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In Vitro Validation of Duct Differentiation in Developing Embryonic Mouse Pancreas
Journal of Surgical Research 90, 126 –130 (2000) doi:10.1006/jsre.2000.5876, available online at http://www.idealibrary.com on
In Vitro Validation of Duct Differentiation in Developing Embryonic Mouse Pancreas 1 Alan S. Kadison, M.D., Thomas S. Maldonado, M.D., Christopher A. Crisera, M.D., Michael T. Longaker, M.D., and George K. Gittes, M.D.* ,2 New York University Medical Center, New York, New York; and *Children’s Mercy Hospital, Kansas City, Missouri 64108 Presented at the Annual Meeting of the Association for Academic Surgery, Philadelphia, Pennsylvania, November 18 –20, 1999
Background. Early embryonic pancreatic epithelia have the capacity for either endocrine or exocrine lineage commitment. Recent studies demonstrated the pluripotential nature of these undifferentiated cells. Isolated pancreatic epithelia grown under the renal capsule formed primarily islets. However, when these same epithelia were grown in a basement-membranerich gel (Matrigel) they formed mostly ducts. Currently, there is no model for in vitro pancreatic duct formation and therefore, the mechanism of duct morphogenesis has never been described. The purpose of this study was to provide such a model by characterizing the expression of two duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR), in isolated undifferentiated pancreatic epithelia grown in vitro. Materials and methods. We microdissected embryonic pancreases at Embryonic Days (E)9.5–11.5 and performed RT-PCR for CAII and CFTR on E9.5 whole pancreases, E10.5 and E11.5 epithelia, as well as E11.5 epithelia grown for 7 days in Matrigel. Next we performed in situ hybridization for CAII and CFTR and immunohistochemistry for CAII on E11.5 epithelia grown for 7 days in Matrigel. Results. Early, undifferentiated embryonic pancreatic epithelium does not express CAII and CFTR by RT-PCR. When E11.5 epithelia were grown for 7 days in Matrigel, however, gene expression for both markers is upregulated as ducts form. Furthermore, CAII was seen by IHC and both CAII and CFTR were seen by in situ hybridization in the ducts after 7 days in Matrigel. Conclusions. These data validate our in vitro system 1 This work was supported by an American College of Surgeons Faculty Research Fellowship and the Juvenile Diabetes Foundation International. 2 To whom correspondence should be addressed at Children’s Mercy Hospital, 2401 Gilham Road, Kansas City, MO 64108.
0022-4804/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
as a model for studying the mechanism of normal pancreatic duct differentiation and may potentially help us to understand the faulty mechanism involved in pancreatic ductal carcinogenesis. © 2000 Academic Press Key Words: pancreas; ducts; development; in vitro. INTRODUCTION
Branching epithelial morphogenesis is an important mechanism of development in many organs, including the pancreas [1– 4]. The embryonic mouse pancreas first develops as an evagination of the foregut endoderm into the surrounding mesenchyme at Embryonic Day 9.5 (E9.5) [5]. The epithelium continues to branch and begins to differentiate into more mature exocrine structures (ducts and acini) by E14.5 [6]. Mesenchymal-epithelial interactions have been shown to be important for pancreatic growth and differentiation [5, 7–11]. Indeed, embryonic pancreas without its mesenchyme fails to develop into islets, ducts, or acini when grown on a filter [7]. Interestingly, epithelia isolated from its surrounding mesenchyme when grown under the renal capsule formed mature islets. In addition, isolated epithelia grown in a basementmembrane-rich gel formed primarily ducts [4]. These studies suggested the pluripotentiality of the early, undifferentiated pancreatic epithelia, and that they may be composed of stem cell precursors that can be driven toward exocrine or endocrine lineage, depending on the signals received from their environment. The concept that this undifferentiated epithelium can be manipulated to become endocrine cells has significant clinical implications with regard to the treatment and potential cure of diabetes mellitus. It is thought that pancreatic stem cells may arise from ducts [6, 12, 13]. Islet morphogenesis has been described as a budding of endocrine cells from ductal
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epithelium [14, 15], and there are many studies that describe the budding of islets and acini from ducts after distal pancreatectomy and pancreatic duct ligation in rats [16, 17]. Understanding the process of normal pancreatic duct differentiation, therefore, may be critical to understanding the biology of pancreatic stem cells. We have recently described the ontogeny of two pancreatic duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR) in the developing embryonic mouse pancreas [18]. In these studies, we demonstrated the absence of these ductal markers in early pancreatic epithelium using immunohistochemistry and in situ hybridization. Carbonic anhydrase II was first detected in the differentiating epithelium by E14.5, and both CAII and CFTR were expressed in the more mature ducts by E16.5. In order to better understand the mechanism of pancreatic duct differentiation, we have grown isolated, undifferentiated E11.5 epithelia in a basementmembrane-rich gel (Matrigel), which is rich in laminin-1. In this system, the epithelia have previously been shown to form primarily ducts [4]. We