Developmental Biology of the Psammomys obesus Pancreas: Cloning and Expression of the Neurogenin-3 Gene

Abstract

The desert gerbil Psammomys obesus, an established model of type 2 diabetes (T2D), has previously been shown to lack pancreatic and duodenal homeobox gene 1 (Pdx-1) expression. Pdx-1 deficiency leads to pancreas agenesis in both mice and humans. We have therefore further examined the pancreas of P. obesus during embryonic development. Using Pdx-1 antisera raised against evolutionary conserved epitopes, we failed to detect Pdx-1 immunoreactivity at any time points. However, at E14.5, Nkx6.1 immunoreactivity marks the nuclei of all epithelial cells of the ventral and dorsal pancreatic buds and the only endocrine cell types found at this time point are glucagon and PYY. At E18.5 the pancreas is well branched and both glucagon- and ghrelin-positive cells are scattered or found in clusters, whereas insulin-positive cells are not found. At E22.5, the acini of the exocrine pancreas are starting to mature, and amylase and carboxypeptidase A immunoreactivity is found scattered and not in all acini. Ghrelin-, glucagon-, PYY-, gastrin-, somatostatin (SS)-, pancreatic polypeptide (PP)-, and insulin-immunoreactive cells are found scattered or in small groups within or lining the developing ductal epithelium as marked by cytokeratin 19. Using degenerate PCR, the P. obesus Neurogenin-3 (Ngn-3) gene was cloned. Nucleotide and amino acid sequences show high homology with known Ngn-3 sequences. Using specific antiserum, we can observe that Ngn-3-immunoreactive cells are rare at E14.5 but readily detectable at E18.5 and E22.5. In conclusion, despite the lack of detection of Pdx-1, the P. obesus pancreas develops similarly to Muridae species, and the Ngn-3 sequence and expression pattern is highly conserved in P. obesus.

Keywords

neurogenin-3 type 2 diabetes developmental biology glucagon insulin pancreas sand rat

The gerbil Psammomys obesus is a promising animal model for the study of type 2 diabetes (T2D) (Hackel et al. 1967; Kalman et al. 2001). The natural habitat of the P. obesus is the arid zone of the Eastern Mediterranean region and North Africa from where it was collected in the 1960s. Our animals are from the colony in Jerusalem that was established in the 1970s when it was collected from the arid shores of the Dead Sea (Shafrir and Ziv 1998). In its native habitat, the P. obesus feeds on succulent leaves of, e.g., the salt bush, which is a low-energy diet (2.98 digestible kcal/g). When transferred to laboratory rodent chow (Purina 5008, 3.31 digestible kcal/g; Charles River Laboratories, Wilmington, MA), which is high in energy to the P. obesus, ∼80% of our population develops diabetes. Development of diabetes begins with hyperinsulinemia, after which hyperglycemia sets in and finally overt T2D occurs. This phenotypic pattern closely resembles the pattern observed in human populations, making the P. obesus a good model for the study of T2D (Walder et al. 2000).

Donev et al. (1989) found that the normoglycemic P. obesus pancreas expresses all the four major pancreatic hormones, and that two types of islets could be observed. These islets were called A islets or PP islets and could be distinguished by whether glucagon or pancreatic polypeptide (PP) was the prevalent cell type in the rims of the islets. PP islets were found to be most prevalent in the head of the pancreas, whereas A-type islets were mostly found in the tail portion of the pancreas (Donev et al. 1989). Interestingly, Pdx-1, a critical transcription factor for the formation of the pancreas and for the maintenance of β-cells, was shown to be deficient in the newborn P. obesus (Leibowitz et al. 2001). Several other well-characterized pancreatic factors, Nkx2.2 (NK2 transcription factor related, locus 2), Nkx6.1 (NK6 transcription factor related, locus 1), and ISL-1 (Lim-homeodomain protein Islet1) were all present in a normal distribution (Leibowitz et al. 2001). Our study is the first to investigate the hormone and transcription factor expression at multiple time points during development of P. obesus. In addition, we report on the cloning of the P. obesus Neurogenin-3 (Ngn-3) gene and localization of Ngn-3 immunoreactivity during development.

