Retinoic acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium

Abstract

Background Retinoic Acid (RA), the active metabolite of Vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. Results Here we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and non-neuronal mesenchyme. By culturing epithelium explants in isolation from other tissues we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. Conclusions RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs.

RDH10-mediated retinol metabolism and RARα-mediated retinoic acid signaling are required for submandibular salivary gland initiation

“…We and others have reported that RA signaling contributes to salivary gland development during branching morphogenesis (Abashev et al, 2017;DeSantis et al, 2017;Wright et al, 2015). We have also shown that RA has a role earlier in gland development, as evidenced by abnormal morphology of SMG initial buds in rare constitutive Rdh10 mutants that remain viable to E12.5 (Wright et al, 2015).…”

“…Initiation could be induced by RA signaling directly within oral epithelium, or by activity within the underlying mesenchyme. We previously showed that RA signaling acts directly within epithelial cells at the pseudoglandular stage of epithelial branching morphogenesis (Abashev et al, 2017;DeSantis et al, 2017). The fact that RA signaling is active within the epithelial cells of the initial pre-bud stage, SMG, suggests that RA acts directly within naïve oral epithelium to direct initiation.…”

“…Immunostaining of frozen sections and whole-mount tissues was performed as previously described (Abashev et al, 2017). Primary antibodies used were anti-E-cadherin (BD Biosciences #610182; 1:50), and anti-SOX9 (Abcam, #185966; 1:200).…”

Salivary gland stem cells: A review of development, regeneration and cancer

“…However, despite this overlap, a population of KIT+ cells remains KRT14-negative in the pre-acinar cells of the SMG (Lombaert et al , 2013), suggesting that KIT and KRT14 diverge to mark distinct progenitor populations in the SG. In support of this segregation, an inverse expression profile to Krt14 was apparent for Kit expression following RA inhibition, where BMS 493 reduces expression of Kit in isolated epithelia explants (Abashev et al , 2017). In addition, KRT14 but not KIT is expressed by emerging SMA+ myoepithelial cells, which in the lacrimal gland give rise only to themselves (Farmer et al , 2017).…”

“…RA signaling occurs in early SMG development (from E10.5), where it plays a role in maintaining SG progenitor cells as well as epithelial morphogenesis. RA-deficient mice exhibit SMG developmental delay (Wright et al , 2015) and similarly, blocking RA signaling in isolated epithelia with the pan-RAR antagonist BMS 493 results in reduced branching morphogenesis (Wright et al , 2015) and repressed cell proliferation (Abashev et al , 2017). Using RAR isoform-specific agonists and inhibitors DeSantis, et al demonstrated isoform-specific roles for retinoic acid receptor (RAR) signaling in maintenance of KRT14+ cells, where RARγ is necessary, but not sufficient, to maintain KRT5+ cells, whereas RARα agonism reduces the number of KRT5+ cells and promotes differentiation (DeSantis et al , 2017).…”