ARTICLE |
Correspondence to: Lars-Inge Larsson, Dept. of Molecular Cell Biology, Statens Seruminstitut, Artillerivej 5, Bldg. 81, DK-2300 Copenhagen S, Denmark.
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Summary |
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We studied the distribution of the homeodomain proteins Pdx-1 and Nkx 6.1 in the developing rat pancreas. During early development, nuclear staining for both Pdx-1 and Nkx 6.1 occurred in most epithelial cells of the pancreatic anlage. Subsequently, Nkx 6.1 became more ß-cell-restricted, and Pdx-1 also occurred in other islet cell types and in the duodenal epithelium. During early pancreatic development, cells co-storing insulin and glucagon were regularly detected. The vast majority of these did not possess nuclear staining for either Pdx-1 or Nkx 6.1. Subsequently, cells storing insulin only appeared. Such cells displayed strongly Pdx-1- and Nkx 6.1-positive nuclei. Therefore, Nkx 6.1, like Pdx-1, may be an important factor in pancreatic development and in mature insulin cell function.
(J Histochem Cytochem 46:707715, 1998)
Key Words: homeodomain proteins, Nkx 6.1, Pdx-1, insulin, pancreatic polypeptide, glucagon
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Introduction |
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The pancreas develops as two buds (the ventral and dorsal anlage) emanating from the primitive foregut, which grow and fuse to form the adult pancreas (
Recently, a mouse homeobox protein, insulin promoter factor 1 (ipf1), was found to be required for development of the murine pancreas (
We decided to study the development of the islet hormone-producing cells in the rat pancreas in relation to the development of Pdx-1- and Nkx 6.1-immunore-active cells to determine the relation of these homeo-box gene products to specific hormonal phenotypes.
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Materials and Methods |
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Animals
Wistar rats (Pan:WIST; The Panum Institute, Copenhagen, Denmark) were mated overnight; noon of the day on which the vaginal plug was discovered was considered as Day 0.5 of gestation (E0.5). Pregnant female rats were sacrificed by CO2 and the fetuses were immersion-fixed overnight in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. With embryos older than E16.5, the abdominal cavity was opened before fixation. Rat embryos were examined at E11.0, E11.5, and at 1-day intervals from E11.5 to E20.5. Pancreases from postnatal rats 113 days of age (P1P13) were immersion-fixed overnight. Adult rats were intracardially perfused with the fixative and the pancreas immersion-fixed for 120 hr. Whole E11.0 and E11.5 embryos, the pancreatic region of E12.5 to E14.5 embryos, and the splenic and duodenal pancreases of E15.5 and older animals were cryoprotected overnight in 30% sucrose, mounted in Tissue-Tek, and frozen in N-hexane cooled by liquid nitrogen. Pancreatic tissue for RNA extraction was immediately transferred to 100 µl RNAzol extraction solution (Cinna/Biotecx; Houston, TX), homogenized, and RNA extracted as described by the manufacturer. At the earliest stage (E13.5), we obtained 2 µg total RNA from one full litter. At later stages, total yield increased.
Multiplex RT-PCR
Random-primed cDNA synthesis and multiplex RT-PCR were performed as described earlier (
Immunocytochemistry
Sections of 35 µm were stained by indirect immunofluorescence using rabbit antisera to recombinant glutathione-S-transferase (GST) fusion proteins incorporating either the C-terminal region of Pdx-1 (stf1) (Ab. 1856-5) or the C-terminal region of Nkx 6.1 (Ab. 174) (
Triple immunostainings for confocal microscopy were performed as above except that Cy5-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used instead of AMCA-labeled antibody. The confocal laserscan microscope (Molecular Dynamics; Sunnyvale, CA) was equipped with a x100, n.a. 1.40 Nikon objective and an argon/krypton laser. Scans were taken using a lateral xy resolution of 0.13 µm and a z resolution of 0.39 µm. In this way, hormone and homeobox protein coexistence could be established in thin optical sections.
