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EXOGENOUS ACTIVIN INCREASES THE PROPORTION OF INSULIN CELLS IN THE DEVELOPING CHICK PANCREAS IN CULTURE
Cell Biology International 2002, Vol. 26, No. 12, 1057–1064 doi:10.1006/cbir.2002.0965, available online at http://www.idealibrary.com on
EXOGENOUS ACTIVIN INCREASES THE PROPORTION OF INSULIN CELLS IN THE DEVELOPING CHICK PANCREAS IN CULTURE CLEM PENNY and BEVERLEY KRAMER* Embryonic Differentiation and Development Research Programme, School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg, 2193 South Africa Received 7 February 2002; accepted 21 August 2002
As activin is believed to be a key signalling factor during early pancreatic development, its influence on the proliferation and/or determination of insulin cells in the developing chick dorsal pancreatic bud was investigated. Dorsal pancreatic buds of 5-day-old chick embryos were explanted on to Matrigel and cultured in serum-free medium (Ham’s F12.ITS), to which 1 or 10 ng/ml activin was added. After 7 days in culture, the explants were processed for immunocytochemistry and the insulin-positive cells were scored and expressed as a proportion of the sum of insulin and glucagon cells. When compared to the control cultures (Hams F12.ITS alone), activin treatment resulted in respective increases in the proportion of insulin cells of 1.6 and 1.9 fold. It is suggested that activin treatment favours differentiation of the insulin cell 2002 Elsevier Science Ltd. All rights reserved. pathway relative to glucagon cells. K: chick pancreas; activin; insulin cells; diabetes mellitus.
INTRODUCTION In humans, the continued production of insulin by the islets of Langerhans of the pancreas is important for maintaining glucose homeostasis. In those individuals in which insulin secretion ceases, as in Type 1 Diabetes, the key to ensuring long-term health would be to provide therapy that would maintain glucose values within normal limits. Ideal treatments would be the implantation of viable, functionally competent islets or stimulation of endocrine progenitor cells to produce insulin cells. The identification and use of factors involved in the differentiation and proliferation of insulin cells could ultimately facilitate the provision of a plentiful supply of insulin cells for individuals with insulin-dependent diabetes. In this regard, we have previously assessed the influence of a number of growth factors and vitamins on insulin cell differentiation using the chick pancreas as a model system (Penny and Kramer, 2000; Mngomezulu and Kramer, 2000; Kramer and Penny, 2001a; Kramer and Penny, 2001b). *To whom correspondence should be addressed: Tel.: 27 11 717-2405; Fax: 27 11 717-2422; E-mail: [email protected] 1065–6995/02/$-see front matter
The activins, members of the transforming growth factor- (TGF-) supergene family, influence many diverse developmental and cellular processes, including the differentiation of erythroid cell lines (Eto et al., 1987), mesoderm induction in Xenopus laevis embryos (Asashima et al., 1990; Green and Smith, 1990; Smith et al., 1990), cartilage formation (Chai et al., 1994), cardiac morphogenesis (Mangiacapra et al., 1995), nerve cell survival (Hashimoto et al., 1990; Schubert et al., 1990) and tooth development Ferguson et al., 1998). During embryogenesis of the chick, activin signalling plays a key role in pancreatic development. The anterior foregut, which lies close to the notochord, receives and responds to activin signals which originate from the notochord. The effect of this inductive signal is to inhibit the expression of the Sonic Hedgehog gene (SHH) within the specific region of the anterior foregut which gives rise to the pancreas (Kim et al., 1997; Hebrok et al., 1998). The down-regulation of SHH subsequently allows for the expression of pancreatic markers within the endoderm, these being insulin and Pdx1 (the pancreatic and duodenal homeobox gene) (Hebrok 2002 Elsevier Science Ltd. All rights reserved.
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et al., 1998), which are required for the outgrowth of the dorsal pancreatic bud (Ohlsson et al., 1993; Jonsson et al., 1994; Guz et al., 1995; Ahlgren et al., 1997; Kim et al., 1997). Another important class of genes involved in pancreatic development which may be regulated by activin signalling is the paired box family (Pax). Pax 4 is essential for the development of insulin and somatostatin cells and the development of mature islets (Sosa-Pineda et al., 1997), while Pax 6 is required for glucagon cell development (St-Onge et al., 1997). Recently, the treatment of a number of -cell lines with activin has been shown to upregulate Pax 4 gene expression in a dose dependent manner (Ueda, 2000), thus suggesting that activin may influence -cell differentiation. As the identity and isolation of endocrine precursor cells has still not been clearly established, we have attempted to stimulate the production of insulin cells from developing chick dorsal pancreatic buds by culturing them some two days after the evagination of the pancreatic bud from the foregut. At this time, few differentiated insulin and glucagon cells are identifiable (Andrew, 1984). However, the inclusion of endocrine progenitor cells is ensured, as the entire endodermal component of the pancreatic bud is explanted. It is well known that permissive signals from both the pancreatic endoderm and mesenchyme are necessary for pancreatic development in mice (Golosow and Grobstein, 1962; Wessels and Cohen, 1967; Spooner et al., 1970) and chick (Dieterlen-Lievre, 1970). In relation to activin signalling, the mRNA for its specific inhibitor follistatin is present in the pancreatic mesenchyme early on in the mouse embryonic pancreas and is later down-regulated (Miralles et al., 1998; Maldonado et al., 2000). Thus in the present study, in order to negate the possible influence of follistatin and other mesenchymeassociated factors on development, and to determine the influence of added factors on the pancreatic endoderm during culture, almost all the mesenchyme was removed from the pancreatic explants. The extracellular matrix (ECM) provides additional signals that influence cellular function during development. Its composition and three dimensional organization affect cell growth, survival and differentiation (Lukashev and Werb, 1998), and the influence of ECM proteins on the in vitro growth and differentiation of the pancreas has been found to be an important factor. Andrew et al. (1994), for example, showed that a collagen gel substrate provided only minimal support for chick insulin cell development, while adequately sustaining that of the other pancreatic endocrine cells.
