cell-dissociation-from-tongue-epithelium-mesenchymeconnective-tissue

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

University of Georgia

P0-Cre and Wnt1-Cre mouse lines have been widely used in combination with loxP-flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1-Cre has been regarded as the gold standard and there have been concerns about the specificity of P0-Cre because it is not clear about the timing and spatial distribution of the P0-Cre transgene in labeling NC cells at early embryonic stages. We re-visited P0-Cre and Wnt1-Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26-lacZ Cre reporter responded to Cre activity more reliably than CAAG-lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0-Cre and reporter (lacZ and RFP) activity in P0-Cre/R26-lacZ and P0-Cre/R26-RFP embryos were detected in the cranial NC and notochord regions in E8.0-9.5 (4-19 somite) embryos. P0-Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0-Cre and Wnt1-Cre were profound in the midbrain and hindbrain regions, i.e., extensive in the midbrain of Wnt1-Cre and in the hindbrain of P0-Cre embryos. The difference between P0-Cre and Wnt1-Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre-driven genetic modifications. This article is protected by copyright. All rights reserved.

Specific and spatial labeling of P0-Cre versus Wnt1-Cre in cranial neural crest in early mouse embryos.

Mammalian taste buds emerge perinatally and most become mature 3-4 weeks after birth. Mature taste bud cells in rodents are known to be renewed by the surrounding K14 basal epithelial cells and potentially other progenitor source(s), but the dynamics between initially developed taste buds and surrounding tissue compartments are unclear. Using the K14-Cre and Dermo1-Cre mouse lines to trace epithelial and mesenchymal cell lineages, we found that early taste buds in E18.5 and newborn mouse tongues are not derived from either lineage. At E11.5 when the tongue primordia (i.e., lingual swellings) emerge, the relatively homogeneous sonic hedgehog-expressing (Shh) epithelial cells express Keratin (K) 8, a marker that is widely used to label taste buds. Mapping lineage of E11.0 Shh epithelium of the tongue rudiment with Shh-CreER/RFP mice demonstrated that both the early taste buds and the surrounding lingual epithelium are from the same population of progenitors - Shh epithelial cells of the tongue primordium. In combination with previous reports, we propose that ShhK8 cells in the homogeneous epithelium of tongue primordium at early embryonic stages are programmed to become taste papilla and taste bud cells. Switching off Shh and K8 expression in the Shh epithelial cells of the tongue primordium transforms the cells to non-gustatory cells surrounding papillae, including K14 basal epithelial cells which will eventually contribute to the cell renewal of mature taste buds.

Early taste buds are from Shh epithelial cells of tongue primordium in distinction from mature taste bud cells which arise from surrounding tissue compartments.

Proper development of taste organs including the tongue and taste papillae requires interactions with the underlying mesenchyme through multiple molecular signaling pathways. The effects of bone morphogenetic proteins (BMPs) and antagonists are profound, however, the tissue-specific roles of distinct receptors are largely unknown. Here, we report that constitutive activation (ca) of ALK2-BMP signaling in the tongue mesenchyme (marked by Wnt1-Cre) caused microglossia-a dramatically smaller and misshapen tongue with a progressively severe reduction in size along the anteroposterior axis and absence of a pharyngeal region. At E10.5, the tongue primordia (branchial arches 1-4) formed in Wnt1-Cre/caAlk2 mutants while each branchial arch responded to elevated BMP signaling distinctly in gene expression of BMP targets (Id1, Snai1, Snai2, and Runx2), proliferation (Cyclin-D1) and apoptosis (p53). Moreover, elevated ALK2-BMP signaling in the mesenchyme resulted in apparent defects of lingual epithelium, muscles, and nerves. In Wnt1-Cre/caAlk2 mutants, a circumvallate papilla was missing and further development of formed fungiform papillae was arrested in late embryos. Our data collectively demonstrate that ALK2-BMP signaling in the mesenchyme plays essential roles in orchestrating various tissues for proper development of the tongue and its appendages in a region-specific manner.

Increased activity of mesenchymal ALK2-BMP signaling causes posteriorly truncated microglossia and disorganization of lingual tissues.

Taste bud cells are specialized epithelial cells that undergo continuous turnover, and thus require active progenitors for their renewal and an intact taste function. Our previous studies suggested that a population of taste bud cells originates from outside of the surrounding tongue epithelium-previously regarded sole source of taste bud progenitors. In this study, we demonstrated that  (SRY-related HMG-box gene 10)-expressing cells, known to be in the migrating neural crest, were also distributed in taste bud-surrounding tissue compartments under the tongue epithelium, that is, the connective tissue core of taste papillae and von Ebner's glands. By lineage tracing of -expressing cells using , a  model driven by the endogenous  promoter, crossing with a  reporter line R26-tdTomato (tdT), we found -labeled tdT cells within taste buds in all three types of taste papillae (fungiform, circumvallate, and foliate) as well as in the soft palate in postnatal mice. The tdT taste bud cells were progressively more abundant along the developmental stages, from virtually zero at birth to over 35% in adults. Most of tdT taste bud cells had a low intensity of immunosignals of Keratin 8 (a widely used taste bud cell marker). In circumvallate taste buds, tdT signals were co-localized principally with a type III taste bud cell marker, less so with type I and II cell makers. Together, our data demonstrate a novel progenitor source for taste buds of postnatal mice--labeled cells in the connective tissue core and/or von Ebner's glands.

Mohamed Ishan

Regenerative Bioscience Center; Department of Animal and Dairy Science,
College of Agricultural and Environmental Sciences,
Regenerative Bioscience Center; Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences

University of Georgia

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice