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WIREs Dev Biol
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Salivary gland organogenesis

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Abstract Our understanding of vertebrate salivary gland organogenesis has been largely informed by the study of the developing mouse submandibular gland (SMG), which will be the major focus of this review. The mouse SMG has been historically used as a model system to study epithelial–mesenchymal interactions, growth factor–extracellular matrix (ECM) interactions, and branching morphogenesis. SMG organogenesis involves interactions between a variety of cell types and their stem/progenitor cells, including the epithelial, neuronal, and mesenchymal cells, and their ECM microenvironment, or niche. Here, we will review recent literature that provides conceptual advances in understanding the molecular mechanisms of salivary gland development. We will describe SMG organogenesis, introduce the model systems used to study development, and outline the key signaling pathways and cellular processes involved. We will also review recent research focusing on the identification of stem/progenitor cells in the SMG and how they are directed along a series of cell fate decisions to form a functional gland. The mechanisms that drive SMG organogenesis provide a template to regenerate functional salivary glands in patients who suffer from salivary hypofunction due to irreversible glandular damage after irradiation or removal of tumors. Additionally, these mechanisms may also control growth and development of other organ systems. WIREs Dev Biol 2012, 1:69–82. doi: 10.1002/wdev.4 This article is categorized under: Vertebrate Organogenesis > From a Tubular Primordium: Branched

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Keratin expression profiles from the SGMAP database. mRNA expression profiles for keratins (K) 5, 15, 14, 19, and 7 at 12 different stages from E11.5 to adult. Interestingly, K5 is expressed at similar levels throughout development and in the adult, while the other keratins have dynamic temporal expression patterns. Gene expression profiles are available online at http://sgmap.nidcr.nih.gov/. Temporal gene expression profiles were obtained using RNA from freshly dissected glands using Agilent 4x44K microarrays. AdtF, adult female; AdtM, adult male; E, embryonic day; P, postnatal day.

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SMG branching morphogenesis in vivo and ex vivo. (a) Whole mount E‐cadherin staining of freshly dissected and fixed submandibular glands (SMGs) and sublingual glands (SLGs) at stages E11.5–E17.5. Briefly, glands were fixed in 4% paraformaldehyde (PFA), and the epithelium was visualized by indirect immunofluorescence after staining with rat anti‐E‐cadherin primary antibody (clone ECCD‐2), biotinylated secondary antibody, and avidin–peroxidase and tyramide histochemistry as previously described.20 (b) Light micrograph images of freshly dissected E13.5 SMG, isolated epithelium (epi), and isolated mesenchyme (mes) using dispase treatment and mechanical dissection. (c) Ex vivo culture of E12.5 SMG and SLG, cultured for a total of 48 h, SMG epithelium is outlined at 0 h. White asterisks indicate the SLG.

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In vivo cell ablation of Ascl3+ progenitor cells. (a) The experimental mice obtained by the cross illustrated carry one copy of the EGFP‐Cre cassette under the control of the Ascl3 promoter and one copy of the silenced diphtheria toxin A (DTA) gene inserted at the Rosa26 locus. Cre‐mediated activation of DTA expression takes place only in Ascl3‐expressing cells. DTA expression results in specific, cell‐autonomous killing of the Ascl3‐expressing cells. (b–e) Histochemical staining of salivary glands from 2‐month‐old female mice using an antibody against SLC12A2 (formerly known as NKCC1), which is a Na–K–Cl cotransporter and an antibody against KCNMA1 (formerly known as Kca1.1), which is a voltage‐ and calcium‐sensitive potassium channel. (b) In control submandibular glands (SMGs), SLC12A2 antibody is localized on the membranes on all acinar cells (brown staining) and on the subpopulation of Ascl3+ progenitor cells in the ducts (arrowheads). (c) In SMGs that carry both the Cre and DTA genes, all SLC12A2‐positive Ascl3‐expressing duct cells are ablated, while SLC12A2+ acinar cells (brown staining) are present. (d) In control SMGs, an antibody to KCNMAI is localized at the apical surface of a subset of duct cells, previously shown to coexpress SLC12A2 and ASCL3 (arrowheads). (e) In SMGs that carry both the Cre and DTA genes, all cells expressing KCNMA1 are ablated, confirming the efficiency of the cell ablation model. Scale bar = 50 µm. (Reprinted with permission from Ref 74. Copyright 2011 Elsevier)

