Irreversible hypofunction of salivary glands is a common side effect of radiotherapy for head and neck cancer and is difficult to remedy. Recent studies indicate that transient activation of Hedgehog signaling rescues irradiation-impaired salivary function in animal models, but the underlying mechanisms are largely unclear. 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. 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. Quantities and activities of salivary gland resident macrophages were substantially and rapidly impaired by irradiation and restored by Hedgehog activation. Conversely, depletion of salivary gland macrophages by clodronate liposomes compromised the restoration of irradiation-impaired salivary function 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. Consistently, expression of these paracrine factors and their receptors in salivary glands decreased following irradiation but were restored by transient Hedgehog activation. 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. Significance: These findings illuminate a novel direction for developing effective treatment of irreversible dry mouth, which is common after radiotherapy for head and neck cancer and for which no effective treatments are available.

Dear Editor, Programmed nuclear degradation (PND) and programmed cell death (PCD) are essential for tissue development and homeostasis in multicellular eucaryotes. PND may be a primitive form of PCD, in which nuclear degradation is uncoupled from plasma membrane events associated with cell death.1, 2 PND occurs in a subset of differentiating mammalian cells (for example, lens fibers), and like PCD is activated by caspases.3, 4 PND and PCD are found in ancient lineages, such as the ciliated protozoan, Tetrahymena thermophila,5, 6 and fundamental underlying mechanisms are conserved. Fas ligand, for example, activates PCD in higher eucaryotes and Tetrahymena,7 and caspase inhibitors effectively block Tetrahymena PND.2, 8 Tetrahymena PND is associated with chromatin condensation, formation of TUNEL-positive nuclei, and DNA cleavage into nucleosome-sized fragments.6, 8 Autophagy ensues as apoptotic nuclei fused with lysosomes.9 PND is an integral part of development in ciliates, which harbor genetically related, but functionally distinct micro- and macronuclei within the same cytoplasm.10 The diploid micronucleus is transcriptionally silent and serves as the reservoir of genetic material that is transmitted during conjugation. Four haploid pronuclei are generated during meiosis in T. thermophila, three of which undergo PND (Figure 1a, upper panel). The remaining nucleus divides to generate two genetically identical pronuclei. Pronuclei are reciprocally exchanged and fuse to form a diploid 'zygotic' micronucleus in each progeny cell. The micronucleus undergoes two post-meiotic divisions, after which two of the four progeny micronuclei differentiate into polyploid, transcriptionally active macronuclei. Hallmarks of macronuclear development include site-specific chromosome fragmentation, DNA rearrangement, selective gene amplification, and extensive chromatin remodeling.10 The commitment to new macronuclear anlagen formation triggers the destruction of the old parental macronucleus by an apoptotic-like autophagic mechanism.6, 8 Thus, PND, nuclear division, and nuclear differentiation are temporally regulated during development, thereby determining the fate of different nuclei that share a common cytoplasm. Since metazoan PCD and Tetrahymena PND utilize the same downstream effectors (caspases),2, 3 these processes may respond to similar signaling molecules. Phosphoinositide 3-kinase (PI 3-kinase) catalyzes the formation of PIP3, and induces autophagic PCD in mammals.11 PI 3-kinase inhibitors block PCD in mammals; however, a role for this pathway in mammalian PND has yet to be reported. Whereas PIP3 has not yet been identified in Tetrahymena, inositol phospholipid signaling is well documented,12 and there is compelling evidence for a PI 3-kinase gene family in the T. thermophila genome.13 To assess the potential role of PI 3-kinase in PND, we examined the effect of well-characterized PI 3-kinase inhibitors on Tetrahymena development. Mating cultures were treated with the PI 3-kinase inhibitors, wortmannin (Calbiochem), 3-methyladenine (3-MA, Sigma), or LY294002 (Calbiochem) at various times during development, and assayed for nuclear acidification and degradation by the pH-sensitive apofluor-staining method.9 Acidified, apoptotic nuclei stain orange/red, whereas healthy, nonacidic nuclei are blue/green (Figure 1a. top panel). Acidification of the (old) parental macronucleus was inhibited when 250 nM wortmannin was added to mating cells prior to pronuclear exchange (Figure 1a, bottom panel). In addition to blocking macronuclear acidification, additional staining structures were detected. The staining compartments correspond to nuclei, since they reacted with the DNA-specific dye DAPI alone (Figure 1b, lower right panel). The sizes and abundance of DAPI/Apofluor staining nuclei suggest that additional micronuclei and macronuclei are generated when PND is blocked. Since high concentrations of wortmannin inhibit myosin light chain kinase (MLCK) in other organisms,14 we assayed macronuclear acidification and nuclear overproliferation in cells treated with the highly specific MLCK inhibitor ML-7. ML-7 (1–10 M) had no effect on nuclear acidification or proliferation (Figure 1b). Higher concentrations (50–100 M) were toxic and therefore uninformative. Conversely, when we reduced the wortmannin concentration from 250 to 25 nM, a comparable block in macronuclear acidification was observed. A similar block in macronuclear acidification was seen with two additional PI 3-kinase inhibitors, 3-MA (10 mM) and LY294002 (100 M), at concentrations comparable to those used with cultured mammalian cells (Figure 1b). 3-MA and to a lesser extent LY294002 also induced nuclear overproliferation. Wortmannin and LY294002 bind to the catalytic subunit of PI 3-kinase, while 3-MA interacts with the regulatory subunit.15 The consistent results obtained with three different PI 3-kinase inhibitors argue that PI 3-kinase activates programmed degradation of the parental macronucleus and unexchanged pronuclei. In an effort to visualize the PI 3-kinase substrate (PIP2) and product (PIP3) directly, mating Tetrahymena cells were radiolabeled with [32]P-orthophosphate, and extracted lipids were separated by thin-layer chromatography.16 No labeled phospholipids were detected when the labeling period was restricted to starved mating cells (data not shown). However, when growing cells were labeled and then starved in preparation for mating, robust phospholipid labeling was observed throughout development. Although no definitive PIP3 signal was evident, its precursor, PIP2, was readily detected (Figure 1c). These results are consistent with data from mammalian cells, where PIP2 and PIP3 represent 5 and 0.005% of the total phosphatidylinositol pool.17 We conclude that the precursor for PIP3 is generated in actively growing cells and maintained throughout development. In combination with strong bioinformatic evidence for a PI...