For quadruple stainings, sections were first stained for Nkx 6.1 or Pdx-1 by the peroxidaseanti-peroxidase (PAP) method using diaminobenzidineH2O2 development (
Controls included conventional staining controls (
All staining and absorption controls were negative at all stages studied. Stainings with the Nkx 6.1 antiserum were eliminated by absorption against recombinant Nkx 6.1 but not with recombinant Pdx-1, whereas the reverse was true for the Pdx-1 antiserum. As a further control, double stainings for glucagon and PP employed poly-L-lysine (Mr 3600; Sigma, St Louis, MO) preabsorption of the primary antibodies (
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Results |
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Nkx 6.1 and Pdx-1 Immunoreactivities Are Present in Most Developing Early Pancreatic Anlage Cells but Show Different Distributions in the Duodenum
At the earliest fetal stage studied (22 somites, approximately E11.0) Pdx-1- and Nkx 6.1-immunoreactive nuclei were detected in epithelial cells of the dorsal (Figure 1AD) and ventral pancreatic buds. The nuclear staining for Pdx-1 was more intense than that for Nkx 6.1. At this stage, few and weakly stained glucagon-immunoreactive cells were observed in the dorsal but not in the ventral bud. The nuclei of the early glucagon cells did not stain for either Pdx-1 or Nkx 6.1, whereas most of the other epithelial cells of the dorsal and ventral buds contained Pdx-1- and Nkx 6.1-positive nuclei. By the 30-somite stage (approximately E11.5) the first insulin-immunoreactive cells were observed. All of the early insulin cells were also glucagon-positive. Only a few of these cells contained weakly Pdx-1- and Nkx 6.1-immunoreactive nuclei. Similarly, at this stage, only a few cells staining for glucagon (but not insulin) possessed weakly Pdx-1- and Nkx 6.1-positive nuclei. With both types of cells the frequency of nuclei staining for Pdx-1 approximated 10%, whereas the frequency of nuclei staining for Nkx 6.1 was somewhat lower. However, because at early stages of development the staining for Nkx 6.1 was weaker than that for Pdx-1, this difference may be more apparent than real. At all ages studied (E13.516.5 and P54) the duodenal epithelial cells contained Pdx-1-positive nuclei, whereas Nkx 6.1 was absent.
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Insulin Cells That Do Not Co-express Glucagon Are Characterized by Intense Nuclear Staining for Pdx-1 and Nkx 6.1
During E11.513.5, progressively more glucagon- and insulin-positive cells appeared. These cells were arranged in clusters of cells immunopositive for both insulin and glucagon or for glucagon only. By E13.5, the first insulin-positive cells that did not also stain for glucagonere observed. Such glucagon-negative insulin cells almost invariably displayed intense nuclear staining for Pdx-1 and Nkx 6.1. These cells were few up to E15.5 (Figure 1EL). Between E15.5 and about E18.5, some major changes in the number and organization of the different cell types were observed (Figure 1EL and Figure 2AC). First, the number of single-positive insulin cells possessing Pdx-1- and Nkx 6.1-immunoreactive nuclei increased dramatically and, concomitantly, the number of cells immunopositive for both glucagon and insulin decreased. Second, the frequency of the (weakly) Pdx-1- and Nkx 6.1-positive glucagon cells decreased to the levels observed in adults (<1%). Third, the nuclear staining for Pdx-1 and Nkx 6.1 in the hormone-negative epithelial cells decreased in intensity, and by E18.5 Pdx-1- and Nkx 6.1-positive nuclei were mainly observed in duct epithelium and in insulin cells (Figure 2AC). Finally, at E15.5 and E16.5 the majority of the glucagon-negative and Pdx-1- and Nkx 6.1-positive insulin cells appeared as single cells, closely associated with the hormone-negative and Pdx-1- and Nkx 6.1-positive epithelial cells, whereas the insulin and glucagon double-positive cells were found mainly within clusters of glucagon cells (Figure 1EL). By E18.5, insulinglucagon double-positive cells were rare, and the frequency of insulin cells now exceeded the frequency of glucagon cells. These insulin cells almost invariably displayed strongly Pdx-1- and Nkx 6.1-immunopositive nuclei and usually occurred in association with Pdx-1- and Nkx 6.1-positive epithelial duct cells (Figure 2AC). At this stage of development, the glucagon clusters assumed an elongated form, as if they were gradually beginning to form a mantle around the insulin cells. Formation of true islet-like structures could be observed during the following 23 days. In the adult pancreas, Pdx-1 and Nkx 6.1 immunoreactivities were restricted to islet cells (Figure 2D). In addition, extremely rare hormone-negative epithelial cells with Nkx 6.1- or Pdx-1-positive nuclei were occasionally detected in the vicinity of the islets, among exocrine cells, or in ducts.
