ABSTRACT
In the present study, we have examined the origin and growth pattern of the β cells in pancreatic islets, to determine whether a single progenitor cell gave rise to all the precursors of the islets, or if each of a few progenitor cells is the founder of a different islet, or if each islet is a mixture of cells originating from a pool of progenitor cells. Aggregation mouse chimaeras where the pancreatic β cells derived from each embryo can be identified in the islets on histological sections were analyzed. In two chimaeras, all the islets contained cells from both the aggregated embryo. This clearly demonstrates that each islet resulted from several independent cells. In addition, the β cells derived from either embryo component were in very small clusters in the islets, suggesting that in situ cell division did not account significantly for islet growth.
Introduction
Pancreas evolves as two évaginations from the duodenum, beginning in the mouse at day 9.5 and 10.5 gestation, respectively, and outgrowing independently as ventral and dorsal pancreas that coalesce later at day 11 (Rugh, 1968). Early endocrine cells emigrate from the cell layer that constitutes the developing exocrine gland and separate from it, most probably by a change in the axis of cell division allowing the escape from the ductule lumen of one of the daughter cells (Pictet and Rutter, 1972). It is still unclear whether Langerhans islets grow through accumulation of cells originating from the exocrine pancreas, or by proliferation of primitive islet cells, or by both processes.
Discrimination between these different possibilities is difficult to make directly in vivo since individual cells cannot be distinguished from their neighbours. To overcome this problem, mouse chimaeras could be very useful, since these experimental animals are made from mixtures of cells contributed by two different embryos with different genotypes that are brought together and allowed to develop. If all the β cells of a given pancreatic islet are derived from a single progenitor cell, each islet in mouse chimaeras will be entirely of one genotype or the other. On the contrary, if an islet is derived from multiple progenitor cells, it will be made of a mixture of cells from both genotypes. For instance, using this method, Thompson et al. (1990) have recently shown, by in situ hybridization combined to immunocytochemistry on stomach sections from mouse chimaeras, that gastrin cells share a common stem cell with the other cells of a gastric gland. Chimaerism of pancreas as a whole has been documented (Williams et al. 1988) but, up to date, no evidence for pancreatic islet chimaerism has been presented. A cell marker well adapted to address the question of pancreatic β cell chimaerism is a human C-peptide/proinsulin product present in certain transgenic mouse lines, because this product is readily detectable in single β cells in histological sections of pancreas (Bucchini et al. 1989). The origin of every β cell in any islet of chimaeric animals can thus be determined.
In the transgenic mice carrying the human proinsulin gene, the human gene product was easily demonstrated to be expressed specifically in all the β cells of the pancreatic islets. This was done on tissue sections by immunocytochemistry using specific antibodies directed against the human C-peptide (Bucchini et al. 1989). In the present study, aggregation chimaeras were made between transgenic and nontransgenic mouse embryos, and islets of these animals were analyzed by immunocytochemistry on pancreas sections, using specific antibodies directed against the human C-peptide. Independent of their embryo of origin, the β cells were identified by their staining with an anti-insulin antibody that did not discriminate between human and mouse insulins. The β cells derived from the transgenic embryo component of the chimaeras stained in addition with a specific anti-human C-peptide antibody that did not bind the mouse C-peptides. It was thus possible to determine whether all the β cells of each pancreatic islet derived from a single progenitor cell. In this study, we show that islets result from several independent cells.
Materials and methods
Mice
The two types of embryos used for the construction of aggregation chimaeras were from crosses of (C57BL/6 × DBA/2)F1 and of Tg74 transgenic mice, respectively. The (C57BL/6 ×DBA/2)F1 parental mice were brown, but at the F2 generation, coat pigmentation is brown, black, or grey. Tg74 are transgenic mice on SJL/J background and have a white coat. They carry about 50 copies of an 11 kilobase pair (kb) human DNA fragment including the insulin gene, integrated at a single locus on chromosome 7 (Michalova et al. 1988). Human proinsulin is synthesized exclusively in pancreatic islet cells and can be demonstrated by immunocytochemistry on pancreas sections, using a specific anti-human C-peptide antibody directed against the other cleavage product of proinsulin. In Tg74 mice, all β cells, as identified by an antiinsulin antibody that does not discriminate between human and mouse insulins, stain with the anti-human C-peptide antibody, i.e., synthesize human proinsulin. The human (pro)insulin was separated from mouse (pro)insulins by reverse-phase HPLC and represents half of the total (pro)insulin in the pancreatic islets (Fromont-Racine et al. 1990). Tg74 mice have normal plasma levels of glucose and total (human+mouse) insulin (Bucchini et al. 1986).
