted into adulthood. In the anterior lobe, PSA-NCAMs appeared at E15 where their distribution was similar to that of the differentiating corticotrophic cells; at subsequent stages, their expression extended to the whole anterior lobe. Only two cell types, corticotrophic and somatotrophic cells, remained labelled in the adult gland. In the intermediate lobe, melanotrophic cells never expressed PSA-NCAMs but these were expressed on folliculo-stellate cells at birth, preceding the onset of innervation. These results suggest that NCAMs and PSA-NCAMs play a role in pituitary histogenesis, cell differentiation and neurointermediate lobe innervation.

Ontogeny of prodynorphin gene expression in the rat hypothalamus.

Opioid peptides, deriving from prodynorphin, proenkephalin and proopiomelanocortin genes, have been shown to modulate brain development. Prodynorphin gene expression was studied here by in situ hybridization in the developing rat hypothalamus using oligodeoxynucleotide probes. Prodynorphin mRNA-synthetizing cells were observed in the ventromedial hypothalamic nucleus, the supraoptic and the paraventricular nuclei from embryonic days 16, 18 and 21, respectively. We detected no transient expression of prodynorphin gene in the rat hypothalamus. Prodynorphin mRNA-containing cells were also observed prenatally in the striatum, the cortex, the hippocampus and the amygdala. When compared with data from the literature, our results suggest that translation may immediately follow transcription of prodynorphin gene in the supraoptic nucleus. The presence of prodynorphin mRNA in the developing rat hypothalamus also raises the possibility of an involvement of prodynorphin-derived peptides in developmental processes and/or in the maturation of adult neural regulations.

Ontogeny of proenkephalin gene expression in the rat hypothalamus.

In the rat hypothalamus, proenkephalin (PE) mRNA synthetizing cells were detected by in situ hybridization, using synthetic oligodeoxy-nucleotides, from embryonic day 14 (E14) in the presumptive anterior hypothalamic area (AHA) and preoptic part of the bed nucleus of the stria terminalis (BST), and from E18 in the developing median preoptic area, perifornical area, suprachiasmatic nucleus, dorsomedial and ventromedial hypothalamic nuclei. In the paraventricular nucleus, cells expressed PE gene in the late prenatal stages; both parvo- and magnocellular neurons synthetized PEmRNA in the early postnatal stages. Cells expressing PE gene were observed after birth in the lateral preoptic area, lateral hypothalamus, medial and lateral parts of the BST. PEmRNA was also found from E14 in the striatum, from E18 in the central and medial amygdaloid nuclei, the medial group of the thalamic nuclei, and postnatally in a second more anterior structure of the thalamus. In the hypothalamus, a clear similarity was observed between adult and developmental distributions of PE gene expressing cells. The early onset of PE gene expression in the developing rat diencephalon suggests an involvement of PE in developmental processes, such as cell proliferation and differentiation; the presence of PE during the perinatal period may also indicate the appearance of adult neural regulations.