have recently shown that the cross-region of the laminin-1 molecule and the ␣6-containing integrin are necessary for pancreatic duct morphogenesis [19]; however, the mechanism of cytodifferentiation and morphogenesis of duct cells has never been characterized. We provide here a model of in vitro duct differentiation wherein we analyzed the expression of the ductal markers CAII and CFTR by PCR, in situ hybridization, and IHC, and show that their temporal expression in vitro parallels their in vivo expression, thus validating our model. MATERIALS AND METHODS Pancreas dissection and culture. Time-dated pregnant CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA). Noon on the day of vaginal plug discovery was considered Day 0.5 of gestation. All procedures were approved by the New York University Medical Center Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institute of Health Publication No. 86-23, revised 1985). Pancreases were harvested at E9.5– 12.5 and epithelia were isolated from surrounding mesenchyme from E10.5 and E11.5 specimens using 1% trypsin in serum-free media as described previously [20]. Isolated E11.5 pancreatic epithelia were grown on filter inserts (Millipore, Bedford, MA) and placed in standard 24-well plates under sterile conditions. Each well was filled with 500 l of filter-sterilized media containing 90% DMEM/F12K, 10% fetal bovine serum, and 1% antibiotic/antimycotic solution (penicillin G 10,000/ml, streptomycin sulfate 10,000 g/ml, Amphotericin B 200 g/ml (Gibco, Grand Island, NY). Filter inserts were filled with 150 l of basement membrane gel (Matrigel, Collaborative Research, Boston, MA), and isolated epithelia were transferred into the gel prior to gel polymerization using sterile siliconized tips. All epithelia were grown at 37° for 7 days. RT-PCR. Embryonic pancreases were microdissected and RNA was extracted from pooled whole pancreas at E9.5, from pooled epithelia at E10.5 and E11.5, and from pooled E11.5 epithelia grown in Matrigel for 7 days, using the S.N.A.P. total RNA isolation kit (Invitrogen, Carlsbad,
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CA). Reverse transcription was performed using the Superscript Preamplification System (Gibco) and PCR for -tubulin was used as control for each RNA extraction. PCR was then performed on the different aged cDNA for CAII and CFTR using the following primer pairs: CAII, 5⬘-GCAAGAGGCCATGTCTGCTC-3⬘ and 5⬘-GGAAGCGTGCGGCCTTTGCT-3⬘; CFTR, 5⬘-CAGTCATCTCTGCCTTGTGG-3⬘ and 5⬘ACGCTGACCTCCACTCAGTG-3⬘. The annealing temperature for both sets of primers was 58°C. Tissue preparation. After 7 days in culture, the E11.5 epithelia were fixed in 4% paraformaldehyde (in PBS) for 2 h and then cryoprotected in 30% sucrose (in PBS) overnight. For in situ hybridization, the dissections were performed in DEPC-treated PBS and RNAse-free conditions were used. Four percent paraformaldehyde was made with DEPC PBS and specimens were fixed for 8 h and then cryoprotected in 30% sucrose (in DEPC PBS) overnight. The epithelia were then excised from the filter inserts under direct microscopic vision, embedded in tissue freezing medium (Triangle Biomedical Science, Durham, NC), frozen in liquid nitrogen, and cut into 6-m sections using a Leica cryostat (Wetzlar, Germany). Immunohistochemistry. Sections were rehydrated with PBS, permeabilized in a Tween-20 solution (0.5%) in PBS for 10 min, and washed with PBS for 5 minutes. Heated sodium citrate (10 mM, pH 3.0) was then used for antigen recovery. The specimens were incubated with 4% blocking serum (donkey serum; Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 8 h. The sections were then incubated in primary antibody CAII (The Binding Site, Co., San Diego, CA) 1:500 in PBS overnight at 4°C followed by secondary antibody (Rhodamine donkey anti-sheep) 1:200 in PBS for 1 h. Specimens were washed in PBS after incubating with the secondary antibody and coverslips were applied using Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL). Mouse salivary gland was used as a positive control for the CAII antibody and nonimmune serum was used as a negative control. In situ hybridization. For probe construction/labeling, PCR was used to generate probes for mouse CAII and mouse CFTR. Whole embryo cDNA was used as the template for the PCR. CAII and CFTR primers (described above) were used to generate a 539- and a 369-bp fragment, respectively. These fragments were subcloned into a 3.9-kb pCRII vector using a TA cloning kit (Invitrogen). The restriction enzyme HindIII was used to linearize the plasmids containing CAII and CFTR, and a phenol/chloroform extraction was performed. The purified linearized plasmids were used to generate labeled RNA probes by performing in vitro transcription using T7 RNA polymerase and digoxigenin-11-dUTP. Mouse salivary gland and lung were used as positive controls for CAII and CFTR, respectively. Sense probes for CAII and CFTR were used as negative controls. Hybridization was performed on the pancreas frozen sections by covering them with 30 l of hybridization buffer containing 10 –30 ng of digoxigenin-labeled probe overnight in a humid chamber at 55°C. Posthybridization, the slides were washed and residual probe was digested with RNaseA. To detect the mRNA signal, the slides were subjected to 2 h of incubation with a 1:500 dilution of sheep anti-DIG-alkaline phosphatase. Finally, the slides were covered with 200 l of developing solution. After the colored signal had optimally developed, the reaction was stopped.