Females were superovulated by an intraperitoneal (ip) injection of 5 IU of pregnant mare serum, followed 46h later by ip injection of 5 IU of human chorionic gonadotropin, placed with males, and checked for vaginal plugs the next morning (day 1). 8-cell-stage embryos were collected on day 3 of pregnancy. Aggregates were produced between (C57BL/6 ×DBA/2)F2 and SJL/J-Tg74 embryos. The zona pellucida was removed by treatment with a 0.5 % pronase solution. Pairs of embryos, one of each type, were aggregated in microdrops and cultured overnight. Those embryos that formed chimaeras and developed into single blastocysts were transferred into uterine horns of day 3 pseudopregnant (CSTBL/6 ×CBA/j)1 recipient females (Hogan et al. 1986).
Isolation and analysis of DNA
The mice were screened by a dot-blot assay, using 20 μg DNA prepared from tail fragments as previously described (Bucchini et al. 1986). The probe was an 11 kb Hriidlll genomic DNA fragment radiolabeled with 32P-dATP and 32P-dCTP by random priming. The hybridization was carried out with 10sctsmin−1ml−1 hybridization buffer. The filters were washed (final wash) in 0.1×SSPE (0.36M NaCl, 0.02M NaH2PO4, pH7.4, 0.002M EDTA, pH7.4) at 65°C and exposed to X-ray films.
Determination of human C-peptide
The mice were analyzed for the presence of human C-peptide in the urine by a radioimmunoassay as described (Bucchini et al. 1986). The assays were carried out with 20 μl urine, ‘bilabelled (specific activity 2000 Ci mmol−1) human C-peptide (CEA-orIS) and an antiserum raised against human C-peptide in the goat. The antiserum was kindly provided by M.A. Root and B.H. Frank, the Eli Lilly Company.
Fixation and tissue preparation
Mice were killed at 1.5 or 4.5 months. The pancreases were rapidly removed, and small fragments were fixed in several mixtures such as glutaraldehyde (2.5% for 1h), or paraformaldehyde (4%) plus glutaraldehyde (0.5%) for 2h, or solution of Bouin-Hollande sublimate without acetic acid (for 6–12 h). The tissues were embedded in Paraplast. Sections 3–5 μm thick were cut and mounted on gelatin-coated slides. The sections were deparaffinized and rehydrated before processing for immunocytochemistry.
Antisera
The following primary antisera were used: guinea pig polyclonal anti-bovine insulin N° 8309 (Novo, Copenhagen, Denmark) diluted 1:1000; rat monoclonal anti-human C-peptide N° GN-ID4 (kindly provided by Dr O. Madsen, Hagedorn Research Laboratory, Gentbfte, Denmark) diluted 1:2000; rabbit polyclonal anti-C-terminal porcine glucagon N° GAN-8 (kindly provided by Dr A. Kervran, CNRS/ INSERM, Unité 264, Montpellier, France) diluted 1/5000; and rabbit polyclonal anti-somatostatin N° 19608 (kindly provided by Dr M.P. Dubois, INRA, Nouzilly, France) diluted 1:2000. Peroxidase-labelled goat anti-guinea pig (or anti-rat) immunoglobulin G (IgG) was purchased from Nordic Immunology (Tilburg, The Netherlands), and peroxidase-labelled goat anti-rabbit IgG from Dako-Immunoglobu-lins (Copenhagen, Denmark).