Materials and Methods

Animals

Animals were acquired from Hadassah Medical School (Jerusalem, Israel). All animals had free access to food (Koffolk 19560; Harlan Laboratories, Jerusalem, Israel) and water and were handled in accordance with the regulations specified under the Protection of Animals Act by the authorities in Denmark, European Union, and Novo Nordisk A/S. Several male and female P. obesus were housed together with one male and one female in each cage and with a barrier between the animals for the first 3 days. During the afternoon of the third day the barriers were removed, and on the fourth day the animals were separated. P. obesus has a gestation time of between 25 and 29 days in our housing environment, counting the day of separation as E0.5, and ∼25% of the females became pregnant. On the day of sacrifice (14.5, 18.5, and 22.5 days of gestation; n = 2, n = 4, n = 2, respectively), the animals were sedated with isoflurane, cervically dislocated, and the tissues removed. The time difference in embryonic days (E14.5 = E10.5, E18.5 = E12, and E22.5 = E17 in mice) in relation to the mouse differ due to the variable gestation period between the litters that we used in our studies.

Immunocytochemistry

The pancreas and parts of the intestines were removed from the embryos and fixed in 4% paraformaldehyde overnight. The tissue was then transferred to 70% ethanol, embedded in paraffin, and sectioned at 3-4 μm using the 2055 Autocut microtome from Leica Instruments GmbH (Wetzlar, Germany). In brief, the sections were deparaffinized and immunostained using the following protocol: 3% hydrogen peroxidase treatment for 5 min followed by microwave oven treatment at 650 W for 4 min and 160 W for 15 min in Tris-EGTA buffer, pH 9. The sections were then blocked in 10% normal donkey serum for 10 min and TNB blocking buffer (PerkinElmer; Hvidovre, Denmark) containing 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent for 30 min and incubated with a mixture of two or three primary antibodies overnight (Table 1). Next, the sections were incubated with secondary biotinylated antibody (1:200) for 30 min and horseradish peroxidase-streptavidin conjugate for 15 min and developed with tyramide signal amplification substrate (Cy3, 1:100; PerkinElmer) for 10 min to visualize one of the antibodies. The one or two other primary antibodies were visualized with Cy2 (1:500) and Cy5 (1:500) using highly cross-absorbed secondary fluorescent antibodies for multiple labeling (Jackson ImmunoResearch Laboratories; West Chester, PA). Between each step the sections were rinsed in PBS. All pictures were taken using Zeiss LSM 510 META scanning laser microscope (Carl Zeiss; Oberkochen, Germany).

Table 1

Antibody table

Antigen	Species	Origin	Dilution
Amylase	Rabbit	Sigma-Aldrich; St Louis, MO	1:250
β-catenin	Goat	Santa Cruz Biotechnology; Santa Cruz, CA	1:100
Carboxypeptidase A	Rabbit	Anawa Trading; Wangen, Switzerland	1:1000
Cytokeratin 19	Sheep	Anil Rustgi, University of Pennsylvania, Philadelphia, PA	1:100
Ghrelin	Goat	Santa Cruz Biotechnology	1:100
Glucagon	Guinea pig	Linco Research; St Charles, MO	1:500/1:1000
Glucagon	Mouse	New England Biolabs; Ipswich, MA	1:25/1:50
Glucagon	Rabbit	Abcam; Cambridge, UK	1:200
Insulin	Guinea pig	Abcam	1:500
ISL-1	Mus	Developmental Studies Hybridoma Bank; University of Iowa, Iowa City, IA	1:1500 TSA
Ki67	Mus	BD Biosciences; Erembodegem, Belgium	1:50
Ki67	Rabbit	Lab Vision; Suffolk, UK	1:50
Neurogenin-3	Mus	Antibody Core, BCBC (Zahn et al. 2004)	1:2000 TSA
Nkx2.2	Rabbit	Thomas M. Jessell, Columbia University, New York, NY	1:2000 TSA
Nkx6.1	Rabbit	Jensen et al. 1996	1:200/1:2000 TSA
Nkx6.1	Mouse	Antibody Core, BCBC (Pedersen et al. 2006)	1:200/1:2000 TSA
Pancreatic polypeptide	Guinea pig	Linco Research	1:1000
Pax6	Rabbit	Turque et al. 1994	1:2000 TSA
Pdx-1	Goat	BCBC, www.betacell.org	1:2000
Pdx-1	Rabbit	BCBC, www.betacell.org	1:5000
Pdx-1 253	Rabbit	Joel Habener, Harvard Medical School, Cambridge, MA	1:5000
Pdx-1 1858	Rabbit	Hagedorn Research Institute, Gentofte, Denmark	1:2000
PYY	Rabbit	Milab; Brønshøj, Denmark	1:1000
Somatostatin	Sheep	Greg Aponte, University of California, Berkeley, CA	1:1000