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Matrigel, a basement membrane extract containing laminin and a number of growth factors, has proven most useful in promoting differentiation in culture of a variety of cells (Reid and Jefferson, 1984), including pancreatic endocrine cell types (Muschel et al., 1986; Hayek et al., 1989; Beattie et al., 1991). More recently, the inclusion of laminin alone has been shown to support differentiation of the foetal mouse pancreas (Jiang et al., 1999; Crisera et al., 2000). The present study assessed the effects of activin at two concentrations, 1 and 10 ng/ml, on the proportion of insulin cells in the chick dorsal pancreatic bud explanted on to Matrigel. As activin was dissolved in trifluoroacetic acid (TFA) and acetonitryl (ACN), control cultures were included with identical volumes of these diluents. However, in view of the reported reduction in cell proliferation caused by increased concentrations of TFAsolubilized growth factors (above 10 9 M), activin was converted from a TFA salt to a biologically compatible HCl salt (Cornish et al., 1999) for use at the higher concentration. In comparing treatments, proportions of insulin cells and not absolute numbers were used. This relates to the possibility that the numbers of progenitor cells and differentiating insulin cells at the time of culture may not be the same in all of the dorsal pancreatic buds used. This proportion is significant in providing an index of proliferation and/or differentiation of both developing insulin and glucagon cells, as these two cell types are believed to derive from a common progenitor (Alpert et al., 1988; Teitelman et al., 1993; Upchurch et al., 1994; Jackerott et al., 1996). Furthermore, the use of proportions makes our findings directly comparable with other results in which changes in the proportion of insulin cells reflect the effects of a variety of nutrients, hormones and growth factors (Rawdon and Andrew, 1997; Rawdon and Andrew, 1998; Mngomezulu and Kramer, 2000; Penny and Kramer, 2000; Kramer and Penny, 2001a; Kramer and Penny, 2001b). MATERIALS AND METHODS Fertile chick Lohman Brown eggs were incubated at 37C for 5 days in a humidified atmosphere. The dorsal pancreatic bud was removed from the embryos at stages 25–26 (Hamburger and Hamilton, 1951) and incubated in 0.04% collagenase type VIII (Sigma Chemical Co., St Louis, U.S.A.) in Tyrode’s solution at 20C for
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20 min. The enveloping pancreatic mesoderm was then manually dissected off. In order to preserve the integrity of the endodermal component, a small amount of residual mesoderm was left in the interstices of the developing lobules. Explants were cultured on Matrigel (Collaborative Research, Bedford, MA, U.S.A.), which is a solubilized basement membrane extract containing various growth factors. It is buffered with sodium bicarbonate and contains gentamycin at 10 g/ml. Three grafts per well were placed on 175 g of Matrigel in 10 or 12 mm diameter wells of Nunclon (Roskilde, Denmark) culture plates. Explants were allowed to adhere to the Matrigel for 30 min and then covered with 1 ml of culture medium. The serum-free medium used was based on Ham’s F12 culture medium, to which 5 g/ml insulin, 5 g/ml transferrin and 10 10 M selenium were added (Ham’s F12.ITS, Highveld Biological, South Africa). Bovine recombinant activin (subtype A) (Research Diagnostics, Flanders, NJ, U.S.A.) was reconstituted in 40% ACN (SMM Chemicals, South Africa) and 0.1% TFA (Merck, Darmstadt, Germany) as a stock solution of 1 mg/ml. Activin at concentrations of 1 ng/ml or 10 ng/ml was added to the Ham’s F12.ITS medium of experimental explants. In order to control for possible diluent effects, control explants were cultured in Ham’s F12.ITS containing 1 l/ml or 10 l/ml of the diluents, ACN (40%) and TFA (0.1%). Explants were also cultured in Ham’s F12.ITS alone. In addition, activin was converted from the TFA salt form to the HCl salt form (Cornish et al., 1999) for use at 10 ng/ml. Activin at a concentration of 1 mg/ml was incubated in 50 l of a 3 mM HCl solution and allowed to stand for 1 h at room temperature, prior to freeze-drying at 60C and 10 2 Torr (1.33 Pa) (Virtis Freezemobile 12, Gardiner, NY, U.S.A.). Before use, activin was redissolved in sterile distilled water (SABAX) with sonication, cooled on ice for 15 s and stored at 4C. On the day of culture, it was diluted in Ham’s F12.ITS. Pancreatic buds were incubated for 7 days at 37C in 5% CO2 in a humidified atmosphere and the medium changed every third day. On retrieval, the explants were quenched in iso-pentane (BDH, Poole, U.K.), cooled in liquid nitrogen and freezedried at 140C and 10 2 Torr. The dried specimens were fixed for 3 h at 60C in vapour generated from re-crystallized parabenzoquinone (Merck) and embedded under vacuum in a 1:1 mixture of Epon and Araldite. The resin blocks were cured for 48 h at 60C. One micron serial sections were cut through the explant and sections mounted on
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polytetrafluoroethylene-coated slides (Rawdon, 1978). An indirect immuno-enzyme procedure was applied to adjacent serial sections to reveal insulin and glucagon cells. Each primary antiserum was applied to seven adjacent sections, spaced so that no cell could be counted more than once. Insulincontaining cells were detected with guinea pig anti-insulin antibody (1:8000) (a gift from L. Orci, Geneva) and glucagon containing cells with rabbit anti-glucagon antibody (1:800) (Milab, Malmo, Sweden) on adjacent serial sections. Incubation with the primary antiserum was carried out for 22 h at 4C. Peroxidase conjugated rabbit anti-guinea pig antiserum at a concentration of 1:20 (Dako, Glostrup, Denmark) was applied to sections stained with anti-insulin antiserum, while swine anti-rabbit peroxidase (Dako) at the same concentration was used following the anti-glucagon antiserum. The site of the antigen was revealed by treatment with a diaminobenzidine (DAB) solution containing hydrogen peroxide (Perhydrol) (Merck). Immunocytochemical control procedures were carried out on sections adjacent to those that contained endocrine cells. These procedures included substitution of the primary antiserum by the primary antiserum pre-absorbed for 24 h at 4C with its own antigen (20 g/ml insulin natural human, Peninsula Labs, California, U.S.A. or 40 g/ml glucagon, natural bovine/porcine, Eurodiagnostica, Malmo, Sweden), diluent alone or non-immune serum of the relevant species. A control section from the pancreas of a hatching chick known to be positive for the antigen concerned was included in each staining run. Counts of insulin and glucagon cells were thus made in 14 serial sections (insulin, n=7; glucagon, n=7) of each explant. Only those cells in which a nucleus was visible or those portions of cells as large or larger than a nucleus were counted. The number of insulin cells was expressed as a proportion (percentage) of the total insulin and glucagon cell count. Proportions of these cell counts under the different conditions of culture were compared by the 2 test. Differences were regarded as significant when P values (one tailed) were less than 0.05. Absolute numbers of insulin cells were not analysed; the counts recorded should not be regarded as such. RESULTS The cultures consisted of solid epithelial masses, sometimes containing epithelial-lined cysts. The majority of the endocrine cells (insulin and
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Fig. 1. Preabsorption with the respective antigen eliminated staining with the relevant antibody. Adjacent sections of a control explant cultured in HamsF12.ITS treated with (1a) a’insulin, (1b) a’insulin absorbed with insulin, (1c) a’glucagon, (1d) a’glucagon absorbed with glucagon. Interference contrast. Magnification, 400.
