Abstract
Gel-forming mucins are macromolecules produced by goblet cells and responsible for the mucus gel formation. Changes in goblet cell density and in gel-forming mucin production have emerged as sensitive indicators for mucosal diseases. A Muc5b-GFP tagged reporter mouse was used to assess Muc5b production in mouse tissues by immunofluorescence microscopy and fluorescent activity using stereromicroscopy and probe-based confocal laser endomicroscopy. Muc5b production was followed longitudinally by recording the fluorescent activity in vagina and in embryonic lung explants under stimulation by interleukin 13. We show that the GFP is easily visualized in the mouse adult ear, nose, trachea, gallbladder, and cervix. Live Muc5b is also easily monitored in the nasal cavity, trachea and vagina where its production varies during the estrus cycle with a peak at the proestrus phase and in pregnant mice. Explant culture of reporter mouse embryonic whole lung shows that interleukin 13 stimulates Muc5b production. The transgenic Muc5b-GFP mouse is unique and suitable to study the mechanisms that regulate Muc5b production/secretion and mucous cell differentiation by live imaging and can be applied to test drug efficacy in mucosal disease models.
Highlights
An intense signal in comparison to immunohistochemistry was observed in the four tissues studied: trachea, middle ear, cervix, and nasal cavity, while no GFP activity was detected in WT mice (Fig. 2b)
The two main reasons for this lack of knowledge are the poor availability of specific antibodies and the difficulty in studying mucins because they are very large and heavily glycosylated molecules with glycoforms and natural variation in glycosylation occurring between organs, tissues and cells[3,17]
RIL13 stimulates Muc5b production in whole embryonic lung culture. This is in agreement with studies from other teams which reported that IL13 induces goblet cell differentiation in the airways[6,18] and stimulates proliferation of goblet cells and expression of mucins in primary conjunctival cell cultures[19]
Summary
Both primary antibodies showed that Muc5b is produced by epithelial cells in the eustachian tube, bulla, trachea, nasal cavity turbinate, gallbladder, bronchi (Fig. 1b), and cervix (see below). We looked for GFP fluorescent activity using epifluorescence stereomicroscopy in excised tissues with an epithelial cavity producing Muc5b and which is accessible to further endomicroscopic studies in living mice (Fig. 2a). An intense signal in comparison to immunohistochemistry was observed in the four tissues studied: trachea, middle ear, cervix, and nasal cavity, while no GFP activity was detected in WT mice (Fig. 2b).
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