Michèle Kedinger

Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium

Endocrine cells of the pancreas and the gastrointestinal tract derive from multipotent endodermal stem cells. We have shown previously that the basic helix– loop–helix (bHLH) transcription factor neurogenin3 (ngn3) is required for the specification of the endocrine lineage in uncommitted progenitors in the developing pancreas. We investigate herein the expression and the function of ngn3 in the control of endocrine cell development in the intestinal and gastric epithelium. Our results indicate that as in the pancreas, gastrointestinal endocrine cells derive from ngn3‐expressing progenitors. Mice homozygous for a null mutation in ngn3 fail to generate any intestinal endocrine cells, and endocrine progenitor cells are lacking. The other main intestinal epithelial cell types differentiate properly. In contrast, in the glandular stomach, the differentiation of the gastrin‐ (G cells) and somatostatin (D cells)‐secreting cells is impaired whereas serotonin‐ (enterochromaffin EC cells), histamine‐ (enterochromaffin‐like ECL cells) and ghrelin (X/A cells)‐expressing cells are still present. Thus, ngn3 is strictly required for endocrine cell fate specification in multipotent intestinal progenitor cells, whereas gastric endocrine development is both ngn3 dependent and independent.

The T3Rα gene encoding a thyroid hormone receptor is essential for post‐natal development and thyroid hormone production

The diverse functions of thyroid hormones are thought to be mediated by two nuclear receptors, T3Rα1 and T3Rβ, encoded by the genes T3Rα and T3Rβ respectively. The T3Rα gene also produces a non‐ligand‐binding protein T3Rα2. The in vivo functions of these receptors are still unclear. We describe here the homozygous inactivation of the T3Rα gene which abrogates the production of both T3Rα1 and T3Rα2 isoforms and that leads to death in mice within 5 weeks after birth. After 2 weeks of life, the homozygous mice become progressively hypothyroidic and exhibit a growth arrest. Small intestine and bones showed a strongly delayed maturation. In contrast to the negative regulatory function of the T3Rβ gene on thyroid hormone production, our data show that the T3Rα gene products are involved in up‐regulation of thyroid hormone production at weaning time. Thus, thyroid hormone production might be balanced through a positive T3Rα and a negative T3Rβ pathway. The abnormal phenotypes observed on the homozygous mutant mice strongly suggest that the T3Rα gene is essential for the transformation of a mother‐dependent pup to an ‘adult’ mouse. These data define crucial in vivo functions for thyroid hormones through a T3Rα pathway during post‐natal development.

The Cdx2 homeobox gene has a tumour suppressor function in the distal colon in addition to a homeotic role during gut development

Background: During development, the homeobox gene Cdx2 exerts a homeotic function, providing the positional information necessary for correct specification of the midgut endoderm. This is illustrated by the non-neoplastic gastric-type heteroplasias present at birth in the pericaecal region of Cdx2+/− mice. Furthermore, intestinal expression of Cdx2 continues throughout life but diminishes in colorectal cancers compared with adjacent normal tissue, suggesting a role in tumorigenesis. Aim: To investigate the consequence of altered Cdx2 expression on colon tumour initiation and/or progression. Methods: Heterozygous Cdx2+/− mice were analysed for spontaneous malignant tumours and for tumour development after treatment with a DNA mutagen, azoxymethane. Results:Cdx2+/− mice did not spontaneously develop malignant tumours. After azoxymethane treatment, the gastric-like heteroplasias in the pericaecal region did not evolve into cancer indicating that they are not precancerous lesions. However, azoxymethane treated Cdx2+/− mice developed tumours specifically in the distal colon 12 weeks after azoxymethane treatment whereas no tumours were found in wild-type littermates at this stage. Histopathological and molecular analyses indicated that these tumours were invasive adenocarcinomas that recapitulated the malignant sequence observed in the majority of sporadic colorectal cancers in human. In addition, we found that the colonic epithelium was less sensitive to radiation induced apoptosis in Cdx2+/− than in wild-type mice. Conclusion: This study provides the first experimental evidence that Cdx2 is a tumour suppressor gene involved in cancer progression in the distal colon. This action in adults is functionally and geographically distinct from its homeotic role during gut development.

Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells

An experimental model was designed to analyze the effect of fetal gut mesenchyme on the cytodifferentiation of crypt cells and of embryonic progenitor cells. The cells used were the rat intestinal crypt cell line, IEC-17, and primary cell cultures prepared form isolated 14-day-old fetal intestinal endoderm (EC). Both cultures prepared from isolated 14-day-old fetal rat intestinal endoderm (EC). Both types of cells were associated with 14-day-old fetal rat gut mesenchyme (Rm) and grafted under the kidney capsule of adult rats. Seventy percent of the Rm/EC and ten percent of the Rm/IEC recombinants, recovered after 9 days, exhibited well-vascularized structures in which the mesenchyme had induced morphogenesis of the cells into a villus epithelium. The four main intestinal epithelial cell types, absorptive, goblet, endocrine, and Paneth cells, were identified using electron microscopy. Biochemical determinations of enzyme activities associated with brush border membranes revealed that alkaline phosphatase, lactase, sucrase, and maltase were expressed in both types of associations. These results were confirmed by immunofluorescence staining using monoclonal antibodies to brush border enzymes. Both enzyme assays and immunocytochemistry showed that the amount of enzymes present in the brush border membrane of Rm/IEC grafts was in general lower than that of the Rm/EC recombinants. The results indicate that fetal rat gut mesenchyme enables morphogenesis and cytodifferentiation of both crypt and embryonic progenitor cells.

