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Imaging Beta Cell Development in Real-Time Using Pancreatic Explants From Mice With Green Fluorescent Protein-Labeled Pancreatic Beta Cells

Posted on: Thursday, 16 June 2005, 03:00 CDT

SUMMARY

We present a convenient method for monitoring pancreatic beta cell development in real-time, through in vitro culture of embryonic pancreatic explants from transgenic mice with a genetic tag for insulin-producing beta cells.

Key words: in vitro culture; pancreatic expiants; mouse; GFP- labeled; real-time.

The mammalian pancreas plays a central role in the regulation of blood glucose levels and intestinal digestion (Kim and MacDonald, 2002; Murtaugh and Melton, 2003). It is a complex organ composed of endocrine and exocrine tissues. The endocrine tissue comprises 1-2% of the cellular mass and produces insulin, glucagon, somatostatin, and pancreatic polypeptide. Insulin and glucagon together function to regulate blood glucose levels in the fed and fasted state. The exocrine pancreas, which represents 98% of the pancreatic mass, is composed of acinar cells that produce digestive enzymes and duct cells that secrete a bicarbonate solution to carry the digestive enzymes to the small intestine.

In the mouse, pancreatic organogenesis begins at the 8-10 somite stage (embryonic day 8 [ES]) and proceeds through a series of well- described stages (Kim and MacDonald, 2002; Murtaugh and Melton, 2003). Although we have a good understanding of pancreatic development at the histological level, we know less regarding the inductive and proliferative signals that control development. Several groups have described conditions for culturing pancreatic rudiments (Gilles et al., 1996; Lammert et al., 2001; Prasadan et al., 2002; Mellitzer et al., 2004; Meneghel-Rozzo et al., 2004; Ogata et al., 2004). They are using these in vitro systems to study pancreatic organogenesis and beta cell formation and to identify factors that control different stages of pancreatic development.

The generation and characterization of a line of transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin 1 promoter (MIP) has been described recently (Hara et al,, 2003). The Iransgene ib only expressed in the insulinproducing beta cells, which show bright green fluorescence when illuminated with blue light. The expression of GFP is evident at E13.5, the earliest stage studied, and the beginning of the secondary transition, a stage of pancreatic organogenesis characterized by a massive wave of cell division and differentiation. Using a modification of previously described methods for cultunng pancreatic buds (Sanvito et al., 1994), we have been able to monitor real-time beta cell formation in buds from MIP- GFP embryos.

The MIP-GFP mice (Hara et al., 2003) and nontransgenic C57BL/6 mice (Charles River Laboratories, Wilmington, DE) were housed in the animal care facility at Vanderbilt University and cared for according to the guidelines of Vanderbilt Institutional Animal Care and Use Committee. The MIP-GFP breeder males (6-12 wk old) heterozygous for the transgene were mated with nonlransgenic females of the same age. The age of the embryos was assumed to be 0.5 d (E0.5) at noon on the d the vaginal plug was found.

Pregnant mice were anesthetized terminally with an intraperitoneal injection of ketamine-xylazine (80:20 mg/kg). Embryos were removed from the uterus, washed in ice-cold Hanks balanced salt solution (HBSS), and placed in ice-cold Roswell Park Memorial Institute (RPMI) 1640 culture media containing 2 mM glutamine (GIBCO-Invitrogen, Gaithersburg, MD) supplemented with 5 mM glucose and 100 U/ml penicillin or streptomycin. The dorsal and ventral pancreatic buds were isolated by dissection using Dumont forceps. Each bud was placed inside a drop of liquid Matrigel (BD Biosciences, San Jose, CA) or collagen gel (100-200 l, prepared as described below) placed on the cover glass lining the 14-mm well of a 35-mm petri dish (MatTek Corporation, Ashland, MA). Pancreatic buds from embryos at E10.5 or younger were isolated attached to a small portion of the gut. The gels containing the buds were allowed to solidify and then covered with 2 ml of RPMI 1640 media (supplemented as described above) and placed in a 37 C incubator gassed with 5% CO2 and 95% O2. The medium was changed every 3-4 d. The collagen gel was prepared using a modification of the method described by Sanvito et al. (1994). In brief, 5 mg of type 1 rat- tail collagen (Sigma, St. Eouis, MO) was dissolved in 3.3 ml of 0.1 N acetic acid. The 1O minimum essential medium (GIBCO-Invitrogen) and 11.76 mg/ml NaHCO^sub 3^ were added, 0.41 ml each. The pH of the solution was adjusted to 7.0 by dropwise addition of 1 N NaOH.