RESULTS
Isolated undifferentiated E11.5 pancreatic epithelium has the capacity to form either exocrine or endocrine structures. We have previously traced the ontog-
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FIG. 1. RT-PCR for CFTR and CAII on whole pancreases at E9.5, on isolated epithelia at E10.5 and E11.5, and on isolated E11.5 epithelia grown in Matrigel for 7 days. Note the absence of CFTR and CAII gene expression in early undifferentiated epithelia with upregulation of gene expression as ducts form in Matrigel.
eny of duct markers in vivo; however, a model of in vitro duct formation is necessary if we are to better characterize this process and potentially manipulate the system in order to control pancreatic lineage selection. We therefore grew isolated E11.5 epithelia in Matrigel for 7 days and then performed PCR, IHC, and in situ hybridization for the duct markers CAII and CFTR in order to validate this culture system as representative of in vivo duct formation. Reverse-transcription PCR revealed the absence of gene expression for both CAII and CFTR in E9.5 whole pancreas and in isolated epithelium at E10.5 and E11.5. After the growth of isolated E11.5 epithelia in Matrigel for 1 week, however, these genes were detectable by PCR in the epithelium (Fig. 1). Control RT-PCR
for -tubulin showed bands for E9.5 whole pancreas, E10.5 epithelia, and E11.5 epithelia (Fig. 2). These data demonstrate that in our culture system, there is upregulation or induction of gene expression for CAII and CFTR as ducts form from undifferentiated epithelia in vitro. Furthermore, in situ hybridization for CAII and CFTR showed strong expression of these markers in the ducts after E11.5 epithelia were grown in Matrigel for 1 week (Fig. 3). This expression supports the PCR data and is consistent with an upregulation or induction of duct marker gene expression during in vitro duct development. Finally, immunohistochemistry for CAII also showed expression in the large cystic ducts formed in culture (Fig. 4). Thus, the temporal expression of duct markers in our culture systems cor-
FIG. 2. -Tubulin control RT-PCR for E9.5 whole pancreases, E10.5 epithelia, and E11.5 epithelia. Note 650-bp band in the Rt ⫹ lane for each age.
KADISON ET AL.: PANCREATIC DUCT DEVELOPMENT
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FIG. 3. In situ hybridization on E11.5 epithelia grown in Matrigel for 7 days for (a) CFTR and (b) CAII (400⫻). Note mRNA signal in the newly formed ducts (d). Hybridization with sense probes to CAII and CFTR yielded no signal (data not shown).