glucagon) were seen in the epithelioid masses (Fig. 2a–h), while few were seen in the walls of the cysts or ducts. Acini were not observed and fibroblasts were few. Explants under the different conditions of culture are represented in Figure 2a–h. Also
represented are the absorption controls in which all immunostaining was eliminated by pre-absorption of the primary antiserum with the appropriate antigen (see Fig. 1a–d). In addition, when the primary antisera were replaced with diluent alone or non-immune serum of the relevant species, no staining resulted. The counts of insulin and glucagon cells and the proportion of insulin cells expressed as a percentage of the sum of insulin and glucagon cell counts are shown in Table 1. In comparing the control cultures, addition of the diluents TFA and ACN to the culture medium increased the proportion of insulin cells from 8.4% to 12.6% (1 ng control) (P<0.0001) and from 8.4% to 21.4% (10 ng control) (P<0.0001) (see Table 1). The diluents thus accounted for respective increases of 4.2% and 13% in the proportion of insulin cells in the controls. The effect of 1 ng/ml activin (plus diluents) was to increase the proportion of insulin cells from 12.6% to 17.7% (P<0.0001) (see Table 1). However, with regards to the effect of diluents, a corrected value of 13.5% was obtained (17.7%– 4.2%) after culture of explants with 1 ng/ml activin. There was also a statistically significant difference in explants treated with the higher concentration of activin together with a raised concentration of diluents (TFA salt of activin). This treatment, however, resulted in a decrease, from 21.4% to 19.4%, in the proportion of insulin cells (see Table 1).
Table 1. Proportions of insulin cells in chick dorsal pancreatic buds cultured for 7 days on Matrigel in Hams F12.ITS to which activin was added Culture condition Ham’s Ham’s Ham’s Ham’s Ham’s Ham’s
F12.ITS alone F12.ITS+1 l/ml diluents† F12.ITS+10 l/ml diluents† F12.ITS+diluents+1 ng/ml activin F12.ITS+diluents+10 ng/ml activin F12.ITS (no diluents)+10 ng/ml activin
Number of Insulin cell Glucagon cell Proportion of explants counts counts insulin cells* 6 6 6 6 6 6
179 228 550 466 350 369
1959 1567 2017 2171 1454 1904
8.4%‡0 12.6%‡§ 21.4%‡¶ 17.7%§ 19.3%¶ 16.2%0
*As a percentage of the sum of insulin and glucagon cell counts. F12.ITS=Ham’s F12 medium supplemented with insulin (5 g/ml), transferrin (5 g/ml) and selenium (10 10 M). †As activin was reconstituted in 40% trifluoracetic acid (TFA) and 0.1% acetonitryl (ACN) (Research Diagnostics), the control cultures had equivalent amounts of TFA and ACN added to F12.ITS (final volumes of 1 l/ml and 10 l/ml TFA+ACN, respectively). ‡F12.ITS+1 l/ml diluents and F12.ITS+10 l/ml significantly different from F12.ITS alone. §F12.ITS+solvents+1 ng/ml activin significantly different from F12.ITS+1 l/ml solvents. ¶F12.ITS+solvent+10 ng/ml activin significantly different from F12.ITS+10 l/ml solvents. 0F12.ITS+10 ng/ml activin significantly different from F12.ITS alone.
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Addition of the HCl salt of activin at 10 ng/ml increased the proportion of insulin cells from 8.4% to 16.2% (P<0.0001) (see Table 1). This proportion of insulin cells was also significantly different from that obtained following culture of dorsal pancreatic buds with activin diluted in TFA and ACN (see Table 1). Figure 2a–j shows representative adjacent sections from each treatment group immunostained for insulin and glucagon respectively. DISCUSSION In the study described here, the increased proportion of insulin cells obtained in cultured chick dorsal pancreatic buds following activin treatment suggests that endocrine cells may respond to activin signalling, and be developmentally focussed along the insulin cell pathway. In this regard, it is known that activin influences pancreatic cell differentiation. For example, in cell culture studies, amylasesecreting pancreatic AR42J cells can be induced to differentiate along an insulin cell pathway following activin treatment (Mashima et al., 1996). When activin signalling is inhibited with the activinbinding protein follistatin, the number of insulin cells is reduced in cultures of rat embryonic pancreas (Miralles et al., 1998). Additionally, impairment of activin signalling in transgenic mice results in lower insulin and glucagon cell numbers (Yamaoka et al., 1998), and the pancreas and islets being smaller than normal (Shiozaki et al., 1999). Previous studies using the present culture system with other selected factors have demonstrated beneficial effects on the development of pancreatic endocrine cells. In particular, Rawdon and Andrew (1997) reported increased proportions of chick insulin cells simply by substituting Matrigel for collagen matrix and by culturing the pancreatic buds in serum-free Hams’s F12.ITS. In further studies, the addition of triiodothyronine (T3) together with raised glucose and essential amino acids further increased proportions of insulin cells (Rawdon and Andrew, 1998). Other factors that have positively influenced chick insulin cell proportions include the substitution of insulin-like growth factor type 1 (IGF-1) for insulin (Rawdon and Andrew, 1998), supplementation of culture medium with nicotinamide (Mngomezulu and Kramer, 2000), the addition of retinoic acid (Penny and Kramer, 2000) and retinoic acid in combination with IGF-1 (Kramer and Penny, 2001a). As the pancreatic explants were initially cultured with activin reconstituted in the protein diluents
Fig. 2. Localization of insulin and glucagon cells in adjacent sections of explants cultured in (2a,b) Ham’s F12.ITS+ 1 ng/ml activin+diluents (TFA-salt), (2c,d) Ham’s F12.ITS+1 l/ml diluents, (2e,f) Ham’s F12.ITS+10 ng/ml activin+diluents (TFA-salt), (2g,h) Ham’s F12.ITS+10 l/ml diluents, (2i,j) Ham’s F12.ITS+10 ng/ml activin (HCl-salt). Interference contrast. Magnification, 400.