FIG. 1. Green fluorescent protein (GFP) expression in situ in the pancreas of an adult mouse insulin I promoter-GFP transgemc mouse (8 wk). Note the GFP fluorescence located throughout the pancreas. The large bright areas, correspond to groups of pancreatic islets, and the scattered dots correspond to single islets and small groups of beta cells. Bar, 4 mm. Inset: isolated islet. Bar, 20 m. Figure is published in color online at http://inva.allenpress.com/invaonline/ ?request -index.html.

Cultured pancreatic buds were examined daily under a fluorescence dissecting microscope (Zeiss M2Bio, Thornwood, NY). A narrow band- pass filter (500-530 nm) allowed the green fluorescence emitting from the developing pancreatic buds to be detected using excitation with blue light at 488 nm.

The pancreas from adult MIP-GFP mice showed areas of bright green fluorescence clearly visible under fluorescence dissecting microscope because of the expression of GFP in the pancreatic beta cells (Hara et al., 2003) (Fig. 1). The large areas of green fluorescence correspond to groups of pancreatic islets and the small areas to single islets or groups of beta cells. The inset of Fig. 1 shows a single islet, isolated using a modification of the collagenase digestion technique described by Lacy and Kostianovsky (1967). Pancreata were removed from terminally anesthetized mice, placed in ice-cold HBSS, and minced with scissors. Collagenase (3 mg/ ml) (Roche, Indianapolis, IN) was added, and the mixture shaken in a 37 C water bath until the tissue was digested. The mixture was then centrifuged, supernatant removed, and the pellet resuspended in HBSS. Centrifugation and resuspension were repeated several times to remove exocrine tissue. The final pellet was resuspended in RPMI 1640 medium. Islets were handpicked under a stereomicroscope and examined under a fluorescence dissecting microscope (Zeiss M2Bio). All islets isolated from adult MIP-GFP mice showed bright green fluorescence (Fig. 1, inset).

We isolated pancreatic buds at different gestational ages ranging from E9.5 to E15.5 (Fig. 2). As expected from the mating of heterozygous MIP-GFP males with nontransgenic females, only 50% of the embryos from each pregnancy showed beta cell GFP expression. The GFP expression was not observed before E13.5 in vivo. Thus, buds isolated on or after E13.5 showed GFP fluorescence whereas those isolated before E13.5 showed no GFP fluorescence.

Buds cultured in three-dimensional collagen gels survived for at least 14 d and differentiated into endocrine and nonendocrine tissue. However, they did not show significant enlargement in size as happens in vivo. Buds removed after day E11.5 did not visibly increase in size at all, whereas some of the buds removed on or before E11.5 showed up to a twofold enlargement. The GFP signal, once expressed, was very bright and easily detectable using a dissecting microscope. The appearance and increase of the GFP signal during culture was monitored daily. The GFP signal was first evident in cells at the center of the pancreatic bud and then spread progressively throughout the bud (Fig. 3). Unlike freshly isolated buds, the time of appearance of GFP fluorescence in cultured buds varied. For example, some E11.5 buds showed GFP-expressing cells after 2 d in culture whereas others showed expression at 4 d. Regardless of the stage at which the buds were isolated, we never observed GFP expression in the cultured buds before a time corresponding to E13.5.

FIG. 2. Green fluorescent protein (GFP) expression in freshly isolated dorsal pancreatic buds. Note the bnghl fluorescence present at the time of isolation. Kars, 200 m; (top), 500 m (bottom). The left panels show bright-field images of the buds, and the right panels show the GFP fluorescence. Figure is published in color online at http://inva.allenpress.coni/invaonline/ ?request=index.html.

We did not observe GFP-expressing cells in any of the pancreatic buds cultured in Matrigel. The buds appeared to differentiate into exocrine pancreatic-like structures (Fig. 4). Moreover, in contrast to the buds cultured in collagen gel, buds in Matrigel progressively enlarged in size. The buds located at the bottom of the gel branched and spread out over the glass surface, differentiating into structures that appeared morphologically similar to acinar tissue (Fig. 4).

Thus, the MIP-GFP transgene genetically tags the insulin- producing pancreatic beta cells with GFP. The pancreatic buds from MIP-GFP transgenic mice can be cultured in collagen gels and beta cell formation monitored in real-time on the basis of the appearance of GFP fluorescence, which appears to mimic that observed in vivo with the fir\st GFP-labeled cells appearing at E13.5 both in vivo and in vitro. We can now begin to use this system to study the effects of extracellular matrix and culture conditions on beta cell formation, which may lead to a better understanding of the inductive and proliferative signals that control pancreatic beta cell development.

