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10x chromium core mgh8/7/2023 ![]() ![]() To determine whether transient activation of Gli-dependent canonical Hedgehog signaling is able to rescue IR-impaired salivary function, we performed 15 Gy local IR to the neck region containing all major SGs in 8- to 10-week-old C57BL/6 mice. Lineage tracing and immunofluorescence stainingīesides the canonical signaling cascade mediated by Gli transcription factors, Hedgehog ligands are capable of triggering several Gli-independent noncanonical signaling cascades ( 20). Normalized data are shown in the form of feature plots. Putative T cells and B cells are abnormally abundant in GFP sample only likely due to blood contamination and are excluded from further analyses. The Uniform Approximation and Projection method was used to visualize the clusters. ![]() The bioinformatics analyses were performed by researchers blinded to treatments with methods described in the Supplementary Methods. Note that 11,048 and 18,333 cells from GFP and Shh groups were sequenced to a depth of 43,924 mean reads per cell and 1,420 median genes per cell. Samples were pooled in equimolar concentrations and sequenced on a single lane of an S4 2 × 150 PE flow cell for an Illumina NovaSeq 6000. Single-cell RNA-seq (scRNA-seq) libraries were generated in TAMU Genomics Core using the 10x Chromium platform and the version 3 Chromium Single Cell 3′ reagent kit following the manufacturer's instructions (10x Genomics). Single-cell suspensions from mouse SMGs were generated as mentioned above. Transient Hedgehog activation ameliorated IR-damage of SG epithelial and endothelial cells, but the direct cellular and molecular targets of Hedgehog activation in SGs remained unclear. ![]() We transiently activated the Hedgehog pathway after IR by inducible expression or adenovirus-mediated intra-SMG transfer of Shh gene, and observed that the long-term salivary function was rescued in both mouse and miniature pig models ( 9–12). Our previous studies revealed that the activity of canonical Hedgehog signaling in adult mouse SMGs is very low in the healthy state, increases following the obstructive damage that induces functional SG regeneration, but is unaffected by IR ( 8). Consequently, the increased ratio of active to repressive form of Gli factors induces the expression of Hedgehog target genes such as Gli1 and Ptch1 (Supplementary Fig. In the canonical Hedgehog signaling cascade, Hedgehog-Ptch binding derepresses the membrane protein Smoothened, which in turn promotes an increase in the active form of Gli transcription factors (full length Gli1/2/3) and decrease in the repressive form of Gli (truncated Gli2/3). Hedgehog signaling is triggered by the binding of Hedgehog ligand proteins such as Sonic hedgehog (Shh) to Patched receptors (Ptch1/2). We found recently that IR-impaired salivary function can be rescued by the transient activation of Hedgehog signaling within SMGs. These findings reveal that resident macrophages and their prorepair paracrine factors are essential for the rescue of irradiation-impaired salivary function by transient Hedgehog activation and are promising therapeutic targets of radiotherapy-induced irreversible dry mouth. ![]() Consistently, expression of these paracrine factors and their receptors in salivary glands decreased following irradiation but were restored by transient Hedgehog activation. Single-cell RNA sequencing and qRT-PCR of sorted cells indicated that Hedgehog activation greatly enhances paracrine interactions between salivary gland resident macrophages, epithelial progenitors, and endothelial cells through Csf1, Hgf, and C1q signaling pathways. Conversely, depletion of salivary gland macrophages by clodronate liposomes compromised the restoration of irradiation-impaired salivary function by transient Hedgehog activation. Quantities and activities of salivary gland resident macrophages were substantially and rapidly impaired by irradiation and restored by Hedgehog activation. Salivary gland cells responsive to Hedgehog/Gli signaling comprised small subsets of macrophages, epithelial cells, and endothelial cells, and their progeny remained relatively rare long after irradiation and transient Hedgehog activation. Here, we show in mice that activation of canonical Gli-dependent Hedgehog signaling by Gli1 gene transfer is sufficient to recover salivary function impaired by irradiation. Recent studies indicate that transient activation of Hedgehog signaling rescues irradiation-impaired salivary function in animal models, but the underlying mechanisms are largely unclear. Irreversible hypofunction of salivary glands is a common side effect of radiotherapy for head and neck cancer and is difficult to remedy. ![]()
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