The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.

Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the “cell cycles” of the diploid micronucleus and polyploid macronucleus are offset. Micronuclear S phase initiates just before cytokinesis and is completed in daughter cells before onset of macronuclear DNA replication. Mitotic micronuclear division occurs mid–cell cycle, while macronuclear amitosis is coupled to cell division. Here we report the first RNA-seq cell cycle analysis of a binucleated ciliated protozoan. RNA was isolated across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the 26,000+ predicted genes were shown to be cell cycle regulated. Proteins present in both nuclei exhibit a single mRNA peak that always precedes their macronuclear function. Nucleus-limited genes, including nucleoporins and importins, are expressed before their respective nucleus-specific role. Cyclin D and A/B gene family members exhibit different expression patterns that suggest nucleus-restricted roles. Periodically expressed genes cluster into seven cyclic patterns. Four clusters have known PANTHER gene ontology terms associated with G1/S and G2/M phase. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication, and cell division.

The Tetrahymena thermophila rDNA exists as a 21 kb palindromic minichromosome with two initiation sites for replication in each half palindrome. These sites localize to the imperfect, repeated 430 bp segments that include the nucleosome-free domains 1 and 2 (D1 and D2). To determine if the D1 and D2 segments act independently or in concert to control initiation, stable DNA transformation assays were performed. Single domain derivatives of the plasmid prD1 failed to support autonomous replication in Tetrahymena. Instead, such constructs propagated exclusively by integration into endogenous rDNA minichromo-somes and displayed weak origin activity as detected by 2D gel electrophoresis. D1/D1 and D2/D2 derivatives also transformed Tetrahymena poorly, showing similar replication defects. Hence, the D1 and D2 segments are functionally non-redundant and cooperate rather than compete to control initiation. The observed replication defect was greatly reduced in a plasmid derivative that undergoes palindrome formation in Tetrahymena, suggesting that a compensatory mechanism overcomes this replication block. Finally, using a transient replication assay, we present evidence that phylogenetically-conserved type I elements directly regulate DNA replication. Taken together, our data support a model in which cooperative interactions between dispersed elements coordinately control the initiation of DNA replication.

Problem: Understanding and communicating medical advances driven by basic research, and acquiring foundational skills in critically appraising and communicating translational basic research literature that affects patient care, are challenging for medical students to develop. Approach: The authors developed a mandatory course from 2012 to 2018 at Texas A&M University College of Medicine to address this problem. Medical Student Grand Rounds (MSGR) trains first-year students to find, critically assess, and present primary research literature about self-selected medically relevant topics. With basic science faculty mentoring, students completed milestones culminating in oral presentations. Students learned to search literature databases and then choose a clinical subject using these skills. They outlined the clinical subject area background and a mechanistic research topic into a clinical problem based on deeper evaluation of primary research literature. “Mechanistic” was defined in this context as providing experimental evidence that explained the “how” and “why” underlying clinical manifestations of a disease. Students received evaluations and feedback from mentors about discerning the quality of information and synthesizing information on their topics. Finally, students prepared and gave oral presentations, emphasizing the primary literature on their topics. Outcomes: In the early stages of the course development, students had difficulty critically assessing and evaluating research literature. Mentored training by research-oriented faculty, however, dramatically improved student perceptions of the MSGR experience. Mentoring helped students develop skills to synthesize ideas from basic research literature. According to grades and self-evaluations, students increased proficiency in finding and interpreting research articles, preparing and delivering presentations, and understanding links among basic and translational research and clinical applications. Next Steps: The authors plan to survey fourth-year students who have completed MSGR about their perceptions of the course in the context of clinical experiences in medical school to guide future refinements.