Immunocytochemistry
The sections were processed at room temperature in a humid chamber by the indirect immunoperoxidase method of Nakane and Pierce (1966), with some modifications (Leduque et al. 1987a). Briefly, sections were sequentially incubated with the following solutions for the times indicated: 1:40 non-immune serum for 15min; primary antiserum for 1h; 1:200 peroxidase-labelled goat anti-species IgG for 30min; and 0.04% diaminobenzidine hydrochloride/0.03% hydrogen peroxide for 30s-lmin, as suggested by Graham and Kamovsky (1966). Sections were dehydrated, cleared and mounted in DPX.
Immunocytochemical specificity tests
The specificity of the rat monoclonal anti-human C-peptide N° GN-ID4 has been checked by a solution phase equilibrium antibody-binding assay (Madsen et al. 1985), and immunofluorescence (Bucchini et al. 1989; Fromont-Racine et al. 1990). The antibody GN-ID4 reacts with both human proinsulin and human C-peptide and is species-specific (Madsen et al. 1985). It stains pancreatic β cells of transgenic mice carrying the human insulin gene, but does not react with nontransgenic mouse pancreas. All the cells positive for insulin also stain for human C-peptide; no glucagon or somatostatin cells give an immunoreaction for human C-peptide (Bucchini et al. 1986). The RIA and immunocytochemical specificities of the antiinsulin, anti-glucagon and anti-somatostatin have been described elsewhere (Leduque et al. 1985; 1986; 19876; 1989).
In the present study, two types of control procedures were used to substantiate specific immunocytochemical staining. Staining was absent when non-immune rat serum replaced the anti-human C-peptide, and when either the anti-human C-peptide, or the goat anti-rat IgG conjugated to peroxidase, or hydrogen peroxide was omitted from the immunocytochemical procedure. This demonstrates the absence of electrostatic and hydrophobic binding of immunocytochemical reagents to tissues (Leduque et al. 1987a). In addition, no cells immunoreactive to anti-human C-peptide antibody were detected in control nontransgenic mouse pancreas.
Results
Obtention of aggregation chimaeras
56 aggregation embryos were obtained and transferred into foster mothers. Five foster females gave rise to 18 newborn (Table 1). Six (of which five were males) had a typical chimaeric coat. The (C57BL/6×DBA/2)F2 mouse coat component was brown, black, or grey. Tg74 component was white, since Tg74 have an albino SJL/J genetic background. Red and black mosaic iris was also found in one chimaera. The Tg74 mouse contribution was further documented in these six chimaeras by two criteria. First, the human proinsulin transgene was demonstrated by a tail DNA dot-blot assay, and second, human C-peptide was found in urine, using a radioimmunoassay. The latter assay indicated that the transgenic embryo component participated in the pancreatic β cells. Six other experimental mice had no white coat component. Among them, one (chimaera 16) had the transgenic human DNA fragment in the DNA prepared from tails and human C-peptide in urine. Since it had the coat from one embryo and the human transgene from the other, it was considered as a true chimaera. The six experimental albino mice were not analyzed further by other markers for demonstrating the presence of any (C57BL/6×DBA/2)F2 component, and were discarded. In total, 7 chimaeric mice were identified, of which one did not have coat colour chimaerism (Table 1). Four of them were used for immunocytochemistry analysis of the pancreas.
Chimaera 11
Staining with anti-human C-peptide antibody allowed identification of the ft cells derived from the transgenic embryo. Human C-peptide was found in all the β cells of all the islets examined (Fig. 1A,B; Table 2). Presence of endocrine β cells was occasionally observed in the duct epithelium by systematic screening of serial sections. These β cells contained also human C-peptide (Fig. 2A,B).
Islet cells contributed by the transgenic component in the mouse chimaeras. Pairs of serial sections from mouse chimaeras were processed for immunochemistry of insulin (left) and human C-peptide (right). (A,B) Chimaera 11. Virtually all the β cells expressed human C-peptide. (C,D) Chimaera 12. Only a few β cells were found to contain human C-peptide (arrows). (E,F) Chimaera 16. None of the β cells contained detectable human C-peptide. (A,B ×170; C,D ×280; E,F ×240).