relates with their expression in vivo and validates our model. DISCUSSION
Early studies on development of the pancreas showed that embryonic pancreatic epithelia, when grown on filters in the absence of its mesenchyme, failed to form islets, ducts, or acini [7]. Other experiments in which pancreatic epithelia and mesenchyme were cultured on opposite sides of a porous filter showed growth and differentiation of the epithelia, suggesting that a soluble “mesenchymal” factor was essential for normal pancreatic development. Unfortunately, subsequent studies have failed to isolate this factor [21] leading investigators to pursue other aspects of mesenchymal-epithelial interactions in pancreatic development. In particular, contact between the epithelium and the mesenchyme has been shown to be critical for exocrine differentiation [9]. More recently, isolated undifferentiated pancreatic epithelia grown under different culture conditions demonstrated the capacity to select different lineages based on these external signals [20]. As previously shown [7], epithelia grown on filters in the absence of
mesenchyme failed to differentiate, both histologically and by immunohistochemical analysis. When these epithelia were grown in a three-dimensional basementmembrane gel, however, they differentiated into large, cystic ducts [20]. Moreover, when the epithelia were grown under the renal capsule, pure clusters of mature islets formed. These results represented the first report of pure islet differentiation and growth from pancreatic precursor cells. We recently investigated the importance of laminin-1, the major noncollagenous protein component of Matrigel and basement membrane, in the induction of exocrine lineage selection by undifferentiated epithelia. Laminin-1 has been shown to be important for branching epithelial morphogenesis in the kidney, lung, and salivary gland [1–3], and we showed by neutralizing antibody-blocking studies that the binding of the cross-region of the laminin-1 molecule to the ␣6-containing integrin was critical for duct morphogenesis in culture [19]. The nature of these in vitro ducts, however, has never been characterized. In order to better understand the mechanism of pancreatic ductal lineage selection, therefore, we wished to define the expression of ductal markers in vitro, in
FIG. 4. Immunofluorescence for CAII on E11.5 epithelia grown in Matrigel for 7 days at (a) 200⫻ and (b) 400⫻. Note strong staining in the newly formed ducts (arrows). Nonimmune serum yielded no staining (data not shown).
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undifferentiated pancreatic epithelia. We harvested embryonic mouse pancreases at Gestational Days 9.5– 11.5 and performed RT-PCR on isolated pooled pancreatic epithelia for CAII and CFTR. Expression of these markers was absent in the epithelia at these early ages. After being grown in Matrigel for 7 days, duct morphogenesis occurred and there was an activation of gene expression for both CAII and CFTR by RT-PCR. Furthermore, in situ hybridization for both ductal markers demonstrated a strong signal exclusively in the ducts, consistent with the PCR data. In addition, immunohistochemistry for CAII revealed intense staining in these ducts. The upregulation or induction of duct marker gene expression in the developing pancreatic epithelium validates our in vitro system as a model for studying embryonic pancreatic duct differentiation because it parallels normal duct development during gestation. We recently showed absence of CAII or CFTR expression by immunohistochemistry and in situ hybridization in pancreatic epithelium at E11.5 and E12.5, respectively, with an upregulation or induction of CAII and CFTR expression in the mature ducts by E16.5 [18]. Our validated in vitro system now allows us access to early, undifferentiated pancreatic precursor cells that can be manipulated in order to study the mechanism of pancreatic duct lineage selection. We are beginning to examine this period of differentiation in vitro in more detail by trying to determine if CAII expression alone is critical for ductal lineage selection, or whether factors controlling CAII expression are responsible for inducing pancreatic duct formation. REFERENCES Klein, G., Langegger, M., Timpl, R., and Ekblom, P. Role of laminin A chain in the development of epithelial cell polarity. Cell 55: 331, 1988. 2. Schuger, L., O’Shea, K. S., Nelson, B. B., and Varani, J. Organotypic arrangement of mouse embryonic lung cells on a basement membrane extract: Involvement of laminin. Development 110: 1091, 1990. 3. Kadoya, Y., Kadoya, K., Durbeej, M., Holmvall, K., Sorokin, L., and Ekblom, P. Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes. J. Cell Biol. 129: 521, 1995. 4. Gittes, G. K., Galante, P. E., Hanahan, D., Rutter, W. J., and Debase, H. T. Lineage-specific morphogenesis in the developing