Gene Cloning

Embryonic pancreatic tissue was placed in RNA-later solution (Ambion; Austin, TX) overnight at 4C. RNA was prepared using the MicroRNA Isolation Kit (ZymoResearch; Hornby, Canada) for the E14.5 and the Qiagen Mini RNA Isolation kit (Qiagen Nordic; West Sussex, UK) for all other samples following the manufacturers' instructions. Cloning and sequencing of the Ngn-3 gene was done by Evrogen (Moscow, Russia). In brief, double-stranded cDNA was synthesized from total RNA extracted from P. obesus E18.5 tissue using the SMART (switching mechanism at the 5′ end of the RNA transcript) method (Clontech Laboratories; Mountain View, CA) (Zhu et al. 2001). Degenerate primers were designed on the basis of nucleotide and amino acid sequences from rat, mouse, and human and used for PCR amplification of the P. obesus Ngn-3 gene. The obtained fragment was cloned and sequenced, and new primers were designed for the subsequent 3′ and 5′ Step-Out rapid amplification of DNA ends (Matz et al. 1999). The resulting PCR products were cloned and sequenced. The Vector NTI (Invitrogen; Carlsbad, CA) software suite was used for sequence analysis and figure generation. The sequence of P. obesus Ngn-3 gene has been deposited in the GenBank database (accession number DQ790002).

In Situ Hybridization

Three-μm paraffin sections from E22.5 were deparaffinized and dehydrated in an ethanol series and rinsed in diethylpyrocarbonate-treated PBS. Sections were permeabilized with proteinase K (10 μg/ml) for 5 min followed by Triton X-100 for 30 min and rinsed in 5X sodium chloride sodium citrate (SSC) buffer. Slides were prehybridized at 60C for 1 hr followed by overnight incubation with antisense, sense, or a rat glucagon-positive control probe. Slides were rinsed with 5X, followed by 2X, followed by 1X SSC, and stored in TBS-Tween 20 (TBST). After 1 hr blocking in 10% normal sheep serum in TBST, the sheep anti-digoxigenin antiserum (1:2500) was added overnight at room temperature. Slides were rinsed in TBST followed by sodium chloride Tris-HCl magnesium chloride Triton X-100 (NTMT) and incubated with substrate nitrotetrazolium blue chloride/5-bromo-4-chloro-3-indolyl phosphate at 37C for 4 hr followed by overnight incubation at room temperature. The reaction was stopped in Tris-EGTA buffer, and the slides were rinsed in PBS and immunostained with guinea pig anti-insulin and rabbit anti-glucagon antibodies.

