Fig 1.
Schematic diagram of general salivary gland structure.
Secretory acinar cells are arranged in clusters, known as acini, which produce primary saliva. The smallest intercalated ducts conduct saliva from the acini to the striated, and excretory ducts. Sites of inducible Cre drivers are indicated, color-coded for each strain. Dcpp1, gene encoding demilune cell and parotid protein; Pip, gene encoding prolactin-inducible protein; Tcf, gene encoding Tcfcp2l1 transcription factor.
Fig 2.
Characterization of PipGCE knock-in allele.
(A) Generation of PipGCE knock-in mice. Pip genomic structure and restriction map is shown at the top. White box represents the non-coding exon sequences and filled boxes, the coding sequences. Thick bars show the sequences used to generate the homologous arms in the targeting vector. Gray box represents 3’ external probe used for Southern blotting. Arrows indicate positions of genotyping PCR primers (An3’ and PipR). (B-E) Analysis of Cre expression in mice after 3 days of tamoxifen treatment, followed by a 3-day chase. (B) Frozen sections were prepared from submandibular gland (SMG); activation of Cre results in expression of Tomato red reporter (TdT) (red); Scale bar = 50 μm. No Cre activity is detected in (C) parotid (Par), (D) lacrimal gland (Lac) or (E) sublingual gland (SLG). Nuclei are stained with DAPI (blue). Scale bars = 25μm. (F) Section from PipGCE/+;R26 TdT/+ SMG at 3 days after tamoxifen treatment. Single labeled acinar cells (red) co-localize with antibody to Nkcc1 (green). Scale bar = 50μm (G) Section from PipGCE/+;R26 TdT/+ SMG at P9, isolated 3 days after tamoxifen administration. Positively labeled acinar cells are red. Nuclei are stained with DAPI. Scale bar = 25μm (H) Section from PipGCE/+;R26 TdT/+ SMG at 3 months after tamoxifen treatment, co-stained with antibody to Nkcc1 (green) to label acinar cells. Labeled acinar cells have expanded to clones (red). Scale bar = 50μm (I) Section from PipGCE/+;R26 TdT/+ SMG after 3 month chase shows expansion of labeled acinar cells into clones (arrowheads). 3d, 3 days chase; 3mos, 3 month chase; d, duct; Scale bar = 50 μm.
Fig 3.
Characterization of Dcpp1GCE knock-in allele.
(A) Generation of Dcpp1GCE knock-in mice. Dcpp1 genomic structure and restriction map is shown at the top. White box represents the non-coding exon sequences and filled boxes, the coding sequences. Thick bars show the sequences used to generate the homologous arms in the targeting vector. Arrows indicate positions of external long-range PCR primers (5’ external primer and GCE primer) and internal primers (An3’ and Dcpp1R) used for genotyping. (B) Analysis of Cre expression in sublingual gland (SLG) of Dcpp1GCE/+;R26 TdT/+ mice after 3 days of tamoxifen treatment, followed by a 3-day chase. Activation of Cre results in expression of Tomato red reporter (TdT) (red). (C) Higher magnification of labeled SLG cells reveals the morphology of serous demilunes (arrowheads). (D) Antibody to Nkcc1 labels SLG acinar cell membranes, and co-localizes with TdT-labeled serous demilune cell (yellow; arrowhead). (E) Analysis of Cre expression in parotid gland (Par) of Dcpp1GCE/+;R26 TdT/+ mice after 3 days of tamoxifen treatment, followed by a 3-day chase. Activation of Cre results in expression of TdT (red) in small clusters of intercalated duct cells (arrowheads). (F) Higher magnification of TdT-labeled (red) intercalated duct cells (arrowhead). Nuclei are stained with DAPI (blue). (G) Antibody to Nkcc1 labels acinar cells (green). TdT-positive cells (red) do not co-localize with acinar cells, but are found within the smallest intercalated ducts (arrowheads). (H) Section from Dcpp1GCE/+;R26 TdT/+ parotid gland after 3 days of tamoxifen treatment, followed by a 3-month chase. TdT-positive cells (red) are clustered in duct-like structures (arrows). (I) At 3 months chase, TdT-labeled cells (red) derived from Dcpp1-expressing cells are clustered in intercalated ducts (arrows). Some Dcpp1-labeled cells may overlap with acinar cells labeled with antibody to Nkcc1 (green; arrowhead). Nuclei are stained with DAPI (blue). 3d, 3 days chase; 3mos, 3 month chase; Scale bars = 50μm (B,E,H); = 25μm (C,D,F,G); = 20μm (I).
Fig 4.
Characterization of TcfGCE knock-in allele.
(A) Generation of TcfGCE knock-in mice. Tcfcp2l1 genomic structure and restriction map is shown at the top. White box represents the non-coding exon sequences and filled boxes, the coding sequences. Thick bars show the sequences used to generate the homologous arms in the targeting vector. Shaded box represents 5’ external probe used for Southern blotting. Arrows indicate positions of genotyping PCR primers (An3’ and TcfcpR). (B) Analysis of Cre expression in TcfGCE/+;R26 TdT/+ mice on frozen sections from submandibular gland (SMG) 1 day after a single tamoxifen gavage shows TdT-positive labeled duct cells (red; arrowheads) and acinar cells (red; arrows). Scale bar = 50μm (C) Section of SMG after 3 days tamoxifen gavage shows TdT-positive Cre activity in ducts, granulated ducts of males (arrowheads) and acinar cells (arrows). Scale bar = 25μm (D) Section of sublingual gland (SLG) after a single tamoxifen gavage at 1 day shows TdT-positive Cre activity in duct (arrowhead) and acinar cells (arrow). (E) SLG after 3 days tamoxifen gavage shows increased TdT-positive labeling of duct (arrowheads) and acinar cells (arrows). (F) Section of parotid (Par) after 3 days tamoxifen gavage shows TdT-positive labeling of both duct (arrowhead) and acinar cells (arrows). (G) Section of lacrimal (Lac) gland after 3 days tamoxifen gavage shows labeling of both duct (arrowhead) and acinar cells (arrow). (H) Section of lung after 3 days tamoxifen gavage shows Cre activity due to TcfGCE expression (red). (I) Section of kidney after 3 days tamoxifen gavage shows Cre activity due to TcfGCE expression (red). Nuclei stained with DAPI (blue). Scale bars = 50μm; 1d, 1 day chase; 3d, 3 days chase.