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pancreas: Role of mesenchymal factors. Development 122: 439, 1996. Wessels, N., and Cohen, J. Early pancreas organogenesis: Morphogenesis, tissue interactions, and mass effects. Dev. Biol. 15: 237, 1967. Slack, J. M. Developmental biology of the pancreas. Development 121: 1569, 1995. Golosow, N., and Grobstein, C. Epitheliomesenchymal interaction in pancreatic morphogenesis. Dev. Biol. 4: 242, 1962. Fell, P. E., and Grobstein, C. The influence of extra-epithelial factors on the growth of embryonic mouse pancreatic epithelium. Exp. Cell Res. 53: 301, 1968. Levine, S., Pictet, R., and Rutter, W. J. Control of cell proliferation and cytodifferentiation by a factor reacting with the cell surface. Nature New Biol. 246: 49, 1973. McEvoy, R. C., Hegre, O. D., Leonard, R. J., and Lazarow, A. Fetal rat pancreas: Differentiation of the acinar cell component in vivo and in vitro. Diabetes 22: 584, 1973. Sakakura, T., Sakagami, Y., and Nishizuka, Y. Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme. Dev. Biol. 72: 201, 1979. Githens, S. The pancreatic duct cell: Proliferative capabilities, specific characteristics, metaplasia, isolation, and culture. J. Pediatr. Gastroenterol. Nutr. 7: 486, 1988. Bouwens, L., and De Blay, E. Islet morphogenesis and stem cell markers in rat pancreas. J. Histochem. Cytochem. 44: 947, 1996. Pictet, R., and Rutter, W. Development of the embryonic endocrine pancreas. Vol. 1, Sect. 7. In Handbook of Physiology. Baltimore: Waverly Press, 1972. Rutter, W. The development of the endocrine and exocrine pancreas. In P. Fitzgerald, and A. Morrison, (Eds.), The Pancreas. Baltimore: Williams & Wilkins, 1980. Bonner-Weir, S., Baxter, L. A., Schuppin, G. T., and Smith, F. E. A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42: 1715, 1993. Wang, R., Kloppel, G., and Bouwens, L. Duct-to-islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 38: 1405, 1995. Kadison, A. S., Crisera, C. A., Maldonado, T. S., Longaker, M. T., and Gittes, G. K. Duct marker ontogeny in developing embryonic mouse pancreas. Submitted for Publication. Crisera, C. A., Kadison, A. S., Breslow, G. D., Maldonado, T. S., Longaker, M. T., and Gittes, G. K. The expression and role of laminin-1 in mouse pancreatic organogenesis. Diabetes, in press. Gittes, G. K., Rutter, W. J., and Debas, H. T. Initiation of gastrin expression during the development of the mouse pancreas. Am. J. Surg. 165: 23; discussion 25, 1993. Ronzio, R. A., and Rutter, W. J. Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia. Dev. Biol. 30: 307, 1973.
In Vitro Validation of Duct Differentiation in Developing Embryonic Mouse Pancreas 1 Alan S. Kadison, M.D., Thomas S. Maldonado, M.D., Christopher A. Crisera, M.D., Michael T. Longaker, M.D., and George K. Gittes, M.D.* ,2 New York University Medical Center, New York, New York; and *Children’s Mercy Hospital, Kansas City, Missouri 64108 Presented at the Annual Meeting of the Association for Academic Surgery, Philadelphia, Pennsylvania, November 18 –20, 1999
Background. Early embryonic pancreatic epithelia have the capacity for either endocrine or exocrine lineage commitment. Recent studies demonstrated the pluripotential nature of these undifferentiated cells. Isolated pancreatic epithelia grown under the renal capsule formed primarily islets. However, when these same epithelia were grown in a basement-membranerich gel (Matrigel) they formed mostly ducts. Currently, there is no model for in vitro pancreatic duct formation and therefore, the mechanism of duct morphogenesis has never been described. The purpose of this study was to provide such a model by characterizing the expression of two duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR), in isolated undifferentiated pancreatic epithelia grown in vitro. Materials and methods. We microdissected embryonic pancreases at Embryonic Days (E)9.5–11.5 and performed RT-PCR for CAII and CFTR on E9.5 whole pancreases, E10.5 and E11.5 epithelia, as well as E11.5 epithelia grown for 7 days in Matrigel. Next we performed in situ hybridization for CAII and CFTR and immunohistochemistry for CAII on E11.5 epithelia grown for 7 days in Matrigel. Results. Early, undifferentiated embryonic pancreatic epithelium does not express CAII and CFTR by RT-PCR. When E11.5 epithelia were grown for 7 days in Matrigel, however, gene expression for both markers is upregulated as ducts form. Furthermore, CAII was seen by IHC and both CAII and CFTR were seen by in situ hybridization in the ducts after 7 days in Matrigel. Conclusions. These data validate our in vitro system 1 This work was supported by an American College of Surgeons Faculty Research Fellowship and the Juvenile Diabetes Foundation International. 2 To whom correspondence should be addressed at Children’s Mercy Hospital, 2401 Gilham Road, Kansas City, MO 64108.