Results

Immunostainings of E14.5

Embryonic tissue from day 14.5, which is approximately equivalent to mouse E10-10.5, was immunostained for the transcription factors Nkx6.1, ISL-1, and Ngn-3 and for β-catenin, glucagon, and PYY. The tissue did not stain with any Pdx-1 antibodies including those raised against N-terminally highly conserved epitopes (data not shown); hence, Nkx6.1 was used to identify the pancreatic epithelium. At E14.5, Nkx6.1 stains the entire pancreatic buds (Figures 1A, 1C, and 1E) including all the cells that will become endocrine, exocrine, and ductal. Nkx2.2 is broadly expressed in most of the dorsal bud and stains most strongly in the glucagon cells (Figure 1B). ISL-1 stains the mesenchymal nuclei surrounding the dorsal pancreatic bud (Figure 1C) as well as the nuclei of endocrine cells (only glucagon cells are detected at E14.5, data not shown), as expected. Most glucagon cells express PYY, but a few single hormone-expressing cells are also observed (Figure 1D). No other endocrine cells can be found at E14.5 (data not shown). β-catenin is involved in cell-cell adhesion and stains the pancreatic epithelium strongly and the surrounding mesenchyme more weakly (Figure 1E). Ngn-3 immunoreactive cells, which are the progenitors for all pancreatic endocrine cells, are quite rare at this time point and do not colocalize with Nkx6.1 (Figure 1E).

Immunostaining of E18.5

The pancreas is well branched at E18.5 (approximately equivalent to mouse E12.5), and the epithelium stains strongly for both cytokeratin 19 and β-catenin (Figures 2A and 2B). Pdx-1 immunoreactivity remained absent. Nkx6.1 cells are now scattered in the epithelium and do not colocalize with glucagon (Figure 2C). Ngn-3-positive cells were readily detectable at this time point (Figures 2A and 2B). Some Ngn-3-positive cells colocalize with Nkx6.1 and mark cells that have become restricted to an endocrine cell fate. The cells that are only Nkx6.1 positive are either less differentiated and earlier in development than the previously mentioned double-positive cells, or they are more differentiated cells that have lost Ngn-3 expression and are on their way to becoming β-cells (Figure 2B). Glucagon- (Figures 2C and 2D) and ghrelin-positive cells (data not shown) are scattered or found in clusters, whereas insulin-, somatostatin-, and PP-positive cells are not found (data not shown). Pax6 (paired box gene 6), an early endocrine cell marker, colocalizes with glucagon (Figure 2D).

Immunostainings of E22.5

At E22.5, which is approximately equivalent to mouse E17, the acini of the exocrine pancreas are starting to mature, and carboxypeptidase A and amylase immunoreactivity are found scattered and not in all acini (Figures 3A and 3B). Insulin-, glucagon-, somatostatin-, PP-, and ghrelin-immunoreactive cells are now found scattered or in small groups within or lining the developing ductal epithelium (Figures 3C and 3D). PYY and gastrin are also observed at this time point (data not shown). Nkx2.2, Pax6, and ISL-1 are expressed in most endocrine cell types including insulin- and glucagon-positive cells (Figures 3E-3G). Nkx6.1 colocalizes now only with the insulin-immunoreactive cells (Figure 3H). Double-staining with Ki67 antibodies in insulin- or glucagon-positive cells shows that endocrine cells do not divide at this time point (Figure 3I).

Figure 1

Analysis of pancreatic markers at E14.5. (A) Nkx6.1 marks the dorsal and ventral pancreatic buds. A few glucagon-positive cells can be observed in the ventral bud. Glucagon cells are not Nkx6.1-positive. (B) Nkx2.2 is more broadly expressed in epithelial cells (dorsal pancreatic bud) and also colocalizes with glucagon. (C) Nkx6.1 immunoreactivity is confined to the epithelium, whereas ISL-1 stains the mesenchyme surrounding the dorsal pancreas. (D) PYY and glucagon are colocalized, but some single hormone-positive cells are also observed (white arrows). (E) β-catenin stains the pancreatic epithelium and rare Ngn-3-positive cells that do not colocalize with Nkx6.1 can be found at this time point. dp, dorsal pancreatic bud; vp, ventral pancreatic bud. Arrows denote non-colocalized cells, whereas arrowheads depict colocalized cells.

Cloning and Expression of Ngn-3

The sequence of the P. obesus Ngn-3 gene was obtained using degenerate primers designed for conserved areas in the human, mouse, and rat Ngn-3 genes. By comparing the obtained sequence of Ngn-3 from the P. obesus with that of the human, mouse and rat, it is found that the P. obesus gene shows 78.7%, 85.7%, and 87.7% nucleotide homology to the previously mentioned species, respectively. The homology is also high examining the amino acid sequences, being 73.1% identical to human, 82.8% to mouse, and 85.6% to the rat proteins (Table 2).

