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Correspondence to: Lars-Inge Larsson, Dept. of Molecular Cell Biology, Statens Seruminstitut, 5, Artillerivej, DK-2300 Copenhagen S, Denmark.
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Summary |
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The homeobox gene product Nkx 6.1 is of unknown function but is expressed in the pancreas and the antropyloric mucosa of the stomach. In the adult pancreas, Nkx 6.1 possesses an insulin cell-restricted distribution, whereas its localization in the stomach is unknown. We now show that the vast majority of serotonin-producing enterochromaffin cells of the antropyloric mucosa contain Nkx 6.1-immunoreactive nuclei. In addition, a subpopulation of cells co-storing serotonin and gastrin display Nkx 6.1-positive nuclei. Such cells have been postulated to represent precursors of mature gastrin and serotonin cells. The nuclei of the co-storing cells have previously also been found to be positive for another homeodomain protein, Pdx-1. Pdx-1-deficient animals were therefore investigated and were found to be devoid of Nkx 6.1-positive nuclei. Our data show that Pdx-1 is needed for Nkx 6.1 expression and suggest a role for Nkx 6.1 in the maturation of gastrin- and serotonin-positive precursor cells. (J Histochem Cytochem 46:717721, 1998)
Key Words: Nkx6.1, serotonin, gastrin, Pdx-1, homeodomain proteins, gastrointestinal tract
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Introduction |
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The gastrointestinal tract contains many different types of endocrine cells producing different regulatory peptides and amines. These cell types are not randomly distributed along the gastrointestinal tract but occur in different patterns and constellations in different specialized regions. This pattern, as it is established in the adult gut, can become disrupted during neoplastic transformation, leading to hormone expression in ectopic sites. Therefore, although gastrin is not expressed in the normal adult pancreas, gastrin-producing tumors are more common in the pancreas than in the stomach (
It is therefore of interest to determine which factors control the expression of gastrointestinal hormones. We have recently identified two homeobox proteins, isl-1 and Pdx-1, as being associated with endocrine cells of the gastric mucosa (
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Materials and Methods |
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Animals
Pdx1-deficient mice were generated by deletion of exon 2, which encodes the homeodomain of Pdx-1 (ipf1), using homologous recombination in embryonal stem cells as described by
Immunocytochemistry
Cryostat or paraffin sections of 35 µm were pretreated by three 5-min cycles in 10 mM citrate buffer, pH 6.0, at full power setting (780 W) in a Miehle PP-780 laboratory microwave oven (Miehle & Cie; Gütersloh, Germany) and were submitted to indirect immunofluorescence staining using rabbit antiserum to a recombinant glutathione-S-transferase (GST) fusion protein incorporating the C-terminal region of rat Nkx 6.1 (Ab.174) (
For some multiple stainings, sections were initially stained for Nkx 6.1 using the peroxidaseanti-peroxidase (PAP) method of
Multiplex RT-PCR
cDNA and multiplex reverse transcriptase polymerase chain reaction (RT-PCR) was performed as described using previously published primer sequences (
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Results |
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The Nkx 6.1 antiserum stained scattered nuclei in the epithelium of the antropyloric glands of the adult mouse and rat gastric mucosa (Figure 1A), but revealed no positive nuclei in the duodenum. Absorption of the antiserum with recombinant Nkx 6.1, but not with recombinant Pdx-1, abolished all staining. Similar results were obtained in mouse and rat stomachs. Therefore, in the following, the description refers to both species.
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Staining for nuclear Nkx 6.1 immunoreactivity and for the gastric hormones gastrin, somatostatin, and serotonin permitted identification of the Nkx 6.1-positive cells as serotonin-immunoreactive enterochromaffin cells (Figure 1A and Figure 1B). The majority of cells (>90%) with Nkx 6.1-positive nuclei were serotonin-immunoreactive, whereas the remaining were negative for serotonin, gastrin, and somatostatin. The majority (>90%) of antropyloric serotonin cells were also Nkx 6.1-positive. All serotonin cells in the duodenum, as well as serotonin-containing mast cells, were Nkx 6.1-negative. No somatostatin cells displayed Nkx 6.1-positive nuclei, but a small subpopulation (<1%) of gastrin-immunoreactive cells possessed Nkx 6.1-positive nuclei. These cells were invariably also serotonin-immunoreactive. Two different subtypes of gastrinserotonin double-positive cells could be defined by their nuclear Nkx 6.1 reactivity. One population was characterized by strong gastrin and weaker serotonin immunoreactivity and no nuclear staining for Nkx 6.1 (Figure 1CF), and the other was characterized by weaker gastrin immunoreactivity, strong serotonin immunoreactivity and nuclear Nkx 6.1 staining (Figure 1GI). Somatostatin cells co-storing serotonin were also detected, albeit infrequently. These cells were Nkx 6.1-negative.
In newborn mice, only very few Nkx 6.1-positive nuclei were seen. Double staining showed that, in newborns, most serotonin cells were Nkx 6.1-negative. Antropyloric mucosa from Pdx-1-deficient and control (wild-type) mice were stained in parallel for Nkx 6.1. No Nkx 6.1-positive nuclei were present in the antropyloric mucosa of any of the eight Pdx-1-deficient mice investigated (eight sections per animal; in all, 99 mm length of mucosa studied), whereas Nkx 6.1-positive nuclei were observed in each of the seven wild-type mice studied (mean ± SD 0.17 ± 0.09 nuclei/mm length of mucosa; eight sections/animal; in all, 89 mm mucosa studied). This difference was significant (p=0.0006, MannWhitney U-test).
Multiplex RT-PCR revealed that no gastrin mRNA could be detected in the Pdx-1-deficient mice, whereas it was present in wild-type mice. Somatostatin and Gtx mRNA levels were the same in both types of animals, but Nkx 6.1 mRNA levels could not be detected. Analyses of adult rat antropyloric specimens revealed that both Nkx 6.1 and Gtx were present at comparable levels.
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Discussion |
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In gastric glands, cell renewal takes place in the regenerative zone (isthmus) by division of a single stem cell. Daughter cells migrate upwards to form surface epithelium and downwards to differentiate into exocrine and endocrine cells. In the antropyloric mucosa, three main types of endocrine cells occur: gastrin, somatostatin, and serotonin cells. We have previously shown that presumptive endocrine stem cells in the isthmus are characterized by co-expression of the gastrin and somatostatin genes (
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Our present results document the presence of Nkx 6.1 immunoreactivity in the majority of antropyloric serotonin cells. Becauase Nkx 6.1 shows sequence similarities to another homeodomain protein, Gtx (
The presence of Nkx 6.1-positive nuclei in the gastric endocrine cell pedigree enables us to elaborate on our scheme (
Because Pdx-1-deficient mice are deficient in gastrin cells but contain increased numbers of serotonin cells (
Our data therefore suggest that a normally functioning Pdx-1 gene is necessary for Nkx 6.1 expression. Interestingly, during pancreatic development we have found Pdx-1 and Nkx 6.1 immunoreactivities in the same developing parenchymal cells. During early development, staining for Nkx 6.1 was weaker than for Pdx-1, and this was parallelled by correspondingly lower mRNA levels (
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Acknowledgments |
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Supported by the Danish National Research Foundation.
Received for publication August 18, 1997; accepted January 7, 1998.
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Literature Cited |
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