0022-4804/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
as a model for studying the mechanism of normal pancreatic duct differentiation and may potentially help us to understand the faulty mechanism involved in pancreatic ductal carcinogenesis. © 2000 Academic Press Key Words: pancreas; ducts; development; in vitro. INTRODUCTION
Branching epithelial morphogenesis is an important mechanism of development in many organs, including the pancreas [1– 4]. The embryonic mouse pancreas first develops as an evagination of the foregut endoderm into the surrounding mesenchyme at Embryonic Day 9.5 (E9.5) [5]. The epithelium continues to branch and begins to differentiate into more mature exocrine structures (ducts and acini) by E14.5 [6]. Mesenchymal-epithelial interactions have been shown to be important for pancreatic growth and differentiation [5, 7–11]. Indeed, embryonic pancreas without its mesenchyme fails to develop into islets, ducts, or acini when grown on a filter [7]. Interestingly, epithelia isolated from its surrounding mesenchyme when grown under the renal capsule formed mature islets. In addition, isolated epithelia grown in a basementmembrane-rich gel formed primarily ducts [4]. These studies suggested the pluripotentiality of the early, undifferentiated pancreatic epithelia, and that they may be composed of stem cell precursors that can be driven toward exocrine or endocrine lineage, depending on the signals received from their environment. The concept that this undifferentiated epithelium can be manipulated to become endocrine cells has significant clinical implications with regard to the treatment and potential cure of diabetes mellitus. It is thought that pancreatic stem cells may arise from ducts [6, 12, 13]. Islet morphogenesis has been described as a budding of endocrine cells from ductal
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epithelium [14, 15], and there are many studies that describe the budding of islets and acini from ducts after distal pancreatectomy and pancreatic duct ligation in rats [16, 17]. Understanding the process of normal pancreatic duct differentiation, therefore, may be critical to understanding the biology of pancreatic stem cells. We have recently described the ontogeny of two pancreatic duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR) in the developing embryonic mouse pancreas [18]. In these studies, we demonstrated the absence of these ductal markers in early pancreatic epithelium using immunohistochemistry and in situ hybridization. Carbonic anhydrase II was first detected in the differentiating epithelium by E14.5, and both CAII and CFTR were expressed in the more mature ducts by E16.5. In order to better understand the mechanism of pancreatic duct differentiation, we have grown isolated, undifferentiated E11.5 epithelia in a basementmembrane-rich gel (Matrigel), which is rich in laminin-1. In this system, the epithelia have previously been shown to form primarily ducts [4]. We have recently shown that the cross-region of the laminin-1 molecule and the ␣6-containing integrin are necessary for pancreatic duct morphogenesis [19]; however, the mechanism of cytodifferentiation and morphogenesis of duct cells has never been characterized. We provide here a model of in vitro duct differentiation wherein we analyzed the expression of the ductal markers CAII and CFTR by PCR, in situ hybridization, and IHC, and show that their temporal expression in vitro parallels their in vivo expression, thus validating our model. MATERIALS AND METHODS Pancreas dissection and culture. Time-dated pregnant CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA). Noon on the day of vaginal plug discovery was considered Day 0.5 of gestation. All procedures were approved by the New York University Medical Center Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institute of Health Publication No. 86-23, revised 1985). Pancreases were harvested at E9.5– 12.5 and epithelia were isolated from surrounding mesenchyme from E10.5 and E11.5 specimens using 1% trypsin in serum-free media as described previously [20]. Isolated E11.5 pancreatic epithelia were grown on filter inserts (Millipore, Bedford, MA) and placed in standard 24-well plates under sterile conditions. Each well was filled with 500 l of filter-sterilized media containing 90% DMEM/F12K, 10% fetal bovine serum, and 1% antibiotic/antimycotic solution (penicillin G 10,000/ml, streptomycin sulfate 10,000 g/ml, Amphotericin B 200 g/ml (Gibco, Grand Island, NY). Filter inserts were filled with 150 l of basement membrane gel (Matrigel, Collaborative Research, Boston, MA), and isolated epithelia were transferred into the gel prior to gel polymerization using sterile siliconized tips. All epithelia were grown at 37° for 7 days. RT-PCR. Embryonic pancreases were microdissected and RNA was extracted from pooled whole pancreas at E9.5, from pooled epithelia at E10.5 and E11.5, and from pooled E11.5 epithelia grown in Matrigel for 7 days, using the S.N.A.P. total RNA isolation kit (Invitrogen, Carlsbad,