Using in situ hybridization, we localized the endogenous Ngn-3 gene at E22.5 in scattered cells in the epithelium (Figures 4A and 4B). Double immunostaining on top of the in situ hybridization showed that the Ngn-3 gene did not colocalize with insulin and glucagon (Figure 4C) and showed the same pattern of scattered cells as was observed for the protein (Figure 4F). Using an antiserum directed against the mouse Ngn-3 protein, a more detailed analysis of Ngn-3 protein immunoreactivity was investigated. Ngn-3 cells are scattered in the epithelium at this time point and most do not express Nkx6.1 (Figure 4D), whose expression is becoming more restricted to the insulin-immunoreactive cells. Immunostaining for Ki67, an s-phase marker of dividing cells, and phosphohistone H3, also a marker of dividing cells, showed that the Ngn-3-immunoreactive cells and the endocrine cells are postmitotic at E22.5 (Figure 4E).

Figure 2

Analysis of pancreatic markers at E18.5. (A,B) Cytokeratin 19 and β-catenin antisera stain the branching epithelium, whereas Nkx6.1 now only labels scattered epithelial cells. Ngn-3-immunoreactive cells are also scattered in the epithelium, and most of these do not colocalize with Nkx6.1-immunoreactive cells. (C,D) Glucagon-immunoreactive cells are scattered in the epithelium and express Pax6 but not Nkx6.1. Arrows denote non-colocalized cells, whereas arrowheads depict colocalized cells.

Discussion

Psammomys obesus belongs to a family of desert gerbils and is used as a model of human T2D. The absence of Pdx-1-like immunoreactivity remains a mystery and inspired us to further study how the pancreas develops in these animals compared with mice and rats. To our knowledge, this is the first comprehensive report on the development of the embryonic pancreas from any of the desert gerbil families (Meriones, Sekeetamys, Desmodillius, Pachyuromys, and Psammomys). We have demonstrated that during early development, E14.5 (equivalent to mouse E10-10.5), the P. obesus pancreas appears morphologically like wild-type mice and rats, and Nkx6.1 and β-catenin are good markers of the early pancreatic epithelium. At this time point, as in mice, we see very few hormone-positive cells, with only glucagon and PYY immunoreactive cells being present. This differs slightly from several mouse species where ghrelin-immunoreactive cells are observed at this time point (Prado et al. 2004; Heller et al. 2005). As previously reported in other rodent species, we also observed that most glucagon cells express PYY (Upchurch et al. 1994; Jackerott et al. 1996). In addition, in agreement with Leibowitz et al. (2001), we were unable to detect Pdx-1 immunoreactivity using several Pdx-1 antisera.

At E18.5 (approximately equivalent to E12.5 in mice), very few endocrine cells are present and most stain for glucagon. Nkx6.1 expression is now scattered in the epithelium as are other markers of endocrine cells, Nkx2.2 and Pax6. No exocrine pancreatic markers were observed. At embryonic day 22.5 (approximately equivalent to E17 in mice), we observed all the other endocrine cell types (insulin, somatostatin, PP, and ghrelin). In addition, we can observe cells immunoreactive for PYY and gastrin, hormones that are transiently expressed in the pancreas during development and are thought to be growth promoting (Gittes et al. 1993; Upchurch et al. 1994). This is the first time when amylase and carboxypeptidase A immunoreactivity is observed in the developing acini, but the pattern is quite heterogeneous, suggesting that the expression is just beginning and is slightly delayed as compared with mice and rats. This difference could also be reflected in the extremely variable gestation time of P. obesus.