ACKNOWLEDGMENTS

This research was supported by U.S. Public Health Service Research Grants DK-53434 to D. W. P. and DK-20595 and DK-61245 to M. H.

FIG. 3. Green fluorescent protein (GFP) expression in embryonic day 11.5 dorsal pancreatic buds cultured in three-dimensional collagen gels. The GFP fluorescence in buds after 2, 3, and 4 d in culture is shown. The left panels show bright-field images of the buds, and the right panels show GFP fluorescence. Bar, 200 m. Figure is published online at http://inva.allenpress.com/invaonline/ ?request-index.html.

FIG. 4. Growth of embryonic day 11.5 dorsal pancreatic buds cultured in Matrigel. Series of bright-field images show llie growth of a bud located at the bottom of the gel touching the glass surface. The pancreatic epithelium is branching out by day 4 of culture, and structures that appear similar to acini are forming by day 5. Bars, 100 m (top), 200 m (bottom). Figure published in color online at http://mva.allenpress.com/invaonline/?request=index.html.

REFERENCES

Gittes, G. K.; Galante, P. E.; Hanahan, D.; Kutter, W. J.; Debas, H. T. Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors. Development 122:439-447; 1996.

Hara, M.; Wang, X.; Kawamura, T.; Bindokas, V. P.; Dizon, R. E.; Alcoser, S. Y.; Magnuson, M. A.; Bell, G. I. Transgenic mice with green fluorescent protein-labeled pancreatic beta-cells. Am. J. Physiol. Endoerinol. Metab. 284:E177-E183; 2003.

Kim, S. K.; MacDonald, R. J. Signaling and transcriptional control oi pancreatic organogenesis. Curr. Opin. Genet. Dev. 12:540- 547; 2002.

Lacy, P. E.; Kostianovsky, M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16:35-39; 1967.

Lammert, E.; Cleaver, 0.; Melton, D. Induction of pancreatic; differentiation by vessels from blood vessels. Science 294:564-567; 2001.

Melhtzer, G.; Martin, M.; Sidhoum-Jenny, M.; Orvain, C.; Earths, J.; Seymour, P. A.; Sander, M.; Gradwohl, G. Pancreatic islet progenitor cells in neurogenin 3-yellow fluorescent protein knock- add-on mice. MoL Endocrinol. 18:2765-2776; 2004.

Meneghel-Rozzo, T.; Rozzo, A.; Poppi, L.; Rupnik, M. In vivo and in vitro development of mouse pancreatic beta-cells in organotypic slices. Cell Tissue Res. 316:295-303; 2004.

Murtaugh, L. G.; Melton, D. A. Genes, signals, and lineages in pancreas development. Annu. Rev. Cell Dev. Biol. 19:71-89; 2003.

Ogata, T.; Li, L.; Yamada, S.; Yamamoto, Y.; Tanaka, Y.; Takei, I.; Umezawa, K.; Kojima, I. Promotion of beta-cell differentiation by conophylline in fetal and neonatal rat panereas. Diabetes 53:2596- 2602; 2004.

Prasadan, K.; Daume, E.; Preuett, B., et al. Glncagon is required for early insulin-positive differentiation in the developing mouse pancreas. Diabetes 51:3229-3236; 2002.

Sanvito, F.; Herrera, P. L.; Huarte, J.; Nichols, A.; Montesano, R.; Orei, L.; Vassalli, J. D. TGF-beta 1 influences the relative development of the exocrine and endocrine pancreas in vitro. Development 120: 3451-3462; 1994.

SUBHADRA C. GUNAWARDANA, MANAMI HARA, GRAEME I. BELL, W. STEVEN HEAD, MARK A. MAGNUSON, AND DAVID W. PISTON1

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 735 Light Hall, Nashville, Tennessee 37232 (S. C. G., W. S. H., M. A. M., D. W. P.) and Department of Medicine, The University of Chicago, Chicago, Illinois 60637 (M. H., G. I. B)

(Received 13 December 2004; accepted 9 February 2005)

1 To whom correspondence should he addressed al E-mail: dave.piston@vanderhilt.edu

Copyright Society for In Vitro Biology Jan/Feb 2005

Source: In Vitro Cellular & Developmental Biology; Animal

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