QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 30 March 2004
doi: 10.1242/jcs.01067

This Article Summary Full Text Full Text (PDF) Movie Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Eyll, J. M. Articles by Rousseau, G. G. Search for Related Content PubMed PubMed Citation Articles by van Eyll, J. M. Articles by Rousseau, G. G. Social Bookmarking                         What's this?

Shh-dependent differentiation of intestinal tissue from embryonic pancreas by activin A

Hormone and Metabolic Research Unit, Institute of Cellular Pathology and Université catholique de Louvain, 75 Avenue Hippocrate, B-1200 Brussels, Belgium

View larger version (53K): [in a new window]   Fig. 1. Expression of activin A signaling components in embryonic mouse pancreas and pancreatic explants. Expression of activin A, follistatin and type I activin receptors ALK2 and ALK4 was assessed by RT-PCR, with TBP mRNA as a reference, on RNA extracted from the pancreas of embryos at the stages indicated (A) or from pancreatic explants cultured for the times (days) indicated (B).

View larger version (58K): [in a new window]   Fig. 2. Differentiation of pancreatic explants in culture. (A) Low magnification microscopy shows rounding of the explant, expansion and branching morphogenesis of the epithelium (e) and redistribution of the mesenchyme (m), for the same explant over the 7 days (d) in culture. (B) Hematoxylin and Eosin staining of sections shows that on day 0 the pancreas consists of epithelial precursors surrounded by mesenchymal cells, and that by day 7 epithelial cells have differentiated into ducts (d) and acini (a). The surrounding mesenchyme has invaded the epithelial structures. (C) Double immunostaining for the pancreatic epithelial cell marker Pdx-1 (green) and the proliferation marker Ki67 (red) in d1 (left) and d7 (right) pancreatic explants. Both epithelial cells and mesenchymal cells are proliferating on day 1 and on day 7. Scale bars, 500 µm for A, 100 µm for B and 50 µm for C.

View larger version (93K): [in a new window]   Fig. 3. Ex vivo differentiation of pancreatic precursors. Double immunostaining for CPA (green) and E-cad (red) (A,C) or for insulin (green) and glucagon (red) (B,D) in pancreatic explants on day 0 (A,B) and on day 7 (C,D) in culture. Nuclei are stained blue with Hoechst in B and D. (A) On day 0, only a few of the epithelial cells stained for E-cad express CPA and most of these are located at the periphery (yellow). These CPA positive cells are not polarized and are not organized into acini. (B) On day 0, there are small clusters of glucagon-expressing cells and very few insulin-expressing cells. (C) On day 7, epithelial cells strongly expressing CPA form acini with a characteristic polarization of the cells and a central lumen (inset). Ductal structures that are negative for CPA (arrowheads in C) are also observed. (D) On day 7, insulin-expressing cells, which have appeared during the culture, and glucagon-expressing cells are organized in large endocrine clusters. Scale bars, 100 µm (A-C); 50 µm (B).

View larger version (91K): [in a new window]   Fig. 4. Morphological and histological changes in pancreatic explants treated with activin A. Activin A, added on day 1 of culture, induces the appearance of a cystic structure within 24 hours (arrow, d2). This structure expands during the following days and peristaltic-like activity of surrounding cells appears on day 5 (see Movie 1, http://jcs.biologists.org/supplemental/). On day 7, Hematoxylin and Eosin staining of sections (bottom panel) shows that the cyst lumen is surrounded by a cylindrical epithelium with villi. Pancreatic tissue is still present, but it is aside of the cyst. Scale bars, 500 µm for upper panels and 100 µm for lower panel.

View larger version (74K): [in a new window]   Fig. 5. Pancreatic and intestinal differentiation in explants treated with activin A. (A) Immunostaining for SMA (red), laminin (green) and nuclei (blue) on day 7 in culture in control explants (left panel) or in explants treated with activin A (right panel). In control explants, thin basement membranes, revealed by laminin staining, surround all the epithelial structures. SMA is not detected. In activin A-treated explants, a thick basal lamina separates the cylindrical epithelial cells layer from the smooth muscle layer, which is stained for SMA. This organization is typical of the intestinal wall structure. Scale bars, 100 µm. (B) Expression of FABP mRNAs, with TBP mRNA as a reference, as detected by RT-PCR in untreated explants (upper panel) and in explants treated with activin A (middle panel) or with a mixture of activin A and follistatin (lower panel) for the times indicated starting on day 1 (0 hours). Activin A induces i-Fabp and l-Fabp, two intestinal markers, and triggers the appearance of an intestinal structure (seen here on day 4 of culture). These effects, which do not occur spontaneously with time in culture, are blocked by follistatin. (C) Immunostaining for E-cad (red) and CPA (green) (left panel) or for glucagon (red), insulin (green) and nuclei (blue) (right panel) on day 7 of culture in activin A-treated explants. Exocrine (E-cad and CPA positive), ductal (E-cad positive and CPA negative) and endocrine (glucagon or insulin positive) cells are found, organized as in control explants (see Fig. 3). Note that the epithelium surrounding the lumen is positive for E-cad, but not for CPA. Scale bars, 100 µm.

View larger version (67K): [in a new window]   Fig. 6. Intestinal differentiation in pancreatic explants is induced by activin A, but not by activin B. Treatment of explants with activin A leads to the appearance of an intestinal structure (A) seen here on day 4 in culture (scale bar, 500 µm) and induces the intestinal markers i-Fabp and l-Fabp, detected by RT-PCR on day 7, as well as Shh, detected by semi-quantitative RT-PCR on day 7 (B). None of these effects are seen when activin B is used instead of activin A.

View larger version (71K): [in a new window]   Fig. 7. Intestinal differentiation in pancreatic explants by activin A depends upon an indirect induction of Shh. (A) Shh expression in pancreatic explants, measured by semi-quantitative RT-PCR as a function of time for 7 days, is induced rapidly after a single addition of activin A (middle panels). This induction of Shh, which does not occur spontaneously with time in culture (upper panels), is inhibited by follistatin (lower panels). (B) Cycloheximide prevents induction of Shh and of the Fabps (measured as described in Fig. 6) by activin A in pancreatic explants. (C) Treatment of pancreatic explants with cyclopamine, a Hedgehog signaling inhibitor, prevents the activin A-induced appearance of the intestinal structure. Treatment of explants with Shh mimics the effect of activin A. Note that the appearance of two cystic structures is not specific of Shh treatment, as it can also be observed after activin A treatment. Pictures were taken on day 4, after a single addition of the agents indicated. Scale bar, 500 µm. (D). Shh induces in pancreatic explants the intestinal markers i-Fabp and l-Fabp. RT-PCR were performed on RNA extracted on day 7 of culture.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter