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Developmental expression of mucin genes ASGP (rMuc4) and rMuc5ac by the rat ocular surface epithelium
1Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
Insights
The study reveals distinct developmental timelines for two key mucins on the rat ocular surface. Gel-forming mucin rMuc5AC appears with goblet cells at 7 days, while membrane-spanning mucin ASGP (rMuc4) emerges later at 14 days, correlating with eyelid opening.
Area of Science:
- Ophthalmology
- Developmental Biology
- Molecular Biology
Background:
- The ocular surface epithelium produces mucins essential for lubrication and protection.
- Two key mucins, ASGP (rMuc4) and rMuc5AC, play distinct roles in ocular surface homeostasis.
- Understanding their developmental expression is crucial for comprehending ocular surface development and disease.
Purpose of the Study:
- To determine the site and timing of initiation for membrane-spanning mucin ASGP (rMuc4) and gel-forming mucin rMuc5AC expression.
- To investigate the developmental patterns of these mucins in the rat ocular surface epithelium.
Main Methods:
- Utilized reverse transcription-polymerase chain reaction (RT-PCR) to detect and quantify ASGP and rMuc5AC mRNA.
- Employed in situ hybridization to localize the expression of ASGP and rMuc5AC mRNA.
- Used periodic acid-Schiff (PAS) staining to identify goblet cells.
Main Results:
- ASGP mRNA was detected at 1 day postpartum, with expression increasing by day 14, localized to the lid margin and conjunctiva.
- rMuc5AC mRNA was detected at 1 day postpartum, with significant increases in expression by adulthood.
- rMuc5AC expression was first observed in goblet cells at 7 days postpartum, correlating with PAS staining.
Conclusions:
- Developmental expression of ASGP (rMuc4) and rMuc5AC is temporally and regionally distinct.
- rMuc5AC expression begins at 7 days postpartum in the fornix, linked to goblet cell differentiation.
- ASGP (rMuc4) expression initiates later at 14 days postpartum at the lid margin, coinciding with eyelid opening.
Purpose:
To determine site and time of initiation of expression of the membrane-spanning mucin ASGP (rMuc4) and the goblet cell-specific, gel-forming mucin rMuc5AC by the developing rat ocular surface epithelium.
Methods:
Newborn Sprague-Dawley rat pups were killed at 1, 7, and 14 days after birth. Adult rats (weight, 200 g) were used as controls. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect ASGP mRNA using beta-actin as an internal control. Competitive RT-PCR was performed to quantitate rMuc5AC mRNA using an rMuc5AC-competitive reference standard (CRS) as an internal control. In situ hybridization was performed to localize ASGP and rMuc5AC mRNA. Goblet cells were detected by staining with periodic acid-Schiff (PAS) reagent.
Results:
ASGP mRNA was detected by RT-PCR at 1 day after birth. Compared with beta-actin, the amount of ASGP mRNA showed a progressive increase from 1 to 14 days of postnatal development. By in situ hybridization, the expression of ASGP was first clearly detected at 14 days after birth at the lid margin, where the most stratification of epithelium was seen, and along the adjacent palpebral conjunctiva. This pattern was seen in rat eyelids that were not yet open but appeared about to open. In rat eyelids already open at 14 days after birth, ASGP mRNA was diffusely spread in the apical cell layer of both conjunctival and corneal epithelia. The expression of rMuc5AC was detected by RT-PCR in ocular surface epithelium in rat pups 1 day after birth. Quantitative RT-PCR showed a low level of rMuc5AC RNA expression in conjunctiva of 1-, 7-, and 14-day-old rats followed by a large increase in expression between 14 days and adulthood. The expression of rMuc5AC was first detected by in situ hybridization in a few goblet cells at 7 days after birth. One or two labeled cells were present in the fornical area; some were on the palpebral side of the fornix; others were present on the bulbar side. The distribution and time of appearance of rMuc5AC correlated with that of PAS staining of goblet cells.
Conclusions:
The developmental expression of the membrane-spanning mucin ASGP (rMuc4) and the gel-forming mucin rMuc5AC are regionally and temporally separated. Expression of the gel-forming mucin begins at the fornix at 7 days after birth and is correlated with the appearance of goblet cells, whereas, expression of the membrane-spanning mucin begins later at the lid margin at day 14. Expression of the membrane-spanning mucin correlates to eyelid opening.