Islet cells contributed by the transgenic component in the mouse chimaeras. Pairs of serial sections from mouse chimaeras were processed for immunochemistry of insulin (left) and human C-peptide (right). (A,B) Chimaera 11. Virtually all the β cells expressed human C-peptide. (C,D) Chimaera 12. Only a few β cells were found to contain human C-peptide (arrows). (E,F) Chimaera 16. None of the β cells contained detectable human C-peptide. (A,B ×170; C,D ×280; E,F ×240).
Presence of immunoreactive cells within the duct epithelium. Same legend as in Fig. 1. (A,B) Chimaera 11. The few cells in the duct epithelium immunoreactive for insulin also contained human C-peptide, i.e., they were of transgenic origin. (C,D) Chimaera 12. The few cells immunoreactive for insulin that were detected in the duct epithelium were not marked by the anti-human C-peptide antibody. (A,B ×250; C,D ×190).
Presence of immunoreactive cells within the duct epithelium. Same legend as in Fig. 1. (A,B) Chimaera 11. The few cells in the duct epithelium immunoreactive for insulin also contained human C-peptide, i.e., they were of transgenic origin. (C,D) Chimaera 12. The few cells immunoreactive for insulin that were detected in the duct epithelium were not marked by the anti-human C-peptide antibody. (A,B ×250; C,D ×190).
Chimaeras 12 and 13
Human C-peptide immunoreactivity was found in all the pancreatic islets. Each of the islets examined (238 out of 241) was a mosaic of β cells, which contained human C-peptide, and β cells, which did not (Fig. 1C,D; Table 2). In all the islets, the cells derived from the transgenic embryo were only a minority. These cells were associated in small clusters scattered within the islets (Fig. ID). Some β cells were detected in the duct epithelium (Fig. 2C), but they were not immunoreactive for human C-peptide (Fig. 2D).
Chimaera 16
We have not been able to detect any human C-peptide immunoreactivity in any β cells (Fig. 1E,F; Table 2). Some β cells were detected in the duct epithelium, and human C-peptide was never observed in these cells (not illustrated).
Serial sections of pancreases were stained with antiinsulin, anti-glucagon and anti-somatostatin antibodies in order to discriminate the β, α, and δ cells in the islets, respectively (Fig. 3).
Islet cells producing each of the pancreatic hormones. Pairs of serial sections from mouse chimaera 11 were processed for immunochemistry of either insulin (A) and glucagon (B), or of insulin (C) and somatostatin (D). The β cells made up the majority of the center of the islets, while the glucagon cells and the somatostatin cells were located around the periphery; furthermore, they were non-overlapping (arrows) (A,B ×200; C,D ×370).
Islet cells producing each of the pancreatic hormones. Pairs of serial sections from mouse chimaera 11 were processed for immunochemistry of either insulin (A) and glucagon (B), or of insulin (C) and somatostatin (D). The β cells made up the majority of the center of the islets, while the glucagon cells and the somatostatin cells were located around the periphery; furthermore, they were non-overlapping (arrows) (A,B ×200; C,D ×370).
Discussion
In the present study, we have examined the origin and the growth of the pancreatic islets, not to determine the developmental lineage of endocrine islet cells (see review in Le Douarin, 1988), but to establish whether (1) a single progenitor cell gave rise to all the precursors of the islets, or (2) if each of a few progenitor cells is the founder of a different islet, or (3) if each islet is a mixture of cells originating from a pool of progenitor cells (Fig. 4). According to the first two hypotheses, one expects to find in mouse chimaeras homogenous islets with only a single cell type, originating from one or the other embryo. According to the third one, individual islets would contain cells from both embryos. In two out of the four chimaeras examined (chimaeras 12 and 13), all the islets contained cells from both aggregated embryos, supporting the third hypothesis (Fig. 4C). This clearly demonstrates that each islet resulted from several independent cells. Because of this conclusion, the homogenous cell population observed in the chimaeras 11 and 16 should be ascribed to the absence or very reduced levels of mosaicism in the endocrine pancreas of these particular chimaeras.