Ngn-3 is the prerequisite gene for endocrine cell development (Gradwohl et al. 2000; Schwitzgebel et al. 2000). We have successfully cloned and characterized the expression of the P. obesus gene. The gene is highly homologous to all published Ngn-3 sequences. Using in situ hybridization, we could demonstrate that the Ngn-3 mRNA appeared in scattered cells in the epithelium and did not colocalize with either insulin or glucagon as has also been observed in mice (Jensen et al. 2000). Using immunohistochemistry, the Ngn-3 protein appears in a scattered pattern throughout the developing epithelium, in agreement with the in situ hybridization. At E14.5 Ngn-3-immunoreactive cells were very rare, but beginning at E18.5 we observed quite a few cells and these never colocalized with hormone-producing cells. Using two different markers of proliferation we could observe that the Ngn-3-positive cells were not proliferating, which is also what is observed in mice (Gradwohl et al. 2000; Jensen et al. 2000; Schwitzgebel et al. 2000).

Figure 3

Analysis of pancreatic markers at E22.5. (A,B) Cytokeratin 19 immunoreactivity is now confined to the developing duct structures and immunoreactivity of exocrine pancreatic markers, carboxypeptidase A and amylase, starts to appear at this time point. (C,D) The hormones glucagon, insulin, somatostatin, PP, and ghrelin are all evident at this time point. PP-immunoreactive cells are weakly detectable. (E-G) The transcription factors Nkx2.2, Pax6, and ISL-1 are immunolocalized in the nucleus of the glucagon and insulin-positive cells. (H) Nkx6.1-immunoreactive cells are scattered in the epithelium and colocalize with insulin but not glucagon cells. (I) Insulin and glucagon cells are very rarely found to be dividing at this time point as marked by Ki67 immunoreactivity. White arrow denotes dividing insulin cell. CPA, carboxypeptidase A; PP, pancreatic polypeptide; SS, somatostatin.

Table 2

Percentage similarity among different species at the protein level

	Homo	Mus	Rattus	Psammomys
Homo sapiens Ngn3	100	75	77	73
Mus musculus Ngn3		100	94	83
Rattus norvegicus Ngn3			100	86
Psammomys obesus Ngn3				100

In conclusion, the P. obesus pancreas shows a strikingly normal development of the endocrine, exocrine, and ductal tissue in the pancreas despite the apparent absence of Pdx-1. All the normal transcription factors we analyzed, except for Pdx-1, are expressed at a similar timing as has been reported in mice and rats. Because the lack of Pdx-1 in mouse (Jonsson et al. 1994; Offield et al. 1996) and human (Stoffers et al. 1997) leads to pancreas agenesis, we hypothesize that either Pdx-1 exists in P. obesus but is sufficiently mutated to escape immunological detection, or alternatively a related homeodomain protein may compensate in P. obesus for a functional deletion of Pdx-1 activity. Such compensation may be partial and restricted primarily to cover Pdx-1 function in pancreas development—and thus in part also explain the diabetic phenotype that may have been instrumental in selective survival in desert-like habitats.

Figure 4

Expression of the Ngn-3 mRNA and protein at E22.5. (A-C) In situ hybridization with Ngn-3 antisense probe shows a pattern of scattered cells. (B) Enlargement of the area boxed in A. (C) Insulin and glucagon immunostaining on the same section as A. Ngn-3 in situ signal is pseudocolored in purple. (D,E) Immunostaining of P. obesus E22.5 tissue with Ngn-3 antiserum shows that these cells sometimes colocalize with Nkx 6.1 at this time point and are not dividing. (F) Insulin, glucagon, and Ngn-3 immunostaining on E22.5. Compare with C. Arrows depict cells with a specific in situ hybridization signal.

Footnotes

Acknowledgements

This work is supported by an ErhvervsPhD award to Louise Vedtofte by Ministeriet for Videnskab, Teknologi og Udvikling, Denmark.

The authors thank Hanne Jensen-Holm, Thorhallur Tomas Buchholz, and Mikkel Schrøder Jørgensen for technical assistance with obtaining time-mated P. obesus, and Susanne Primdahl for assistance with tissue processing and embedding. The authors also thank Dr. Ole D. Madsen for critical feedback on this manuscript. The ISL-1 monoclonal antibody developed by Thomas M. Jessell was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA.

