Localization of AQP5 during development of the mouse submandibular salivary gland

Abstract

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5-18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.

Fig. 1

AQP5 expression pattern during prenatal development of the mouse submandibular gland (SMG). A Pseudoglandular stage (~E14): no AQP5 staining is detectable in the terminal bud (t), epithelial stalk (e) or mesechyme (m). B Early canalicular stage (~E14-15): AQP5 negative cells in the terminal bud (t), presumptive duct (pd), and mesenchyme (m). C Late canalicular stage (~E15-E16): scattered positive pro-acinar cells are present (arrow). No AQP5 is found in the presumptive duct (pd) or mesenchyme (m). D Early terminal bud stage (~E16-E17): all pro-acinar cells are AQP5 positive (arrow). In the intralobular duct (IAD), cells proximal to the pro-acini show apical AQP5 staining (arrowhead). E and F Late terminal bud stage (~E17-18): a similar expression pattern is observed as in D. Additionally, the rest of the intralobular duct (IAD) is also positive, and the interlobular duct (IED) is negative. Scale bar (A–E) 50 μm and (F) 100 μm

Fig. 2

AQP5 expression pattern during postnatal development of the mouse submandibular gland (SMG). A At birth (postnatal day 0, P0), the pro-acinar cells (arrow) and the intercalated ducts (ID) are AQP5 positive. No AQP5 is detected in the intralobular duct (IAD) or interlobular duct (IED). B Pre-weaning (P5), both pro-acini (arrow) and intercalated ducts (ID) are AQP5 positive. No AQP5 is detected in the striated duct (SD). C Young adult females (P25): acini (arrow) and the proximal part of the intercalated duct (ID) are positive, while the granulated convoluted tubule (GCT) is negative. D Adult females (P60) show the same AQP5 pattern as in C. In addition, no AQP5 is detected in the transition from GCT to striated duct (arrowhead). E Adult males (P60): acini (arrow) and entire intercalated ducts (ID) are positive while the granulated convoluted tubule (GCT) is negative. F IgG negative control in young adult female (P25) tissue shows no unspecific staining in the acini (arrow), intercalated duct (ID), or in the granulated convoluted tubule (GCT). Granules in the granulated convoluted tubule are not seen using this method. A–F Scale bar 50 μm

Fig. 3

Confocal imaging of the adult SMG (P60). Aquaporin 5 (AQP5) demonstrates an apical, lateral, and basal localization in the SMG acini. A Localization of ZO-1 (green) near luminal membranes of acinar cells. B Localization of AQP5 (red) within the same cells as in A. C Merged image of A and B confirms the apical localization of AQP5. Arrow heads luminal membrane (scale bar 20 μm). D Localization of E-cadherin (green) within lateral membranes in both acinar (arrow heads) and GCT cells (*). E Localization of AQP5 (red) in the acinar cells. F Merged image of D and E demonstrates a lateral localization of AQP5 (yellow/orange), designated by arrowheads. An AQP5 and E-cadherin non-overlapping pattern of expression in the luminal canaliculi is evident (arrow). Non-overlapping patterns are also seen in the GCT cells (*) (scale bar, 50 μm). G Immunostaining of the basement membrane protein coll IV (blue). H Localization of AQP5 (red) in the acinar cells. I Merged image confirms a basal localization (arrow head) of AQP5 distinct from coll IV localization (scale bar 20 μm). J Localization of E-cadherin (green) within lateral membranes in acinar cells in a tissue immunostained with preabsorbed AQP5. K Preabsorption of the AQP5 antibody using its cognate peptide, showing complete elimination of the AQP5 staining pattern in the same cells as in J (scale bar 20 μm). A–I images are of male P60, J–K images are of female P60

Fig. 4

Gold labeling of AQP5 in the SMG of adult animals (P60). A Overview of AQP5 gold staining in the basal membrane, membranes of intercellular canaliculi, as well as the lateral membrane (LM). CL canalicular lumen, BD basal digits (scale bar, 1 μm). B Gold labeling of AQP5 was detected in the apical membrane of acinar cells (scale bar, 1 μm). C Longitudinal section of an intercellular canaliculus showing gold labeling of AQP5 in the membrane (scale bar, 0.5 μm). D AQP5 was localized in the basal membrane in areas where digits could be seen, while no AQP5 was detectable in the basement membrane (scale bar 0.2 μm). Non-linear adjustments were applied to entire images