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Neuronal signaling increases progenitor cell proliferation in an EGFR‐dependent manner. (a) E13 epithelia were cultured in media supplemented with either FGF10 (400 ng/mL), CCh (10 nM), HBEGF (2 ng/mL), CCh + HBEGF or PD168393 (an inhibitor of EGFR signaling, 10 µM). After 24 h of treatment, the epithelia were fixed and immunostained with antibodies to K5 and K19 and costained with DAPI, and ducts were imaged by confocal analysis. Addition of CCh, an analog of the neurotransmitter ACh, increases the percentage of proliferating cells expressing K5 (green), whereas HBEGF increases the K5+K19+ (yellow) and K5K19+ (red) cell populations. PD (PD168393), an inhibitor of EGFR signaling, reduces the number of K5+K19 cells and inhibits their proliferation. (b) The number of proliferating cells (Ki67+) in an intact submandibular gland decreases after DAMP (a muscarinic receptor inhibitor, 10 µM) or PD (10 µM) treatment. (c) Proliferating epithelial cells were further analyzed by FACS for their K5 and K19 expression. DAMP and PD inhibit K5+K19 cell proliferation compared to DMSO‐treated controls, confirming that their maintenance by proliferation is both M1‐ and EGFR‐dependent. Proliferation of the K5+K19+ (15%) and the K5K19+ (80%) cells with DAMP treatment was similar to control, suggesting that these cells can proliferate independent of muscarinic activation. PD treatment increased the proliferation of K5+K19+ cells (from 15 to 30%) and decreased proliferation of K5K19+ cells (from 81 to 68%), indicating K5+K19+ and K5K19+ cell proliferation is not solely dependent on EGFR signaling. All graphs are mean ± standard error of the mean from three independent experiments; analysis of variance with post hoc Dunnett's test, **P < 0.01, ***P < 0.001. (Reprinted with permission from Ref 66. Copyright 2010 American Association for the Advancement of Science)

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Keratin 5+ progenitor cell population in the E14 submandibular gland (SMG). (a and b) Light micrograph and confocal analysis of K5+ cells (green) in the E14 SMG. K5+ cells are localized in the end buds and ducts of the epithelium (red, E‐cadherin). Image is a single confocal section (5 µM), scale bar = 100 µm. (c) FACS analysis of E14 SMGs using antibodies to E‐cadherin, and K5 quantifies the number of K5+ epithelial cells. K5+ cells are 9.6 ± 1.3% of the E‐cadherin+ epithelial cells in an E14 SMG. Lineage tracing analysis confirmed that K5+ cells are progenitor cells in the SMG. (Reprinted with permission from Ref 66. Copyright 2010 American Association for the Advancement of Science)

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Cell types involved in submandibular gland (SMG) development. Whole mount antibody immunostaining of an E13.5 SMG and sublingual gland (SLG). (a) Low‐power (10×) images of an E13.5 SMG and SLG and (b) high‐power (60×) images of an end bud (lower panel) stained with antibodies specific to the epithelium (laminin, blue), nerves (Tubb3, green, clone TUJ1), and blood vessels (PECAM, red). At high power (b, green nerve staining), evidence of neuronal–epithelial interactions are seen by the abundant neuronal varicosities (arrowhead) in proximity to the developing epithelium. White asterisk indicates the SLG.

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Cleft formation in wild type and Lama5−/− submandibular glands (SMGs). (a) Cleft formation in the SMG. The columnar cell morphology of the outer epithelial cell layer at E12.5 becomes less organized locally near the base of clefts after 5 and 10 h in ex vivo culture. Epithelia were immunostained with anti‐E‐cadherin antibody (red, clone ECCD‐1) and DAPI (blue) and imaged by confocal microscopy. The basement membrane is marked by a white solid line and the outer epithelial cells by a white dashed line. Open triangles show cleft position. Scale bar, 10 µm. (Reprinted with permission from Ref 59. Copyright 2010 American Association for the Advancement of Science) (b) Immunostaining showing both laminin alpha 1 (LAMA1) and laminin alpha 5 (LAMA5) are present in the early cleft (white arrowheads). (Reprinted with permission from Ref 31. Copyright 2007 Elsevier) (c) Ex vivo cultured SMGs from control and Lama5 null embryos. Loss of Lama5 results in a delay in cleft formation, although the end bud continues to enlarge and undergo proliferation. Cleft formation begins after 24 h of culture, but the SMG of the Lama5−/− mice are hypoplastic and the sublingual gland does not form. (Reprinted with permission from Ref 31. Copyright 2007 Elsevier)

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