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Quantitation of Insulin, Pdx-1, Nkx 6.1, and Gtx mRNA Expression During Pancreatic Development
Pancreatic specimens from E13.5E17.5 were analyzed by multiplex RT-PCR. At the earliest stage studied (E13.5) and during the following 2 days of development, low levels of insulin gene expression were detected (Figure 3A). From E15.5 to E17.5, the levels of insulin mRNA increased about 100-fold (inset in Figure 3A). Subsequently, from E17.5 to E19.5, no major changes in insulin gene expression were observed (Figure 3A). Two days after birth (P2), the levels of insulin mRNA were about 10 times the levels observed at E19.5 (Figure 3A). By E13.5, low levels of Nkx 6.1, Pdx-1, and Gtx mRNAs were detected (Figure 3B and Figure 3C). By E14.5 the levels of Pdx-1 and Nkx 6.1 mRNAs increased and then remained relatively constant until E16.5. During this period, the levels of Pdx-1 and Nkx 6.1 mRNAs were four to five times higher than those of Gtx (Figure 3C). By E17.5 the levels of all three mRNAs fell and then rose again by E18.5. Postnatally, significant expression of Nkx 6.1 and Pdx-1 was present in the pancreas but fell to very low levels in adult pancreas. Insulin expression followed a similar pattern, reflecting the development of the exocrine portion of the pancreas. In isolated islets from new-born rats, high levels of Pdx-1 and Nkx 6.1 mRNAs were detected, whereas Gtx mRNA was undetactable.
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Developing and Mature Somatostatin Cells Are Devoid of Nkx 6.1 but Show Labeling for Pdx-1
By E16.5, somatostatin cells were first observed. During E16.517.5, only a few somatostatin cells occurred and some of these cells also stored insulin (data not shown). No cells co-storing somatostatin and glucagon were detected. Pdx-1-positive nuclei were detected in some but not all somatostatin cells irrespective of whether or not they also stored insulin. No somatostatin cells containing Nkx 6.1-positive nuclei were found. By E18.5, somatostatin cells became much more numerous. At this stage, very few cells staining for both somatostatin and insulin were found. Almost all (>90%) somatostatin cells had Pdx-1-positive nuclei, whereas none had Nkx 6.1-positive nuclei. In addition, during the ensuing development no Nkx 6.1-positive somatostatin cells were seen. After E18.5, the frequency of Pdx-1-positive somatostatin cells decreased to the 1020% seen in adult pancreas. No cells staining for both insulin and somatostatin were detected after E19.5.
Developing PP Cells Frequently Contain Glucagon and Are Occasionally Positive for Pdx-1 but Rarely Positive for Nkx 6.1
By E20.5, the first PP-immunoreactive cells were detected. Occasional cells immunopositive for both insulin and PP were seen, but after P3 no such cells could be detected. In contrast, many cells were immunopositive for both glucagon and PP (Figure 2EG). During E21.5P5, counts revealed that the proportion of such double-positive cells increased from about 2030% of all PP cells (E21.5P2) to 6075% at P3P5. Subsequently, the frequency of double-positive cells decreased again. In adult pancreatic islets, variable numbers of cells immunopositive for both glucagon and PP (1738% of PP-positive cells) were seen in the duodenal portion and tail of the pancreas. At all stages of development, weak nuclear Pdx-1 staining could be detected in a few (<5%) PP cells. Most of these Pdx-1-positive PP cells were negative for both glucagon and insulin. Only extremely rare (<1%) PP cells contained weakly Nkx 6.1-positive nuclei. These were seen only in the perinatal period and not in adults. About half of the Nkx 6.1-positive PP cells were also immunoreactive for glucagon, but none stained for insulin.
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Discussion |
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This is the first report on the developmental expression of Nkx 6.1 in the pancreas. This homeodomain gene product was previously found to be present in the insulin cells of the adult pancreas and in RNA extracted from antropyloric mucosa of the stomach (
Against this background, it is very interesting to note the great parallels in Nkx 6.1 and Pdx-1 gene expression in the developing pancreas. During early development the majority of the parenchymal cells were immunopositive for both Pdx-1 and Nkx 6.1, and later during development both immunoreactivities became virtually restricted to islet cells. Pdx-1 showed a wider expression pattern than Nkx 6.1 and was also detected in the duodenal epithelium and in many non-ß-cells of the developing and adult pancreas. This pattern agrees with the hypothesis that Pdx-1, together with other factors, may activate Nkx 6.1 gene expression (cf.
We found the majority of the earliest glucagon- and insulin-immunopositive cells to be devoid of Nkx 6.1 and Pdx-1 staining. This contrasts to the report by
All the insulin cells that we observed during early development co-stored glucagon. The majority of these insulinglucagon double-positive cells were devoid of nuclear staining for Pdx-1 and Nkx 6.1 and occurred intermingled with cells positive for glucagon only. According to
It has been suggested that the insulinglucagon double-positive cells of the developing pancreas represent precursors of mature ß-cells (
In contrast to data obtained in the developing mouse pancreas (
Pdx-1 has also been characterized as an insulin gene transcription factor (
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Acknowledgments |
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Supported by grants from the Danish National Research Fund (Center for Gene Regulation and Plasticity in the Neuroendocrine Network), the Danish MRC, and the Danish Biotechnology Program (Research Center for Medical Biotechnology).
Received for publication August 18, 1997; accepted January 7, 1998.
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