References

Donev

Petkov

Marquie

Duhault

Jablenska

(1989) Immunohistochemical investigations of the endocrine pancreas in normoglycemic sand rats (Psammomys obesus). Acta Diabetol Lat 26:309–313

Gittes

Rutter

Debas

(1993) Initiation of gastrin expression during the development of the mouse pancreas. Am J Surg 165:23–25

Gradwohl

Dierich

LeMeur

Guillemot

(2000) Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci USA 97:1607–1611

Hackel

Mikat

Lebovitz

Schmidt-Nielsen

Horton

Kinney

(1967) The sand rat (Psammomys obesus) as an experimental animal in studies of diabetes mellitus. Diabetologia 3:130–134

Heller

Jenny

Collombat

Mansouri

Tomasetto

Madsen

Mellitzer

. (2005) Genetic determinants of pancreatic epsilon-cell development. Dev Biol 286:217–224

Jackerott

Oster

Larsson

(1996) PYY in developing murine is let cells: comparisons to development of islet hormones, NPY, and BrdU incorporation. J Histochem Cytochem 44:809–817

Jensen

Heller

Funder-Nielsen

Pedersen

Lindsell

Weinmaster

Madsen

. (2000) Independent development of pancreatic alpha- and beta-cells from neurogenin 3-expressing precursors: a role for the notch pathway in repression of premature differentiation. Diabetes 49:163–176

Jensen

Serup

Karlsen

Nielsen

Madsen

(1996) mRNA profiling of rat islet tumors reveals nkx 6.1 as a beta-cell-specific homeodomain transcription factor. J Biol Chem 271:18749–18758

Jonsson

Carlsson

Edlund

(1994) Insulin-promoterfactor 1 is required for pancreas development in mice. Nature 371:606–609

10.

Kalman

Ziv

Shafrir

Bar-On

Perez

(2001) Psammomys obesus and the albino rat—two different models of nutritional insulin resistance, representing two different types of human populations. Lab Anim 35:346–352

11.

Leibowitz

Ferber

Apelqvist

Edlund

Gross

Cerasi

Melloul

. (2001) IPF1/PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal with type 2 diabetes. Diabetes 50:1799–1806

12.

Matz

Shagin

Bogdanova

Britanova

Lukyanov

Diatchenko

Chenchik

(1999) Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res 27:1558–1560

13.

Offield

Jetton

Labosky

Ray

Stein

Magnuson

Hogan

. (1996) PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983–995

14.

Pedersen

Klinck

Hecksher-Sorensen

Zahn

Madsen

Serup

Jorgensen

(2006) Generation and characterization of monoclonal antibodies against the transcription factor Nkx6.1. J Histochem Cytochem 54:567–574

15.

Prado

Pugh-Bernard

Elghazi

Sosa-Pineda

Sussel

(2004) Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci USA 101:2924–2929

16.

Schwitzgebel

Scheel

Conners

Kalamaras

Lee

Anderson

Sussel

. (2000) Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 127:3533–3542

17.

Shafrir

Ziv

(1998) Cellular mechanism of nutritionally induced insulin resistance: the desert rodent Psammomys obesus and other animals in which insulin resistance leads to detrimental outcome. J Basic Clin Physiol Pharmacol 9:347–385

18.

Stoffers

Zinkin

Stanojevic

Clarke

Habener

(1997) Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 15:106–110

19.

Turque

Plaza

Radvanyi

Carriere

Saule

(1994) Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells. Mol Endocrinol 8:929–938

20.

Upchurch

Aponte

Leiter

(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

21.

Walder

Fahey

Morton

Zimmet

Collier

(2000) Characterization of obesity phenotypes in Psammomys obesus (Israeli sand rats). Int J Exp Diabetes Res 1:177–184

22.

Zahn

Hecksher-Sorensen

Pedersen

Serup

Madsen

(2004) Generation of monoclonal antibodies against mouse neurogenin 3: a new immunocytochemical tool to study the pancreatic endocrine progenitor cell. Hybrid Hybridomics 23:385–388

23.

Zhu

Machleder

Chenchik

Siebert

(2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30:892–897