A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary glands

Fibroblast growth factor 10 (FGF10) is well established as a mesenchyme-derived growth factor and a critical regulator of fetal organ development in mice and humans. Using a single-cell RNA sequencing (RNA-seq) atlas of salivary gland (SG) and a tamoxifen inducible Fgf10CreERT2:R26-tdTomato mouse, we show that FGF10pos cells are exclusively mesenchymal until postnatal day 5 (P5) but, after P7, there is a switch in expression and only epithelial FGF10pos cells are observed after P15. Further RNA-seq analysis of sorted mesenchymal and epithelial FGF10pos cells shows that the epithelial FGF10pos population express the hall- marks of ancient ionocyte signature Forkhead box i1 and 2 (Foxi1, Foxi2), Achaete-scute homolog 3 (Ascl3), and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10pos cells are specialized SG ionocytes located in ducts and important for the ionic modification of saliva. In addition, they maintain FGF10-dependent gland homeostasis via communication with FGFR2bpos ductal and myoepithelial cells

Localization of AQP5 during development of the mouse submandibular salivary gland

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

Limited Regeneration of Adult Salivary Glands after Severe Injury Involves Cellular Plasticity

Summary: In the adult salivary glands, the origin of replacement and regenerated acinar cells remains unclear. Although many reports describe the identification of stem cells in adult salivary glands, we have shown that differentiated acinar cells can be maintained and regenerated through self-duplication. Here, we have used genetic mouse models to further investigate acinar cell replacement and regeneration during homeostasis and after injury. Under normal conditions or after duct ligation, replacement of duct and acinar cells occurs through lineage-restricted progenitors. In contrast, after irradiation, in vivo lineage tracing shows that acinar, as well as duct, cells contribute to acinar cell regeneration, revealing that cellular plasticity is involved in salivary gland repair. Our results also indicate that even after radiation damage, several cell populations have regenerative potential for restoring salivary gland function. : Using in vivo lineage tracing, Weng et al. demonstrate that salivary gland acinar and duct lineages are maintained separately under homeostasis and after duct ligation, but after irradiation, both duct and acinar cells generate acini. This indicates that several cell populations have the potential to restore salivary gland function

FGFR2 is essential for salivary gland duct homeostasis and MAPK-dependent seromucous acinar cell differentiation

Exocrine acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific populations later in development and during acinar differentiation are unknown. Here, we use scRNAseq and conditional deletion of murine FGFRs in vivo to identify essential roles for FGFRs in craniofacial, early SG development and progenitor function during duct homeostasis. Importantly, we also discover that FGFR2 via MAPK signaling is critical for seromucous acinar differentiation and secretory gene expression, while FGFR1 is dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activates the FGFR2-dependent seromucous transcriptional program. Here, we propose a model where MEC-derived FGF7 drives seromucous acinar differentiation, providing a rationale for targeting FGFR2 signaling in regenerative therapies to restore acinar function.ISSN:2041-172

Cell-Specific Cre Strains For Genetic Manipulation in Salivary Glands

<div><p>The secretory acinar cells of the salivary gland are essential for saliva secretion, but are also the cell type preferentially lost following radiation treatment for head and neck cancer. The source of replacement acinar cells is currently a matter of debate. There is evidence for the presence of adult stem cells located within specific ductal regions of the salivary glands, but our laboratory recently demonstrated that differentiated acinar cells are maintained without significant stem cell contribution. To enable further investigation of salivary gland cell lineages and their origins, we generated three cell-specific Cre driver mouse strains. For genetic manipulation in acinar cells, an inducible Cre recombinase (Cre-ER) was targeted to the prolactin-induced protein (<i>Pip</i>) gene locus. Targeting of the <i>Dcpp1</i> gene, encoding demilune cell and parotid protein, labels intercalated duct cells, a putative site of salivary gland stem cells, and serous demilune cells of the sublingual gland. Duct cell-specific Cre expression was attempted by targeting the inducible Cre to the <i>Tcfcp2l1</i> gene locus. Using the R26^{Tomato Red} reporter mouse, we demonstrate that these strains direct inducible, cell-specific expression. Genetic tracing of acinar cells using Pip^GCE supports the recent finding that differentiated acinar cells clonally expand. Moreover, tracing of intercalated duct cells expressing Dcpp^GCE confirms evidence of duct cell proliferation, but further analysis is required to establish that renewal of secretory acinar cells is dependent on stem cells within these ducts.</p></div

Schematic diagram of general salivary gland structure.