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TFA and ACN, the effects of the combined diluents were determined at the two different concentrations of activin. The diluents alone accounted for overall increases in the proportion of insulin cells of 4.2% and 13%, respectively. While the lower concentration of activin (1 ng/ml plus diluents) increased the proportion of insulin cells, the higher concentration (10 ng/ml plus diluents) caused a relative reduction. It is suggested that this effect is due to the raised concentration of TFA, as Cornish et al. (1999) similarly reported a reduction in cell proliferation caused by increased concentrations of TFA together with the growth factors amylin, amylin (1–8) and calcitonin. Moreover, this effect of TFA was found to be neither species- nor cell-specific, as it reduced cell proliferation in cultured foetal rat osteoblasts, adult canine chondrocytes and neonatal mice calvariae (Cornish et al., 1999). Conversion of the peptides amylin, amylin (1-8) and calcitonin from TFA to HCl salt clearly stimulated cell proliferation in all these cell types. As a result of this, Cornish et al. (1999) emphasized that care should be taken in working with TFA solubilized peptides, as a proliferative effect may not be detected, or an incorrect impression of an inhibitory effect on cells and tissues may be obtained. Thus, in the present study it was deemed necessary to convert TFA-dissolved activin into the biologically compatible HCl salt. Following this procedure, it was found that the higher concentration of the HCl salt of activin caused an increase in the proportion of insulin cells. In the present study, we reported a 1.6-fold increase in the proportion of insulin cells at the lower concentration of activin, and a 1.9-fold increase at the higher concentration. Rawdon and Andrew (1998) similarly obtained a 1.9-fold increase with the addition of increased glucose and amino acids in the presence of T3. Using the same culture system and following treatment with nicotinamide (Mngomezulu and Kramer, 2000), a 2.5fold increase in the proportion of insulin cells was attained. Subsequently, we have found that retinoic acid stimulates a 3.5-fold increase in the insulin cell population of the cultured chick dorsal pancreatic bud (Penny and Kramer, 2000). Although these results are collectively very promising, we have yet to attain the physiological percentage of insulin cells (34% in the 12 day chick splenic lobe; Andrew et al, 1994) within our culture system. Further understanding of how these hormones and nutrients influence the regulation of insulin cell-specific transcription factors should allow this figure to be reached and even exceeded.
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In relation to activin, there are a number of activin-responsive, lineage-restricted transcription factors required for pancreatic development. With the inhibition of SHH by activin, Pdx1, which is homologous to the activin-modulated Xenopus homeoprotein Xlhbox8 (Wright et al., 1988), is up-regulated. Pdx1 is of particular interest in insulin cell neogenesis and pancreatic regeneration, as it is expressed within the ducts of the embryonic mouse pancreas (Guz et al., 1995) and is also associated with proliferating duct cells during regeneration of the adult rat pancreas (Sharma et al., 1999). Furthermore, it has been suggested that new islets may arise from Pdx1 positive stem cells within the adult rat pancreas (Sharma et al., 1999). With regard to the paired box genes Pax4 and Pax 6, although pancreatic-associated levels of gene expression following activin treatment have not yet been determined, Pax4 expression has recently been shown to be induced by activin A in NIT1 and INS-1E -cell lines (Ueda, 2000). As Pax4 is able to inhibit the transcriptional activity of glucagon cell-associated Pax 6 in a competitive manner, it has been suggested that such interactions may regulate embryonic insulin cell development (Fujitani et al., 1999; Smith et al., 1999). Thus in the present study, if the expression levels of Pax4 had been increased and Pax 6 decreased, this could conceivably have favoured the increased proportion of insulin cells in the developing pancreas. This possibility is reflected in the increased proportion of insulin cells and the resultant decrease in glucagon cells after treatment with the HCl salt of activin. Previous studies of the proliferative ability of insulin-positive cells in cultured rat (Miralles et al., 1998) and chick pancreatic buds (Mngomezulu and Kramer, 2000) have suggested that differentiated endocrine cells have little proliferative ability and that it is the proliferation and subsequent differentiation of endocrine precursors which lead to increased insulin cell numbers. In the present study, we would thus suggest that activin may have stimulated an increased proliferation of endocrine precursor cells, ultimately leading to an increased pool of insulin cells. It would seem that activin can directly influence the differentiation of endocrine precursors, as it is expressed in putative endocrine progenitor cells at E12 in the foetal rat pancreatic anlage, prior to the appearance of either insulin- or glucagon-positive cells. Later in development, at E13.5, cells expressing activin are co-localized with both glucagon- and insulin-positive cells (Furukawa et al., 1995).
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It still remains to be determined whether activin has a direct effect on the expression of Pdx1 and Pax4 within our culture system. In view of the function of Pdx1 and Pax4 in modulating pancreatic, and more specifically, insulin cell development, understanding how these genes are regulated could provide a replenishable source of insulin cells for the treatment of diabetes.
