Phenotypic Diversity Caused by Differential Expression of SFTPC-Cre-Transgenic Alleles

Abstract

Type II alveolar epithelial cells (AEC2s) play an essential role in the function and maintenance of the pulmonary epithelium. Several transgenic mice have been developed to study the function of these cells in vivo by using the human SFTPC promoter to drive expression of Cre recombinase. The precise activity of each of these transgenic alleles has not been studied, and previous reports suggest that their activity can depend on breeding strategies. We bred mice with a conditional allele of the essential telomere capping protein TRF2 with two different SFTPC-Cre-transgenic strains and observed opposite phenotypes (100% lethality vs. 100% viability). We characterized the Cre recombinase activity in these two transgenic lines and found that the contrasting phenotypes were driven by difference in embryonic expression of the two transgenes, likely due to position effects or differences in the transgenic constructs. We also tested if SFTPC-Cre activity was dependent on maternal or paternal inheritance. When paternally inherited, both SFTPC-Cre alleles produced offspring with constitutive reporter activity independent of the inheritance of the Cre allele, suggesting that Cre recombinase was expressed in the male germline before meiosis. Immunohistochemical analysis of the testis showed reporter activity during spermatogenesis. Analysis of single-cell RNA sequencing data from murine and human testis demonstrated SFTPC expression uniquely during human spermatogenesis, suggesting that use of the human promoter in these constructs is responsible for male germline activity. Our data highlight the importance of careful analysis of transgenic allele activity and identify an SFTPC-Cre allele that is useful for panepithelial targeting in the mouse.

Keywords: SFTPC; lineage tracing; lung development.

Figure 1.

Divergent phenotypes and embryonic expression of SFTPC-Cre–transgenic mice. (A) Kaplan-Meier survival analysis of Trf2^Fl/Fl;SFTPC-cre^Yo and Trf2^Fl/Fl;SFTPC-cre^Blh mice. Groups were compared using the log rank test. (B) Schematics of the transgenic constructs that were used to make SFTPC-cre^Yo and SFTPC-cre^Blh mice, respectively, and the mT (membrane-targeted TdTomato)/mG (membrane-targeted GFP) reporter that was used in our experiments (–7). (C) Representative photomicrographs of Embryonic Day (E) 14.5 lungs from the indicated mice. GFP⁺ and epithelial cells were localized with antibodies directed toward GFP and E-Cadherin, respectively. Scale bars: 50 μm. (D) Quantitation of the fraction of epithelial cells that were labeled with GFP in the two strains of mice. Plot shows mean (±SD); groups were compared with Mann-Whitney test. ****P < 0.0001. Tg = transgenic.

Figure 2.

Male germline activity in SFTPC-Cre–transgenic mice. (A) Schematic of the test cross that was used to measure male germline activity. Embryos were scored based on the expression pattern of GFP. (B) Proportion of offspring that were born with constitutive GFP expression. (C) Representative photomicrographs of testis from mice of the indicated genotypes. Sections were stained with hematoxylin and eosin (H&E; top panels) or immunohistochemistry (IHC) was performed with an antibody specific for GFP (bottom panels). Scale bars: 200 μm in H&E panels, 100 μm in IHC panels, and 50 μm in insets. (D) Uniform manifold approximation and projection (UMAP) of single-cell RNA sequencing data from human testis with cells expressing SFTPC highlighted in purple. Gray line depicts the direction of differentiation of spermatogenesis from spermatogonial stem cells to spermatids, with supporting marker genes shown in Figure E3. nf = none found; WT = wild-type.

Figure 3.

Lineage tracing of SFTPC-Cre–transgenic mice in adult lung and compensation in Trf2^Fl/Fl;SFTPC-Cre mice. (A and C) Representative photomicrographs of adult lungs from indicated strains of mice. Club cells, type II alveolar epithelial cells (AEC2s), and GFP⁺ cells are localized with antibodies to SCGB1A1, pro-SFTPC, and GFP, respectively. Scale bars: 50 μm. (B and D) Quantitation of the proportion of lineage-labeled cells in the adult lung. (E) Proportion of lineage-labeled epithelial cells (E-Cadherin⁺ and GFP⁺) in E14.5 lungs from the indicated genotypes. (F and G) Proportion of lineage-labeled club cells (F) and AEC2s (G) in adult mice from the indicated genotypes. All plots show means (±SD); groups were compared with the Mann-Whitney test. **P < 0.01 and ****P < 0.0001.