As mentioned above, three SCGB proteins namely SCGB1A1, SCGB3A1, and SCGB3A2 are highly expressed in airway epithelial club cells. However, very little is known about whether there are any redundancies in the expressions and/or functions among these three proteins, and whether and/or how they are related to each other. In order to gain insight into the possible functional relationships among the three SCGBs, their protein and mRNA expression patterns were examined in lungs during gestation and in adult mice by immunohistochemistry and by mRNA analysis, respectively. Specificities of anti-SCGB3A1 and anti-SCGB3A2 antibodies were determined by immunohistochemistry using lung sections obtained from Scgb3a1- and Scgb3a2-knockout mice as negative controls. The histochemical and qRT-PCR analyses revealed that the three SCGBs exhibit unique spatiotemporal expression patterns during embryogenesis; SCGB1A1 and SCGB3A2 are both expressed in the large (bronchi) and small (bronchiole) airways, however, the expression of SCGB1A1 starts at embryonic day (E) 16.5 while SCGB3A2 starts being expressed at E14.5, 2 days earlier. In contrast, SCGB3A1 expression was observed only in large airways at E16.5 and thereafter, but not in small airways. Further, the lack of Scgb3a1 or Scgb3a2 expression did not have any effect on those of the other Scgb genes as determined by mRNA measurements. Previously, SCGB1A1 deficiency was shown to attenuate pulmonary neuroepithelial body (NEB) differentiation in mice lacking Scgb1a1. Further, it was reported that Scgb3a2 expression is enriched in clusters in the NEB microenvironment. In order to understand the possible roles for SCGB3A1 and SCGB3A2 in NEB differentiation, calcitonin gene-related peptide (CGRP) immunostaining that specifically detects NEBs was carried out to determine the size of NEBs and the number of NEB foci per airway as indicators for neuroendocrine differentiation using Scgb3a1(-/-) and Sgb3a2(-/-) mice. The distribution of ciliated cells in the airway was also examined using immunostaining for beta-tubulin, a marker for ciliated cells. The results demonstrate that the lack of Scgb3a1 or Scgb3a2 did not affect development of the NEBs or ciliated cells during embryogenesis. These results suggest that SCGB1A1, SCGB3A1, and SCGB3A2 each may possess its own unique biological function in the lung.
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