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CA). Reverse transcription was performed using the Superscript Preamplification System (Gibco) and PCR for -tubulin was used as control for each RNA extraction. PCR was then performed on the different aged cDNA for CAII and CFTR using the following primer pairs: CAII, 5⬘-GCAAGAGGCCATGTCTGCTC-3⬘ and 5⬘-GGAAGCGTGCGGCCTTTGCT-3⬘; CFTR, 5⬘-CAGTCATCTCTGCCTTGTGG-3⬘ and 5⬘ACGCTGACCTCCACTCAGTG-3⬘. The annealing temperature for both sets of primers was 58°C. Tissue preparation. After 7 days in culture, the E11.5 epithelia were fixed in 4% paraformaldehyde (in PBS) for 2 h and then cryoprotected in 30% sucrose (in PBS) overnight. For in situ hybridization, the dissections were performed in DEPC-treated PBS and RNAse-free conditions were used. Four percent paraformaldehyde was made with DEPC PBS and specimens were fixed for 8 h and then cryoprotected in 30% sucrose (in DEPC PBS) overnight. The epithelia were then excised from the filter inserts under direct microscopic vision, embedded in tissue freezing medium (Triangle Biomedical Science, Durham, NC), frozen in liquid nitrogen, and cut into 6-m sections using a Leica cryostat (Wetzlar, Germany). Immunohistochemistry. Sections were rehydrated with PBS, permeabilized in a Tween-20 solution (0.5%) in PBS for 10 min, and washed with PBS for 5 minutes. Heated sodium citrate (10 mM, pH 3.0) was then used for antigen recovery. The specimens were incubated with 4% blocking serum (donkey serum; Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 8 h. The sections were then incubated in primary antibody CAII (The Binding Site, Co., San Diego, CA) 1:500 in PBS overnight at 4°C followed by secondary antibody (Rhodamine donkey anti-sheep) 1:200 in PBS for 1 h. Specimens were washed in PBS after incubating with the secondary antibody and coverslips were applied using Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL). Mouse salivary gland was used as a positive control for the CAII antibody and nonimmune serum was used as a negative control. In situ hybridization. For probe construction/labeling, PCR was used to generate probes for mouse CAII and mouse CFTR. Whole embryo cDNA was used as the template for the PCR. CAII and CFTR primers (described above) were used to generate a 539- and a 369-bp fragment, respectively. These fragments were subcloned into a 3.9-kb pCRII vector using a TA cloning kit (Invitrogen). The restriction enzyme HindIII was used to linearize the plasmids containing CAII and CFTR, and a phenol/chloroform extraction was performed. The purified linearized plasmids were used to generate labeled RNA probes by performing in vitro transcription using T7 RNA polymerase and digoxigenin-11-dUTP. Mouse salivary gland and lung were used as positive controls for CAII and CFTR, respectively. Sense probes for CAII and CFTR were used as negative controls. Hybridization was performed on the pancreas frozen sections by covering them with 30 l of hybridization buffer containing 10 –30 ng of digoxigenin-labeled probe overnight in a humid chamber at 55°C. Posthybridization, the slides were washed and residual probe was digested with RNaseA. To detect the mRNA signal, the slides were subjected to 2 h of incubation with a 1:500 dilution of sheep anti-DIG-alkaline phosphatase. Finally, the slides were covered with 200 l of developing solution. After the colored signal had optimally developed, the reaction was stopped.
RESULTS
Isolated undifferentiated E11.5 pancreatic epithelium has the capacity to form either exocrine or endocrine structures. We have previously traced the ontog-
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FIG. 1. RT-PCR for CFTR and CAII on whole pancreases at E9.5, on isolated epithelia at E10.5 and E11.5, and on isolated E11.5 epithelia grown in Matrigel for 7 days. Note the absence of CFTR and CAII gene expression in early undifferentiated epithelia with upregulation of gene expression as ducts form in Matrigel.
eny of duct markers in vivo; however, a model of in vitro duct formation is necessary if we are to better characterize this process and potentially manipulate the system in order to control pancreatic lineage selection. We therefore grew isolated E11.5 epithelia in Matrigel for 7 days and then performed PCR, IHC, and in situ hybridization for the duct markers CAII and CFTR in order to validate this culture system as representative of in vivo duct formation. Reverse-transcription PCR revealed the absence of gene expression for both CAII and CFTR in E9.5 whole pancreas and in isolated epithelium at E10.5 and E11.5. After the growth of isolated E11.5 epithelia in Matrigel for 1 week, however, these genes were detectable by PCR in the epithelium (Fig. 1). Control RT-PCR
for -tubulin showed bands for E9.5 whole pancreas, E10.5 epithelia, and E11.5 epithelia (Fig. 2). These data demonstrate that in our culture system, there is upregulation or induction of gene expression for CAII and CFTR as ducts form from undifferentiated epithelia in vitro. Furthermore, in situ hybridization for CAII and CFTR showed strong expression of these markers in the ducts after E11.5 epithelia were grown in Matrigel for 1 week (Fig. 3). This expression supports the PCR data and is consistent with an upregulation or induction of duct marker gene expression during in vitro duct development. Finally, immunohistochemistry for CAII also showed expression in the large cystic ducts formed in culture (Fig. 4). Thus, the temporal expression of duct markers in our culture systems cor-
FIG. 2. -Tubulin control RT-PCR for E9.5 whole pancreases, E10.5 epithelia, and E11.5 epithelia. Note 650-bp band in the Rt ⫹ lane for each age.