Mesenchyme-dependent differentiation of epithelial progenitor cells in the gut

The digestive tract and the gut as a paradigm represents an attractive system for the study of mechanisms involved in the differentiation of two types of progenitor cells: the endodermal cells during embryonic life and the undifferentiated crypt cells during epithelial renewal of the adult intestine. The morphological and functional events that accompany the differentiation processes of progenitor cells into the polarized epithelial cell types characteristic of the intestine appear comparable in both situations (1,2). During organogenesis of the gut, histological observations underlined a close relationship between epithelial cells and their underlying mesenchymal cells (3,4). Developmental biologists have emphasized experimentally the importance of interactions between the endoderm and mesenchyme during organogenesis of the digestive tract. In the adult intestine, gastroenterologists have focused their attention on a specialized mesenchymal cell type (the pericryptal fibroblasts) that displays, like epithelial cells, proliferative activities and migrating properties. The aim of this review is to provide current knowledge on epithelial-mesenchymal interactions during ontogenesis of the digestive tract and also to relate some experiments supporting the view of the perpetuation of epithelial-mesenchymal interactions beyond embryonic life.

Intestinal Epithelial-Mesenchymal Cell Interactions

ABSTRACT: Intestinal morphogenesis, as well as maintenance of the stem cell population and of the steady state between cell proliferation and differentiation, results from controlled cell interactions. There is growing evidence that the mesenchymal cells control epithelial cell behavior via their own expression and induction in the epithelial cells of key regulatory genes. This heterologous cross talk involves basement membrane molecules and paracrine factors. New in vitro/in vivo cellular models allowed us to analyze various mesenchymal cell phenotypes and to show that they exhibit different inductive properties on epithelial cells and that their proliferation and metabolic properties are differentially modulated by cytokines. Finally the epithelial‐mesenchymal unit is controlled by hormonal and exogenous factors.

Involvement of T3Rα- and β-receptor subtypes in mediation of T3 functions during postnatal murine intestinal development ☆ ☆☆ ★

Abstract Background & Aims: Thyroid hormones are implicated in intestinal development. Their effects are mediated by nuclear receptors, which are transcriptional regulators activated upon binding of triiodothyronine. The aim of this study was to define the involvement of the receptor subtypes during intestinal development. Methods: We used strains of knockout mice lacking T3Rα, T3Rβ, or both receptors, encoded by T3Rα and T3Rβ genes. Results: Morphological features and expression of digestive enzymes and of two intestinal regulators, Cdx-1 and Cdx-2, were compared in wild-type and T3Rα, T3Rβ, and T3Rαβ knockout animals. T3Rα−/− mice had abnormal intestinal morphology, assessed by a decrease in the number of epithelial cells along the crypt-villus axis and a decrease in proliferating crypt cells. Expression of Cdx-1 and Cdx-2, and of the digestive enzymes, was down-regulated. These parameters can be partially reversed by T3 injection. A similar (jejunum) or more severe (ileum) phenotype was found in T3Rαβ double mutants. In contrast, no changes occurred in T3Rβ mice. Conclusions: These data describe for the first time a direct effect of TH through the T3Rα-receptor subtypes on postnatal intestinal mucosa maturation. They also suggest that T3Rβ receptors are dispensable but can partially substitute for T3Rα. GASTROENTEROLOGY 1999;116:1367-1378

Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes☆

The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of 5-12-day-old chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones.

Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat

SummaryA modification of Weisers (1973) cell isolation method was used in order to study the developmental pattern of various intestinal enzyme activities in villus and crypt cells of normal rats from 5 days after birth until 8 weeks. Alkaline phosphatase and enterokinase activities were always located in the upper villus zone during postnatal development. Enterokinase activity was higher in the upper villus cells during the third week of life than after this period. Aminopeptidase activity was located in the crypt cells during the first week, its maximum activity remained in this area until the third week. At this time, sucrase activity appeared in the crypt cells, then aminopeptidase and sucrase activities rose to the villus zone during the fourth week. Amylase activity was detected along the entire crypt-villus axis 5 days after birth, reaching maximum activity in crypt cells at the end of the first week and in the upper villus cells after the fourth week. In contrast with the other enzymes studied almost all amylase activity was soluble in the youngest animals whereas at weaning most of the activity appeared in a particulate form in the villus cells. But in the crypt cells the ratio between particulate and soluble form remained unchanged until the adult stage. Various hypotheses are advanced to explain the patterns of evolution of the different enzymes.

Epithelial-mesenchymal interactions in intestinal epithelial differentiation.

The complex morphogenetic events and the concomitant structural and functional differentiation of intestinal progenitor cells are dependent on tissue interactions. Several experimental models of hetero-species or -topic recombinants between epithelial and mesenchymal anlagen are described. They enabled us to elucidate the respective roles of these tissue components in morphogenesis, epithelial differentiation, and hormone-elicited responses. Among the mechanisms of tissue interactions, the possible mediation of permissive and instructive information via the extracellular matrix is postulated. Arguments in favor of this are provided by the observation of compositional changes in matrix molecules during intestinal development and differentiation. On the other hand, in vitro experimental data emphasize the role of actual contacts between epithelial and mesenchymal cell populations and the importance of the mesenchyme for basement membrane formation.