ACKNOWLEDGEMENTS The gift of the anti-insulin antiserum from Dr L. Orci, Geneva, is gratefully acknowledged. Our thanks to the South African Medical Research Council and the Research Committee of the Council of the University of the Witwatersrand, Johannesburg for financial support. Mrs L. York, Mrs J. Dickinson and Mrs G. de Wet are thanked for their skilled technical assistance. This study was approved by the Animal Ethics Committee of the University of the Witwatersrand, Johannesburg (AEC No. 98-93-1). REFERENCES A U, Pff SL, J TM, E T, E H, 1997. Independent requirement for Isl1 in formation of pancreatic mesenchyme and islet cells. Nature 385: 257–260. A S, H D, T G, 1988. Hybrid insulin genes reveal a developmental lineage for pancreatic endocrine cells and imply a relationship with neurons. Cell 53: 295–308. A A, 1984. The development of the gastro-enteropancreatic neuroendocrine system in birds. In: Falkmer S, Hackanson R, Sundler F, eds. Evolution and Tumour Pathology of the Neuroendocrine System. Amsterdam, Elsevier Science. 91–109. A A, R BB, A BC, 1994. Failure of insulin cells to develop in cultured embryonic chick pancreas: a model system for the detection of factors supporting insulin cell differentiation. In Vitro Cell Dev Biol 30A: 664–670. A M, N H, S K, K K, I K, S H, U M, 1990. Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor). Roux’s Arch Dev Biol 198: 330–335. B GM, L DA, H A, 1991. Functional impact of attachment and purification in the short term culture of human pancreatic islets. J Clin Endocrinol Metab 73: 93–98. C Y, M A, C C, G S, B P J, L T, S V, S HC, 1994. Specific transforming-beta subtypes regulate embryonic mouse Meckel’s cartilage and tooth development. Dev Biol 162: 85–103. C J, C KE, L Q-X, X CL, M TB, C GJS, R IR, 1999. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am J Physiol 277: E779–E783. C CA, K AS, B GD, M TS, L MT, G GK, 2000. Expression and role of
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The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 48: 507–513. S D, K H, L M, V J, K D, F WH, 1990. Activin is a nerve cell survival molecule. Nature 344: 868–870. S S, T T, Z Y-Q, F M, N Y, K I, 1999. Impaired differentiation of endocrine and exocrine cells in transgenic mouse expressing the truncated type II activin receptor. Biochim Biophys Acta 1450: 1–11. S JC, P BM, V N K, H D, 1990. Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A. Nature 345: 729–731. S SB, E HC, C JR, G MS, 1999. Pairedhomeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development. Mol Cell Biol 19: 8272–8280. S-P B, C M, T M, O G, G P, 1997. The Pax4 gene is essential for differentiation of insulin-producing cells in the mammalian pancreas. Nature 386: 399–402. S BS, W BT, R WJ, 1970. The development of the dorsal and ventral mammalian pancreas in vivo and in vitro. J Cell Biol 47: 235–246. S-O L, S-P B, C K, M A, G P, 1997. Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas. Nature 387: 406–409. T G, A S, P JM, M A, H D, 1993. Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and neuronal proteins tyrosine hydrolase and neuropeptide Y, but not pancreatic polypeptide. Development 118: 1031–1039. U Y, 2000. Activin A increases Pax4 gene expression in pancreatic cell lines. FEBS Letts 480: 101–105. U BH, A GW, L AB, 1994. Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor. Development 120: 245–252. W NK, C JH, 1967. Early pancreas organogenesis: morphogenesis, tissue interactions, and mass effects. Dev Biol 15: 237–270. W CVE, S P, D EM, 1988. XlHbox 8: a novel Xenopus homeoprotein restricted to a narrow band of endoderm. Development 104: 787–794. Y T, I C, Y M, M T, Y T, L S, M M, H J, S H, N S, I M, 1998. Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants. J Clin Invest 102: 294–301.
EXOGENOUS ACTIVIN INCREASES THE PROPORTION OF INSULIN CELLS IN THE DEVELOPING CHICK PANCREAS IN CULTURE CLEM PENNY and BEVERLEY KRAMER* Embryonic Differentiation and Development Research Programme, School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg, 2193 South Africa Received 7 February 2002; accepted 21 August 2002
As activin is believed to be a key signalling factor during early pancreatic development, its influence on the proliferation and/or determination of insulin cells in the developing chick dorsal pancreatic bud was investigated. Dorsal pancreatic buds of 5-day-old chick embryos were explanted on to Matrigel and cultured in serum-free medium (Ham’s F12.ITS), to which 1 or 10 ng/ml activin was added. After 7 days in culture, the explants were processed for immunocytochemistry and the insulin-positive cells were scored and expressed as a proportion of the sum of insulin and glucagon cells. When compared to the control cultures (Hams F12.ITS alone), activin treatment resulted in respective increases in the proportion of insulin cells of 1.6 and 1.9 fold. It is suggested that activin treatment favours differentiation of the insulin cell 2002 Elsevier Science Ltd. All rights reserved. pathway relative to glucagon cells. K: chick pancreas; activin; insulin cells; diabetes mellitus.
INTRODUCTION In humans, the continued production of insulin by the islets of Langerhans of the pancreas is important for maintaining glucose homeostasis. In those individuals in which insulin secretion ceases, as in Type 1 Diabetes, the key to ensuring long-term health would be to provide therapy that would maintain glucose values within normal limits. Ideal treatments would be the implantation of viable, functionally competent islets or stimulation of endocrine progenitor cells to produce insulin cells. The identification and use of factors involved in the differentiation and proliferation of insulin cells could ultimately facilitate the provision of a plentiful supply of insulin cells for individuals with insulin-dependent diabetes. In this regard, we have previously assessed the influence of a number of growth factors and vitamins on insulin cell differentiation using the chick pancreas as a model system (Penny and Kramer, 2000; Mngomezulu and Kramer, 2000; Kramer and Penny, 2001a; Kramer and Penny, 2001b). *To whom correspondence should be addressed: Tel.: 27 11 717-2405; Fax: 27 11 717-2422; E-mail: [email protected] 1065–6995/02/$-see front matter
The activins, members of the transforming growth factor- (TGF-) supergene family, influence many diverse developmental and cellular processes, including the differentiation of erythroid cell lines (Eto et al., 1987), mesoderm induction in Xenopus laevis embryos (Asashima et al., 1990; Green and Smith, 1990; Smith et al., 1990), cartilage formation (Chai et al., 1994), cardiac morphogenesis (Mangiacapra et al., 1995), nerve cell survival (Hashimoto et al., 1990; Schubert et al., 1990) and tooth development Ferguson et al., 1998). During embryogenesis of the chick, activin signalling plays a key role in pancreatic development. The anterior foregut, which lies close to the notochord, receives and responds to activin signals which originate from the notochord. The effect of this inductive signal is to inhibit the expression of the Sonic Hedgehog gene (SHH) within the specific region of the anterior foregut which gives rise to the pancreas (Kim et al., 1997; Hebrok et al., 1998). The down-regulation of SHH subsequently allows for the expression of pancreatic markers within the endoderm, these being insulin and Pdx1 (the pancreatic and duodenal homeobox gene) (Hebrok 2002 Elsevier Science Ltd. All rights reserved.