<p>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. <i>Dcpp1</i>, gene encoding demilune cell and parotid protein; <i>Pip</i>, gene encoding prolactin-inducible protein; <i>Tcf</i>, gene encoding Tcfcp2l1 transcription factor.</p

Characterization of <i>Dcpp1</i>^<i>GCE</i> knock-in allele.

<p><b>(A)</b> Generation of <i>Dcpp1</i>^<i>GCE</i> knock-in mice. <i>Dcpp1</i> 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>(B)</b> Analysis of Cre expression in sublingual gland (SLG) of <i>Dcpp1</i>^<i>GCE/+</i><i>;R26</i>^<i>TdT/+</i> 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). <b>(C)</b> Higher magnification of labeled SLG cells reveals the morphology of serous demilunes (arrowheads). <b>(D)</b> Antibody to Nkcc1 labels SLG acinar cell membranes, and co-localizes with TdT-labeled serous demilune cell (yellow; arrowhead). <b>(E)</b> Analysis of Cre expression in parotid gland (Par) of <i>Dcpp1</i>^<i>GCE/+</i><i>;R26</i>^<i>TdT/+</i> 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). <b>(F)</b> Higher magnification of TdT-labeled (red) intercalated duct cells (arrowhead). Nuclei are stained with DAPI (blue). <b>(G)</b> 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). <b>(H)</b> Section from <i>Dcpp1</i>^<i>GCE/+</i><i>;R26</i>^<i>TdT/+</i> 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). <b>(I)</b> 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). <i>3d</i>, 3 days chase; <i>3mos</i>, 3 month chase; Scale bars = 50<i>μ</i>m (B,E,H); = 25<i>μ</i>m (C,D,F,G); = 20<i>μ</i>m (I).</p

Characterization of <i>Pip</i>^<i>GCE</i> knock-in allele.

<p><b>(A)</b> Generation of <i>Pip</i>^<i>GCE</i> knock-in mice. <i>Pip</i> 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>(B-E)</b> Analysis of Cre expression in mice after 3 days of tamoxifen treatment, followed by a 3-day chase. <b>(B)</b> Frozen sections were prepared from submandibular gland (SMG); activation of Cre results in expression of Tomato red reporter (TdT) (red); Scale bar = 50 <i>μ</i>m. No Cre activity is detected in <b>(C)</b> parotid (Par), <b>(D)</b> lacrimal gland (Lac) or <b>(E)</b> sublingual gland (SLG). Nuclei are stained with DAPI (blue). Scale bars = 25<i>μ</i>m. <b>(F)</b> Section from <i>Pip</i>^<i>GCE/+</i><i>;R26</i>^<i>TdT/+</i> SMG at 3 days after tamoxifen treatment. Single labeled acinar cells (red) co-localize with antibody to Nkcc1 (green). Scale bar = 50<i>μ</i>m <b>(G)</b> Section from <i>Pip</i>^<i>GCE/+</i><i>;R26</i>^<i>TdT/+</i> SMG at P9, isolated 3 days after tamoxifen administration. Positively labeled acinar cells are red. Nuclei are stained with DAPI. Scale bar = 25<i>μ</i>m <b>(H)</b> Section from <i>Pip</i>^<i>GCE/+</i><i>;R26</i>^<i>TdT/+</i> 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 = 50<i>μ</i>m <b>(I)</b> Section from <i>Pip</i>^<i>GCE/+</i><i>;R26</i>^<i>TdT/+</i> SMG after 3 month chase shows expansion of labeled acinar cells into clones (arrowheads). <i>3d</i>, 3 days chase; <i>3mos</i>, 3 month chase; <i>d</i>, duct; Scale bar = 50 <i>μ</i>m.</p