Schematic representation of the possible expansion of the cells from which the islet fi cells derive. (A) A single progenitor cell gives rise to all the precursors of all the islets. (B) There are several independent progenitor cells for the β cells, but a single progenitor gives rise to all the β cells in an islet. (C) There are several progenitor cells, and mixed progenies make up each islet. The results obtained with chimaeras 12 and 13 are relevant to the latter hypothesis.
Schematic representation of the possible expansion of the cells from which the islet fi cells derive. (A) A single progenitor cell gives rise to all the precursors of all the islets. (B) There are several independent progenitor cells for the β cells, but a single progenitor gives rise to all the β cells in an islet. (C) There are several progenitor cells, and mixed progenies make up each islet. The results obtained with chimaeras 12 and 13 are relevant to the latter hypothesis.
In the two animals with chimaeric pancreatic islets (chimaeras 12 and 13), endocrine fi cells derived from the transgenic embryo component were in small clusters of very few cells in any given islet (Fig. 1C,D). This suggests that the islets were made of many cell clones resulting either from 1–4 cell divisions of individual cells, or from recruitment of clustered small progenies of endocrine cells, i.e., smaller islets. Our present results exclude the monoclonal origin of an islet, and also suggest that islet cell proliferation does not significantly account for islet growth. The number of pancreatic endocrine cells increases strikingly between day 13 and day 18 of fetal development, while the mitotic activity is very modest in this cell population. In contrast, there are more DNA-synthesizing cells around the growing fetal islets than in the islets themselves (Hellerstrom and Swenne, 1985). Altogether, these observations suggest that fetal islet growth mainly results from recruitment of precursors located in the vicinity of the islets. In contrast, large patches of exocrine pancreatic cells with a single genotype have been reported in mouse chimaeras (Dewey and Mintz, 1978), which implies a quite different pattern of growth, i.e., the in situ proliferation of the exocrine cells.
In the four mice examined, insulin-containing cells were found in the duct epithelium (Fig. 2). This suggests that islet cells are originating from the epithelial layer of the pancreatic diverticulum. A very few pancreatic endocrine cells are found at 20–21 somites (day 9.5 of gestation) in the mouse (Wessells and Evans, 1968). At that time, they are often found in the region lining the ductules, between the exocrine cell layer and the basal lamina, which separates the exocrine cell compartment from the surrounding mesenchymal cells. It seems that they arise by division of epithelial cells by a change in the axis of cell division allowing the escape from the ductule lumen of one of the daughter cells (Pictet and Rutter, 1972). In the following days, the first islets are seen in this space, and it is only later in fetal development (day 15) that typical islets with no obvious connexion with the exocrine tissue are found. Insulin-containing cells have been already observed in vivo in the duct epithelium of newborn rats (Leduque et al. 1987a), and upon culture (Leduque et al. 1989; Teitelman and Lee, 1987). There are also two experimental conditions where similar observations were made. Destruction of β cells after streptozotocin treatment of 1-day-old rats is followed by signs of repair characterized by the appearance of insulin-positive cells in the acinar parenchyma and within the duct epithelium (Leduque et al. 1987a), and budding of islets from ducts was a prominent feature (Cantenys et al. 1981; Dutrillaux et al. 1982). More recently, it was shown that cellophane wrapping of the hamster pancreas led to endocrine cell differentiation and islet formation by cell budding from proliferating ductular epithelium (Rosenberg and Vinik, 1989). Both phenomenons might mimic normal islet ontogeny. Whether these putative islet precursor cells might contribute to postnatal islet growth remains to be demonstrated.
ACKNOWLEDGEMENTS
We thank O. Madsen for the anti-human C-peptide monoclonal antibody, M. A. Root and B. H. Frank for the anti-human C-peptide antiserum. A. Paldi was a recipient of a fellowship from the Institut National de la Santé et de la Recherche Médicale. This work was supported by grants from the Fondation pour la Recherche Médicale, the Ligue Nationale contre le Cancer, the Département de Biologie Humaine, Université Claude Bernard Lyon I (985-387), and the Centre National de la Recherche Scientifique.