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et al., 1998), which are required for the outgrowth of the dorsal pancreatic bud (Ohlsson et al., 1993; Jonsson et al., 1994; Guz et al., 1995; Ahlgren et al., 1997; Kim et al., 1997). Another important class of genes involved in pancreatic development which may be regulated by activin signalling is the paired box family (Pax). Pax 4 is essential for the development of insulin and somatostatin cells and the development of mature islets (Sosa-Pineda et al., 1997), while Pax 6 is required for glucagon cell development (St-Onge et al., 1997). Recently, the treatment of a number of -cell lines with activin has been shown to upregulate Pax 4 gene expression in a dose dependent manner (Ueda, 2000), thus suggesting that activin may influence -cell differentiation. As the identity and isolation of endocrine precursor cells has still not been clearly established, we have attempted to stimulate the production of insulin cells from developing chick dorsal pancreatic buds by culturing them some two days after the evagination of the pancreatic bud from the foregut. At this time, few differentiated insulin and glucagon cells are identifiable (Andrew, 1984). However, the inclusion of endocrine progenitor cells is ensured, as the entire endodermal component of the pancreatic bud is explanted. It is well known that permissive signals from both the pancreatic endoderm and mesenchyme are necessary for pancreatic development in mice (Golosow and Grobstein, 1962; Wessels and Cohen, 1967; Spooner et al., 1970) and chick (Dieterlen-Lievre, 1970). In relation to activin signalling, the mRNA for its specific inhibitor follistatin is present in the pancreatic mesenchyme early on in the mouse embryonic pancreas and is later down-regulated (Miralles et al., 1998; Maldonado et al., 2000). Thus in the present study, in order to negate the possible influence of follistatin and other mesenchymeassociated factors on development, and to determine the influence of added factors on the pancreatic endoderm during culture, almost all the mesenchyme was removed from the pancreatic explants. The extracellular matrix (ECM) provides additional signals that influence cellular function during development. Its composition and three dimensional organization affect cell growth, survival and differentiation (Lukashev and Werb, 1998), and the influence of ECM proteins on the in vitro growth and differentiation of the pancreas has been found to be an important factor. Andrew et al. (1994), for example, showed that a collagen gel substrate provided only minimal support for chick insulin cell development, while adequately sustaining that of the other pancreatic endocrine cells.
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Matrigel, a basement membrane extract containing laminin and a number of growth factors, has proven most useful in promoting differentiation in culture of a variety of cells (Reid and Jefferson, 1984), including pancreatic endocrine cell types (Muschel et al., 1986; Hayek et al., 1989; Beattie et al., 1991). More recently, the inclusion of laminin alone has been shown to support differentiation of the foetal mouse pancreas (Jiang et al., 1999; Crisera et al., 2000). The present study assessed the effects of activin at two concentrations, 1 and 10 ng/ml, on the proportion of insulin cells in the chick dorsal pancreatic bud explanted on to Matrigel. As activin was dissolved in trifluoroacetic acid (TFA) and acetonitryl (ACN), control cultures were included with identical volumes of these diluents. However, in view of the reported reduction in cell proliferation caused by increased concentrations of TFAsolubilized growth factors (above 10 9 M), activin was converted from a TFA salt to a biologically compatible HCl salt (Cornish et al., 1999) for use at the higher concentration. In comparing treatments, proportions of insulin cells and not absolute numbers were used. This relates to the possibility that the numbers of progenitor cells and differentiating insulin cells at the time of culture may not be the same in all of the dorsal pancreatic buds used. This proportion is significant in providing an index of proliferation and/or differentiation of both developing insulin and glucagon cells, as these two cell types are believed to derive from a common progenitor (Alpert et al., 1988; Teitelman et al., 1993; Upchurch et al., 1994; Jackerott et al., 1996). Furthermore, the use of proportions makes our findings directly comparable with other results in which changes in the proportion of insulin cells reflect the effects of a variety of nutrients, hormones and growth factors (Rawdon and Andrew, 1997; Rawdon and Andrew, 1998; Mngomezulu and Kramer, 2000; Penny and Kramer, 2000; Kramer and Penny, 2001a; Kramer and Penny, 2001b). MATERIALS AND METHODS Fertile chick Lohman Brown eggs were incubated at 37C for 5 days in a humidified atmosphere. The dorsal pancreatic bud was removed from the embryos at stages 25–26 (Hamburger and Hamilton, 1951) and incubated in 0.04% collagenase type VIII (Sigma Chemical Co., St Louis, U.S.A.) in Tyrode’s solution at 20C for
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20 min. The enveloping pancreatic mesoderm was then manually dissected off. In order to preserve the integrity of the endodermal component, a small amount of residual mesoderm was left in the interstices of the developing lobules. Explants were cultured on Matrigel (Collaborative Research, Bedford, MA, U.S.A.), which is a solubilized basement membrane extract containing various growth factors. It is buffered with sodium bicarbonate and contains gentamycin at 10 g/ml. Three grafts per well were placed on 175 g of Matrigel in 10 or 12 mm diameter wells of Nunclon (Roskilde, Denmark) culture plates. Explants were allowed to adhere to the Matrigel for 30 min and then covered with 1 ml of culture medium. The serum-free medium used was based on Ham’s F12 culture medium, to which 5 g/ml insulin, 5 g/ml transferrin and 10 10 M selenium were added (Ham’s F12.ITS, Highveld Biological, South Africa). Bovine recombinant activin (subtype A) (Research Diagnostics, Flanders, NJ, U.S.A.) was reconstituted in 40% ACN (SMM Chemicals, South Africa) and 0.1% TFA (Merck, Darmstadt, Germany) as a stock solution of 1 mg/ml. Activin at concentrations of 1 ng/ml or 10 ng/ml was added to the Ham’s F12.ITS medium of experimental explants. In order to control for possible diluent effects, control explants were cultured in Ham’s F12.ITS containing 1 l/ml or 10 l/ml of the diluents, ACN (40%) and TFA (0.1%). Explants were also cultured in Ham’s F12.ITS alone. In addition, activin was converted from the TFA salt form to the HCl salt form (Cornish et al., 1999) for use at 10 ng/ml. Activin at a concentration of 1 mg/ml was incubated in 50 l of a 3 mM HCl solution and allowed to stand for 1 h at room temperature, prior to freeze-drying at 60C and 10 2 Torr (1.33 Pa) (Virtis Freezemobile 12, Gardiner, NY, U.S.A.). Before use, activin was redissolved in sterile distilled water (SABAX) with sonication, cooled on ice for 15 s and stored at 4C. On the day of culture, it was diluted in Ham’s F12.ITS. Pancreatic buds were incubated for 7 days at 37C in 5% CO2 in a humidified atmosphere and the medium changed every third day. On retrieval, the explants were quenched in iso-pentane (BDH, Poole, U.K.), cooled in liquid nitrogen and freezedried at 140C and 10 2 Torr. The dried specimens were fixed for 3 h at 60C in vapour generated from re-crystallized parabenzoquinone (Merck) and embedded under vacuum in a 1:1 mixture of Epon and Araldite. The resin blocks were cured for 48 h at 60C. One micron serial sections were cut through the explant and sections mounted on
