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Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage

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ABSTRACT

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt’s base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus–surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt–villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

Electronic supplementary material: The online version of this article (doi:10.1007/s00418-016-1453-4) contains supplementary material, which is available to authorized users.

No MeSH data available.


Guca2a and Guca2b expression by cells of the secretory lineage in the duodenum and colon. aGuca2a expression in Paneth cells in duodenum. bGuca2a expression in cells at the base and the neck region of the crypts in colon. cGuca2a expression in a duodenal brush cell. dGuca2a expression in a duodenal goblet cell. e Costaining of Guca2a transcript (dark brown) and lectin UEA1-binding fucose glycoproteins (bright red) in duodenum. fGuca2b expression in Paneth cells in duodenum. gGuca2b expression in cells at the base of the crypts in colon. hGuca2b expression in a duodenal brush cell. No Guca2b transcript was observed in goblet cells. i, jGuca2b levels were generally low in columnar cells, but comparatively high in columnar cells adjoining goblet cells. B brush cell, G goblet cell, P Paneth cell
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Fig5: Guca2a and Guca2b expression by cells of the secretory lineage in the duodenum and colon. aGuca2a expression in Paneth cells in duodenum. bGuca2a expression in cells at the base and the neck region of the crypts in colon. cGuca2a expression in a duodenal brush cell. dGuca2a expression in a duodenal goblet cell. e Costaining of Guca2a transcript (dark brown) and lectin UEA1-binding fucose glycoproteins (bright red) in duodenum. fGuca2b expression in Paneth cells in duodenum. gGuca2b expression in cells at the base of the crypts in colon. hGuca2b expression in a duodenal brush cell. No Guca2b transcript was observed in goblet cells. i, jGuca2b levels were generally low in columnar cells, but comparatively high in columnar cells adjoining goblet cells. B brush cell, G goblet cell, P Paneth cell

Mentions: In the small intestine, the Guca2a- and Guca2b-expressing cells at the base of the crypt were found to contain secretory granules and (in part) colocalized with Paneth cells (Fig. 5a, f). In contrast, Guca2a or Guca2b expression was not observed in the adjacent stem cells. A previous in situ hybridization study suggested that apparent Guca2b localization at the base of the crypts was a result of autofluorescence of the Paneth cells (Whitaker et al. 1997), but since RNAscope uses an enzymatic detection method, this cannot have confounded our observations. Moreover, our detection of Guca2a and Guca2b in Paneth cells is consistent with studies using a fluorescence-activated cell sorting technique to isolate specific epithelial cell lineages (Sato et al. 2011; Cash et al. 2006). This showed that Guca2a and Guca2b rank in the 90th percentile of most highly expressed genes in murine Paneth cells, and that Paneth cells are highly enriched in these transcripts, relative to stem cells (NCBI GEO datasets GSE5156 and GSE25109). In addition, these datasets reveal that Gucy2c is expressed at near-equal levels in Paneth and stem cells, congruent with the relatively uniform distribution of Gucy2c transcript in crypt epithelium (Fig. 4k–n).Fig. 5


Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage
Guca2a and Guca2b expression by cells of the secretory lineage in the duodenum and colon. aGuca2a expression in Paneth cells in duodenum. bGuca2a expression in cells at the base and the neck region of the crypts in colon. cGuca2a expression in a duodenal brush cell. dGuca2a expression in a duodenal goblet cell. e Costaining of Guca2a transcript (dark brown) and lectin UEA1-binding fucose glycoproteins (bright red) in duodenum. fGuca2b expression in Paneth cells in duodenum. gGuca2b expression in cells at the base of the crypts in colon. hGuca2b expression in a duodenal brush cell. No Guca2b transcript was observed in goblet cells. i, jGuca2b levels were generally low in columnar cells, but comparatively high in columnar cells adjoining goblet cells. B brush cell, G goblet cell, P Paneth cell
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Fig5: Guca2a and Guca2b expression by cells of the secretory lineage in the duodenum and colon. aGuca2a expression in Paneth cells in duodenum. bGuca2a expression in cells at the base and the neck region of the crypts in colon. cGuca2a expression in a duodenal brush cell. dGuca2a expression in a duodenal goblet cell. e Costaining of Guca2a transcript (dark brown) and lectin UEA1-binding fucose glycoproteins (bright red) in duodenum. fGuca2b expression in Paneth cells in duodenum. gGuca2b expression in cells at the base of the crypts in colon. hGuca2b expression in a duodenal brush cell. No Guca2b transcript was observed in goblet cells. i, jGuca2b levels were generally low in columnar cells, but comparatively high in columnar cells adjoining goblet cells. B brush cell, G goblet cell, P Paneth cell
Mentions: In the small intestine, the Guca2a- and Guca2b-expressing cells at the base of the crypt were found to contain secretory granules and (in part) colocalized with Paneth cells (Fig. 5a, f). In contrast, Guca2a or Guca2b expression was not observed in the adjacent stem cells. A previous in situ hybridization study suggested that apparent Guca2b localization at the base of the crypts was a result of autofluorescence of the Paneth cells (Whitaker et al. 1997), but since RNAscope uses an enzymatic detection method, this cannot have confounded our observations. Moreover, our detection of Guca2a and Guca2b in Paneth cells is consistent with studies using a fluorescence-activated cell sorting technique to isolate specific epithelial cell lineages (Sato et al. 2011; Cash et al. 2006). This showed that Guca2a and Guca2b rank in the 90th percentile of most highly expressed genes in murine Paneth cells, and that Paneth cells are highly enriched in these transcripts, relative to stem cells (NCBI GEO datasets GSE5156 and GSE25109). In addition, these datasets reveal that Gucy2c is expressed at near-equal levels in Paneth and stem cells, congruent with the relatively uniform distribution of Gucy2c transcript in crypt epithelium (Fig. 4k–n).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt’s base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus–surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt–villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

Electronic supplementary material: The online version of this article (doi:10.1007/s00418-016-1453-4) contains supplementary material, which is available to authorized users.

No MeSH data available.