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An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

Bohórquez DV, Samsa LA, Roholt A, Medicetty S, Chandra R, Liddle RA - PLoS ONE (2014)

Bottom Line: However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist.We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells.These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

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3D ultrastructure reveals axonal process escorted by enteric glia.A. Enteroendocrine cells compared to other intestinal epithelial cells express neurofilaments light and medium (top panel). Neurofilament proteins light and medium are expressed in 22% and 47% of Pyy-GFP cells, respectively (bottom panel). This quantification was performed using immunohistochemistry with neurofilament-specific antibodies. B. Top panel is a representative image showing that neurofilament heavy is expressed in subepithelial myofibroblasts but not in enteroendocrine cells. Neurofilament light is contained within the Pyy-GFP cell basal process (bottom panel). C. Enteroendocrine cells contain neurofilament medium within the neuropod. Inset shows the position of the cell in the epithelium of the ileum. 3D reconstruction of confocal z-stacks depicts the neurofilament medium contained within the Pyy-GFP cell neuropod. D. The SBEM data also revealed the relationship between the neuropod in the Pyy-GFP cell and enteric glia. Enteric glia trespass the basal lamina and penetrate into the epithelium (inset). SBEM data segmentation revealed that the enteric glia extends a cytoplasmic process into the epithelium that contacts the enteroendocrine cell neuropod. Bars in B and C = 10 µm, in D = 1 µm.
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pone-0089881-g003: 3D ultrastructure reveals axonal process escorted by enteric glia.A. Enteroendocrine cells compared to other intestinal epithelial cells express neurofilaments light and medium (top panel). Neurofilament proteins light and medium are expressed in 22% and 47% of Pyy-GFP cells, respectively (bottom panel). This quantification was performed using immunohistochemistry with neurofilament-specific antibodies. B. Top panel is a representative image showing that neurofilament heavy is expressed in subepithelial myofibroblasts but not in enteroendocrine cells. Neurofilament light is contained within the Pyy-GFP cell basal process (bottom panel). C. Enteroendocrine cells contain neurofilament medium within the neuropod. Inset shows the position of the cell in the epithelium of the ileum. 3D reconstruction of confocal z-stacks depicts the neurofilament medium contained within the Pyy-GFP cell neuropod. D. The SBEM data also revealed the relationship between the neuropod in the Pyy-GFP cell and enteric glia. Enteric glia trespass the basal lamina and penetrate into the epithelium (inset). SBEM data segmentation revealed that the enteric glia extends a cytoplasmic process into the epithelium that contacts the enteroendocrine cell neuropod. Bars in B and C = 10 µm, in D = 1 µm.

Mentions: Neurofilaments are a family of intermediate filaments formed by the following three members: NF heavy (200–220 kDa), NF medium (145–160 kDa), and NF light (68–70 kDa) [24]. Using fluorescence activated cell (FAC) sorting and qRT-PCR, we tested the expression of neurofilaments genes in Pyy-GFP enteroendocrine cells compared to non-GFP epithelial cells. RNA from dissociated sensory neurons served as positive control. Although no expression of NF heavy was observed in enteroendocrine cells, there was significant expression of NF medium and light in Pyy-GFP enteroendocrine cells compared to non-GFP epithelial cells (Figure 3A).


An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

Bohórquez DV, Samsa LA, Roholt A, Medicetty S, Chandra R, Liddle RA - PLoS ONE (2014)

3D ultrastructure reveals axonal process escorted by enteric glia.A. Enteroendocrine cells compared to other intestinal epithelial cells express neurofilaments light and medium (top panel). Neurofilament proteins light and medium are expressed in 22% and 47% of Pyy-GFP cells, respectively (bottom panel). This quantification was performed using immunohistochemistry with neurofilament-specific antibodies. B. Top panel is a representative image showing that neurofilament heavy is expressed in subepithelial myofibroblasts but not in enteroendocrine cells. Neurofilament light is contained within the Pyy-GFP cell basal process (bottom panel). C. Enteroendocrine cells contain neurofilament medium within the neuropod. Inset shows the position of the cell in the epithelium of the ileum. 3D reconstruction of confocal z-stacks depicts the neurofilament medium contained within the Pyy-GFP cell neuropod. D. The SBEM data also revealed the relationship between the neuropod in the Pyy-GFP cell and enteric glia. Enteric glia trespass the basal lamina and penetrate into the epithelium (inset). SBEM data segmentation revealed that the enteric glia extends a cytoplasmic process into the epithelium that contacts the enteroendocrine cell neuropod. Bars in B and C = 10 µm, in D = 1 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3935946&req=5

pone-0089881-g003: 3D ultrastructure reveals axonal process escorted by enteric glia.A. Enteroendocrine cells compared to other intestinal epithelial cells express neurofilaments light and medium (top panel). Neurofilament proteins light and medium are expressed in 22% and 47% of Pyy-GFP cells, respectively (bottom panel). This quantification was performed using immunohistochemistry with neurofilament-specific antibodies. B. Top panel is a representative image showing that neurofilament heavy is expressed in subepithelial myofibroblasts but not in enteroendocrine cells. Neurofilament light is contained within the Pyy-GFP cell basal process (bottom panel). C. Enteroendocrine cells contain neurofilament medium within the neuropod. Inset shows the position of the cell in the epithelium of the ileum. 3D reconstruction of confocal z-stacks depicts the neurofilament medium contained within the Pyy-GFP cell neuropod. D. The SBEM data also revealed the relationship between the neuropod in the Pyy-GFP cell and enteric glia. Enteric glia trespass the basal lamina and penetrate into the epithelium (inset). SBEM data segmentation revealed that the enteric glia extends a cytoplasmic process into the epithelium that contacts the enteroendocrine cell neuropod. Bars in B and C = 10 µm, in D = 1 µm.
Mentions: Neurofilaments are a family of intermediate filaments formed by the following three members: NF heavy (200–220 kDa), NF medium (145–160 kDa), and NF light (68–70 kDa) [24]. Using fluorescence activated cell (FAC) sorting and qRT-PCR, we tested the expression of neurofilaments genes in Pyy-GFP enteroendocrine cells compared to non-GFP epithelial cells. RNA from dissociated sensory neurons served as positive control. Although no expression of NF heavy was observed in enteroendocrine cells, there was significant expression of NF medium and light in Pyy-GFP enteroendocrine cells compared to non-GFP epithelial cells (Figure 3A).

Bottom Line: However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist.We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells.These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

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Related in: MedlinePlus