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polytetrafluoroethylene-coated slides (Rawdon, 1978). An indirect immuno-enzyme procedure was applied to adjacent serial sections to reveal insulin and glucagon cells. Each primary antiserum was applied to seven adjacent sections, spaced so that no cell could be counted more than once. Insulincontaining cells were detected with guinea pig anti-insulin antibody (1:8000) (a gift from L. Orci, Geneva) and glucagon containing cells with rabbit anti-glucagon antibody (1:800) (Milab, Malmo, Sweden) on adjacent serial sections. Incubation with the primary antiserum was carried out for 22 h at 4C. Peroxidase conjugated rabbit anti-guinea pig antiserum at a concentration of 1:20 (Dako, Glostrup, Denmark) was applied to sections stained with anti-insulin antiserum, while swine anti-rabbit peroxidase (Dako) at the same concentration was used following the anti-glucagon antiserum. The site of the antigen was revealed by treatment with a diaminobenzidine (DAB) solution containing hydrogen peroxide (Perhydrol) (Merck). Immunocytochemical control procedures were carried out on sections adjacent to those that contained endocrine cells. These procedures included substitution of the primary antiserum by the primary antiserum pre-absorbed for 24 h at 4C with its own antigen (20 g/ml insulin natural human, Peninsula Labs, California, U.S.A. or 40 g/ml glucagon, natural bovine/porcine, Eurodiagnostica, Malmo, Sweden), diluent alone or non-immune serum of the relevant species. A control section from the pancreas of a hatching chick known to be positive for the antigen concerned was included in each staining run. Counts of insulin and glucagon cells were thus made in 14 serial sections (insulin, n=7; glucagon, n=7) of each explant. Only those cells in which a nucleus was visible or those portions of cells as large or larger than a nucleus were counted. The number of insulin cells was expressed as a proportion (percentage) of the total insulin and glucagon cell count. Proportions of these cell counts under the different conditions of culture were compared by the 2 test. Differences were regarded as significant when P values (one tailed) were less than 0.05. Absolute numbers of insulin cells were not analysed; the counts recorded should not be regarded as such. RESULTS The cultures consisted of solid epithelial masses, sometimes containing epithelial-lined cysts. The majority of the endocrine cells (insulin and
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Fig. 1. Preabsorption with the respective antigen eliminated staining with the relevant antibody. Adjacent sections of a control explant cultured in HamsF12.ITS treated with (1a) a’insulin, (1b) a’insulin absorbed with insulin, (1c) a’glucagon, (1d) a’glucagon absorbed with glucagon. Interference contrast. Magnification, 400.
glucagon) were seen in the epithelioid masses (Fig. 2a–h), while few were seen in the walls of the cysts or ducts. Acini were not observed and fibroblasts were few. Explants under the different conditions of culture are represented in Figure 2a–h. Also
represented are the absorption controls in which all immunostaining was eliminated by pre-absorption of the primary antiserum with the appropriate antigen (see Fig. 1a–d). In addition, when the primary antisera were replaced with diluent alone or non-immune serum of the relevant species, no staining resulted. The counts of insulin and glucagon cells and the proportion of insulin cells expressed as a percentage of the sum of insulin and glucagon cell counts are shown in Table 1. In comparing the control cultures, addition of the diluents TFA and ACN to the culture medium increased the proportion of insulin cells from 8.4% to 12.6% (1 ng control) (P<0.0001) and from 8.4% to 21.4% (10 ng control) (P<0.0001) (see Table 1). The diluents thus accounted for respective increases of 4.2% and 13% in the proportion of insulin cells in the controls. The effect of 1 ng/ml activin (plus diluents) was to increase the proportion of insulin cells from 12.6% to 17.7% (P<0.0001) (see Table 1). However, with regards to the effect of diluents, a corrected value of 13.5% was obtained (17.7%– 4.2%) after culture of explants with 1 ng/ml activin. There was also a statistically significant difference in explants treated with the higher concentration of activin together with a raised concentration of diluents (TFA salt of activin). This treatment, however, resulted in a decrease, from 21.4% to 19.4%, in the proportion of insulin cells (see Table 1).
Table 1. Proportions of insulin cells in chick dorsal pancreatic buds cultured for 7 days on Matrigel in Hams F12.ITS to which activin was added Culture condition Ham’s Ham’s Ham’s Ham’s Ham’s Ham’s
F12.ITS alone F12.ITS+1 l/ml diluents† F12.ITS+10 l/ml diluents† F12.ITS+diluents+1 ng/ml activin F12.ITS+diluents+10 ng/ml activin F12.ITS (no diluents)+10 ng/ml activin
Number of Insulin cell Glucagon cell Proportion of explants counts counts insulin cells* 6 6 6 6 6 6
179 228 550 466 350 369
1959 1567 2017 2171 1454 1904
8.4%‡0 12.6%‡§ 21.4%‡¶ 17.7%§ 19.3%¶ 16.2%0
*As a percentage of the sum of insulin and glucagon cell counts. F12.ITS=Ham’s F12 medium supplemented with insulin (5 g/ml), transferrin (5 g/ml) and selenium (10 10 M). †As activin was reconstituted in 40% trifluoracetic acid (TFA) and 0.1% acetonitryl (ACN) (Research Diagnostics), the control cultures had equivalent amounts of TFA and ACN added to F12.ITS (final volumes of 1 l/ml and 10 l/ml TFA+ACN, respectively). ‡F12.ITS+1 l/ml diluents and F12.ITS+10 l/ml significantly different from F12.ITS alone. §F12.ITS+solvents+1 ng/ml activin significantly different from F12.ITS+1 l/ml solvents. ¶F12.ITS+solvent+10 ng/ml activin significantly different from F12.ITS+10 l/ml solvents. 0F12.ITS+10 ng/ml activin significantly different from F12.ITS alone.