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FIG. 3. In situ hybridization on E11.5 epithelia grown in Matrigel for 7 days for (a) CFTR and (b) CAII (400⫻). Note mRNA signal in the newly formed ducts (d). Hybridization with sense probes to CAII and CFTR yielded no signal (data not shown).
relates with their expression in vivo and validates our model. DISCUSSION
Early studies on development of the pancreas showed that embryonic pancreatic epithelia, when grown on filters in the absence of its mesenchyme, failed to form islets, ducts, or acini [7]. Other experiments in which pancreatic epithelia and mesenchyme were cultured on opposite sides of a porous filter showed growth and differentiation of the epithelia, suggesting that a soluble “mesenchymal” factor was essential for normal pancreatic development. Unfortunately, subsequent studies have failed to isolate this factor [21] leading investigators to pursue other aspects of mesenchymal-epithelial interactions in pancreatic development. In particular, contact between the epithelium and the mesenchyme has been shown to be critical for exocrine differentiation [9]. More recently, isolated undifferentiated pancreatic epithelia grown under different culture conditions demonstrated the capacity to select different lineages based on these external signals [20]. As previously shown [7], epithelia grown on filters in the absence of
mesenchyme failed to differentiate, both histologically and by immunohistochemical analysis. When these epithelia were grown in a three-dimensional basementmembrane gel, however, they differentiated into large, cystic ducts [20]. Moreover, when the epithelia were grown under the renal capsule, pure clusters of mature islets formed. These results represented the first report of pure islet differentiation and growth from pancreatic precursor cells. We recently investigated the importance of laminin-1, the major noncollagenous protein component of Matrigel and basement membrane, in the induction of exocrine lineage selection by undifferentiated epithelia. Laminin-1 has been shown to be important for branching epithelial morphogenesis in the kidney, lung, and salivary gland [1–3], and we showed by neutralizing antibody-blocking studies that the binding of the cross-region of the laminin-1 molecule to the ␣6-containing integrin was critical for duct morphogenesis in culture [19]. The nature of these in vitro ducts, however, has never been characterized. In order to better understand the mechanism of pancreatic ductal lineage selection, therefore, we wished to define the expression of ductal markers in vitro, in
FIG. 4. Immunofluorescence for CAII on E11.5 epithelia grown in Matrigel for 7 days at (a) 200⫻ and (b) 400⫻. Note strong staining in the newly formed ducts (arrows). Nonimmune serum yielded no staining (data not shown).
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undifferentiated pancreatic epithelia. We harvested embryonic mouse pancreases at Gestational Days 9.5– 11.5 and performed RT-PCR on isolated pooled pancreatic epithelia for CAII and CFTR. Expression of these markers was absent in the epithelia at these early ages. After being grown in Matrigel for 7 days, duct morphogenesis occurred and there was an activation of gene expression for both CAII and CFTR by RT-PCR. Furthermore, in situ hybridization for both ductal markers demonstrated a strong signal exclusively in the ducts, consistent with the PCR data. In addition, immunohistochemistry for CAII revealed intense staining in these ducts. The upregulation or induction of duct marker gene expression in the developing pancreatic epithelium validates our in vitro system as a model for studying embryonic pancreatic duct differentiation because it parallels normal duct development during gestation. We recently showed absence of CAII or CFTR expression by immunohistochemistry and in situ hybridization in pancreatic epithelium at E11.5 and E12.5, respectively, with an upregulation or induction of CAII and CFTR expression in the mature ducts by E16.5 [18]. Our validated in vitro system now allows us access to early, undifferentiated pancreatic precursor cells that can be manipulated in order to study the mechanism of pancreatic duct lineage selection. We are beginning to examine this period of differentiation in vitro in more detail by trying to determine if CAII expression alone is critical for ductal lineage selection, or whether factors controlling CAII expression are responsible for inducing pancreatic duct formation. REFERENCES Klein, G., Langegger, M., Timpl, R., and Ekblom, P. Role of laminin A chain in the development of epithelial cell polarity. Cell 55: 331, 1988. 2. Schuger, L., O’Shea, K. S., Nelson, B. B., and Varani, J. Organotypic arrangement of mouse embryonic lung cells on a basement membrane extract: Involvement of laminin. Development 110: 1091, 1990. 3. Kadoya, Y., Kadoya, K., Durbeej, M., Holmvall, K., Sorokin, L., and Ekblom, P. Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes. J. Cell Biol. 129: 521, 1995. 4. Gittes, G. K., Galante, P. E., Hanahan, D., Rutter, W. J., and Debase, H. T. Lineage-specific morphogenesis in the developing
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