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Addition of the HCl salt of activin at 10 ng/ml increased the proportion of insulin cells from 8.4% to 16.2% (P<0.0001) (see Table 1). This proportion of insulin cells was also significantly different from that obtained following culture of dorsal pancreatic buds with activin diluted in TFA and ACN (see Table 1). Figure 2a–j shows representative adjacent sections from each treatment group immunostained for insulin and glucagon respectively. DISCUSSION In the study described here, the increased proportion of insulin cells obtained in cultured chick dorsal pancreatic buds following activin treatment suggests that endocrine cells may respond to activin signalling, and be developmentally focussed along the insulin cell pathway. In this regard, it is known that activin influences pancreatic cell differentiation. For example, in cell culture studies, amylasesecreting pancreatic AR42J cells can be induced to differentiate along an insulin cell pathway following activin treatment (Mashima et al., 1996). When activin signalling is inhibited with the activinbinding protein follistatin, the number of insulin cells is reduced in cultures of rat embryonic pancreas (Miralles et al., 1998). Additionally, impairment of activin signalling in transgenic mice results in lower insulin and glucagon cell numbers (Yamaoka et al., 1998), and the pancreas and islets being smaller than normal (Shiozaki et al., 1999). Previous studies using the present culture system with other selected factors have demonstrated beneficial effects on the development of pancreatic endocrine cells. In particular, Rawdon and Andrew (1997) reported increased proportions of chick insulin cells simply by substituting Matrigel for collagen matrix and by culturing the pancreatic buds in serum-free Hams’s F12.ITS. In further studies, the addition of triiodothyronine (T3) together with raised glucose and essential amino acids further increased proportions of insulin cells (Rawdon and Andrew, 1998). Other factors that have positively influenced chick insulin cell proportions include the substitution of insulin-like growth factor type 1 (IGF-1) for insulin (Rawdon and Andrew, 1998), supplementation of culture medium with nicotinamide (Mngomezulu and Kramer, 2000), the addition of retinoic acid (Penny and Kramer, 2000) and retinoic acid in combination with IGF-1 (Kramer and Penny, 2001a). As the pancreatic explants were initially cultured with activin reconstituted in the protein diluents
Fig. 2. Localization of insulin and glucagon cells in adjacent sections of explants cultured in (2a,b) Ham’s F12.ITS+ 1 ng/ml activin+diluents (TFA-salt), (2c,d) Ham’s F12.ITS+1 l/ml diluents, (2e,f) Ham’s F12.ITS+10 ng/ml activin+diluents (TFA-salt), (2g,h) Ham’s F12.ITS+10 l/ml diluents, (2i,j) Ham’s F12.ITS+10 ng/ml activin (HCl-salt). Interference contrast. Magnification, 400.
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TFA and ACN, the effects of the combined diluents were determined at the two different concentrations of activin. The diluents alone accounted for overall increases in the proportion of insulin cells of 4.2% and 13%, respectively. While the lower concentration of activin (1 ng/ml plus diluents) increased the proportion of insulin cells, the higher concentration (10 ng/ml plus diluents) caused a relative reduction. It is suggested that this effect is due to the raised concentration of TFA, as Cornish et al. (1999) similarly reported a reduction in cell proliferation caused by increased concentrations of TFA together with the growth factors amylin, amylin (1–8) and calcitonin. Moreover, this effect of TFA was found to be neither species- nor cell-specific, as it reduced cell proliferation in cultured foetal rat osteoblasts, adult canine chondrocytes and neonatal mice calvariae (Cornish et al., 1999). Conversion of the peptides amylin, amylin (1-8) and calcitonin from TFA to HCl salt clearly stimulated cell proliferation in all these cell types. As a result of this, Cornish et al. (1999) emphasized that care should be taken in working with TFA solubilized peptides, as a proliferative effect may not be detected, or an incorrect impression of an inhibitory effect on cells and tissues may be obtained. Thus, in the present study it was deemed necessary to convert TFA-dissolved activin into the biologically compatible HCl salt. Following this procedure, it was found that the higher concentration of the HCl salt of activin caused an increase in the proportion of insulin cells. In the present study, we reported a 1.6-fold increase in the proportion of insulin cells at the lower concentration of activin, and a 1.9-fold increase at the higher concentration. Rawdon and Andrew (1998) similarly obtained a 1.9-fold increase with the addition of increased glucose and amino acids in the presence of T3. Using the same culture system and following treatment with nicotinamide (Mngomezulu and Kramer, 2000), a 2.5fold increase in the proportion of insulin cells was attained. Subsequently, we have found that retinoic acid stimulates a 3.5-fold increase in the insulin cell population of the cultured chick dorsal pancreatic bud (Penny and Kramer, 2000). Although these results are collectively very promising, we have yet to attain the physiological percentage of insulin cells (34% in the 12 day chick splenic lobe; Andrew et al, 1994) within our culture system. Further understanding of how these hormones and nutrients influence the regulation of insulin cell-specific transcription factors should allow this figure to be reached and even exceeded.
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In relation to activin, there are a number of activin-responsive, lineage-restricted transcription factors required for pancreatic development. With the inhibition of SHH by activin, Pdx1, which is homologous to the activin-modulated Xenopus homeoprotein Xlhbox8 (Wright et al., 1988), is up-regulated. Pdx1 is of particular interest in insulin cell neogenesis and pancreatic regeneration, as it is expressed within the ducts of the embryonic mouse pancreas (Guz et al., 1995) and is also associated with proliferating duct cells during regeneration of the adult rat pancreas (Sharma et al., 1999). Furthermore, it has been suggested that new islets may arise from Pdx1 positive stem cells within the adult rat pancreas (Sharma et al., 1999). With regard to the paired box genes Pax4 and Pax 6, although pancreatic-associated levels of gene expression following activin treatment have not yet been determined, Pax4 expression has recently been shown to be induced by activin A in NIT1 and INS-1E -cell lines (Ueda, 2000). As Pax4 is able to inhibit the transcriptional activity of glucagon cell-associated Pax 6 in a competitive manner, it has been suggested that such interactions may regulate embryonic insulin cell development (Fujitani et al., 1999; Smith et al., 1999). Thus in the present study, if the expression levels of Pax4 had been increased and Pax 6 decreased, this could conceivably have favoured the increased proportion of insulin cells in the developing pancreas. This possibility is reflected in the increased proportion of insulin cells and the resultant decrease in glucagon cells after treatment with the HCl salt of activin. Previous studies of the proliferative ability of insulin-positive cells in cultured rat (Miralles et al., 1998) and chick pancreatic buds (Mngomezulu and Kramer, 2000) have suggested that differentiated endocrine cells have little proliferative ability and that it is the proliferation and subsequent differentiation of endocrine precursors which lead to increased insulin cell numbers. In the present study, we would thus suggest that activin may have stimulated an increased proliferation of endocrine precursor cells, ultimately leading to an increased pool of insulin cells. It would seem that activin can directly influence the differentiation of endocrine precursors, as it is expressed in putative endocrine progenitor cells at E12 in the foetal rat pancreatic anlage, prior to the appearance of either insulin- or glucagon-positive cells. Later in development, at E13.5, cells expressing activin are co-localized with both glucagon- and insulin-positive cells (Furukawa et al., 1995).
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It still remains to be determined whether activin has a direct effect on the expression of Pdx1 and Pax4 within our culture system. In view of the function of Pdx1 and Pax4 in modulating pancreatic, and more specifically, insulin cell development, understanding how these genes are regulated could provide a replenishable source of insulin cells for the treatment of diabetes.
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