The DNA damage response pathway is required for multiciliated cell differentiation.

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Published in final edited form as: Curr Biol. 2026 Apr 6;36(8):2156–2166.e3. doi: 10.1016/j.cub.2026.03.027 Search in PMC Search in PubMed View in NLM Catalog Add to search The DNA damage response pathway is required for multiciliated cell differentiation Cayla E Jewett Cayla E Jewett 1 Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, North Wolfe Street, Baltimore, MD, 21205, USA 2 Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, East 17 th Avenue, Aurora, CO, 80045, USA Find articles by Cayla E Jewett 1, 2, * , Andrew J Holland Andrew J Holland 1 Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, North Wolfe Street, Baltimore, MD, 21205, USA 3 Current position: Oncology Research, Amgen Research, One Amgen Center Drive, Thousand Oaks, CA, 91320, USA Find articles by Andrew J Holland 1, 3, * , Chad G Pearson Chad G Pearson 2 Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, East 17 th Avenue, Aurora, CO, 80045, USA Find articles by Chad G Pearson 2, * Author information Article notes Copyright and License information 1 Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, North Wolfe Street, Baltimore, MD, 21205, USA 2 Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, East 17 th Avenue, Aurora, CO, 80045, USA 3 Current position: Oncology Research, Amgen Research, One Amgen Center Drive, Thousand Oaks, CA, 91320, USA Author contributions Conceptualization, CEJ, AJH, CGP; Investigation, CEJ; Formal Analysis, CEJ; Writing – Original Draft, CEJ, CGP; Writing –Review & Editing, CEJ, AJH, CGP; Funding Acquisition, CEJ, AJH, CGP. * Corresponding authors: [email protected] , [email protected] , [email protected] Issue date 2026 Apr 20. PMC Copyright notice PMCID: PMC13058275  NIHMSID: NIHMS2157284  PMID: 41946360 The publisher's version of this article is available at Curr Biol Previous version available: This article is based on a previously available preprint posted on bioRxiv on July 14, 2025: " The DNA damage response pathway is required for multiciliated cell differentiation ". Summary Multiciliated cells (MCCs) lining the airways, reproductive tracts, and brain ventricles, construct hundreds of motile cilia, each anchored by a centriole 1 . This necessitates the production of hundreds of centrioles in a post-mitotic state, yet centriole duplication is normally restricted to the S and G2 phases of the cell cycle 2 . During their differentiation, MCCs utilize an alternative cell cycle repurposing many of the Cyclin-CDKs used in a canonical cell cycle 3 , 4 , yet how this alternative cell cycle bypasses numerical and temporal constraints governing centriole duplication remains unclear. DNA damage can result from external sources or occur during programmed genome rearrangements in processes like immunity or meiosis 5 . To maintain genome integrity, cells activate DNA repair pathways that alter the cell cycle to prevent harmful outcomes such as cancer or immune dysfunction 6 . Here, we uncover an unexpected role for DNA damage during the terminal differentiation of MCCs. We show that differentiating MCCs accumulate extensive double-strand DNA breaks during centriole amplification, with DNA damage levels scaling with centriole number. DNA damage response (DDR) kinases are required to support centriole biogenesis and ciliogenesis. Moreover, we find that the high transcriptional output needed to express centriole and cilia genes generates RNA-DNA hybrids (R-loops) that co-localize with sites of DNA damage. These findings suggest that transcription-coupled DNA damage engages DDR signaling to permit centriole amplification in MCCs. Together, our findings reveal a developmental program that harnesses physiological DNA damage and DDR signaling to adaptively rewire the canonical cell cycle. Keywords: DNA damage, H2AX, ATM, DNA-PK, R-loop, centriole amplification, cilia, cell cycle eTOC: Jewett et al. find that multiciliated cells exhibit abundant DNA damage during differentiation and require the DNA damage response kinases. These results reveal how DNA damage signaling can be used to rewire a canonical cell cycle into a developmental process that supports centriole overduplication. Graphical Abstract Results and Discussion DNA damage occurs during centriole amplification in MCCs To address how MCCs undergo a unique cell cycle allowing for overduplication of centrioles in the absence of other S and G2 process, we screened for events that regulate the cell cycle. Surprisingly, differentiating cells that expressed the MCC transcription factor FOXJ1 produced abundant double-strand DNA breaks labeled by γH2AX stained foci ( Figure 1A , cyan nuclei label MCCs). These DNA breaks were not present in neighboring FOXJ1-negative stem cells ( Figure 1A ). γH2AX foci in MCCs colocalized with the DNA damage marker 53BP1 ( Figure 1B ), suggesting that DNA repair machinery was recruited to damage sites. To confirm the DNA damage observed with immunofluorescence, an alkaline comet assay was performed. We first validated this assay in our primary culture system by treating stem cells prior to differentiation and MCCs with etoposide to exogenously induce double-strand DNA breaks and observed an elongated comet tail compared to the comet head, indicating the migration of smaller chromosome fragments ( Figure 1C bottom panel, 1D , S1A ). Differentiating MCCs also showed an elongated comet tail when compared to the comet head, indicating DNA breaks ( Figure 1C top right panel, 1D , S1A ). In contrast, this comet tail was not present in undifferentiated stem cells ( Figure 1C top left panel, 1D , S1A ). Together this shows that DNA damage occurs during MCC differentiation. Figure 1. DNA damage occurs during centriole amplification in MCCs. Open in a new tab ( A ) Primary mouse trachea epithelia cells differentiated at an air-liquid interface for 7 days, fixed, and stained for the MCC transcription factor (FOXJ1, cyan), DNA damage (γH2AX, magenta), and DNA (DAPI, blue). ( B ) Single nucleus of an MCC from primary mouse trachea epithelia cells fixed at differentiation day 7 and stained for DNA damage (γH2AX, magenta), DNA damage signaling (53BP1, gray), and DNA (DAPI, blue). Arrows denote examples of colocalization between γH2AX and 53BP1 foci. ( C ) DMSO treated (top panel) and Etoposide treated (bottom panel) nuclei in comet assay showing stem cells prior to differentiation (left) and MCCs at differentiation day 5 (right). Images were pseudo-colored into a heat map so that warmer colors represent higher fluorescence intensity. Comet tails indicate shorter DNA fragments from breaks that have migrated in the electrophoresis direction. ( D ) Quantitation of comet assay measuring ratio of tail to head distance from centroid of nucleus. See also Figure S1 . Nuclei with DNA breaks will have a longer comet tail. Note that ALI cultures at differentiation day 5 include both MCCs and stem cells without DNA breaks, contributing to the large variance. Each dot represents a single nucleus, and 3 biological replicates are represented. Welch’s t-test was used to determine P-values. ( E ) Primary mouse trachea epithelia cells fixed at differentiation days 3-14 and stained for the MCC transcription factor (FOXJ1, cyan), DNA damage (γH2AX, magenta), microtubules, centrioles, and cilia (β-tubulin, green), and DNA (DAPI, blue). MCCs were grouped into differentiation stages based on FOXJ1 and β-tubulin staining. See also Figure S1B , C . ( F-G ) Fold-change in FOXJ1 ( F ) and γH2AX ( G ) nuclear intensities compared to neighboring stem cells. Each dot represents the nuclear average from a field of view, and 3 biological replicates are represented. Mann-Whitney test was used to determine P-values. ( H ) Primary mouse trachea epithelia cells fixed at differentiation day 7 and stained for the MCC transcription factor (FOXJ1, cyan), DNA damage (γH2AX, magenta), and DNA (DAPI, blue). Cells were treated with etoposide either during transcription activation (differentiation days 0-2) or during centriole amplification (differentiation days 3-5). ( I,J ) Percent of FOXJ1+ cells ( I ) and cells with motile cilia ( J ) for etoposide treatment in (H). Each dot represents a field of view, and 3 biological replicates are represented. Mann-Whitney test was used to determine P-values. All graphs show mean ± SD. We next investigated when during the process of MCC differentiation DNA damage arises. MCC differentiation occurs over two weeks in culture and can be asynchronous among a population of cells. Broadly, differentiation can be grouped into three stages. The first stage begins in the nucleus with transcription activation of an MCC-specific transcription program. The second stage is cytoplasmic centriole amplification, when cells assemble hundreds of centrioles. The third stage is ciliation, during which cells nucleate motile cilia from these newly assembled centrioles. We assessed DNA damage using γH2AX alongside markers for the stages of MCC differentiation ( Figure 1E , S1B , C , see methods ). γH2AX nuclear intensity increased slightly during transcription activation, peaked at 4-fold during centriole amplification, and persisted throughout ciliation, although decreasing in intensity ( Figure 1E – G ). This timing of DNA damage during centriole amplification was consistent in both cultured primary mouse trachea epithelia cells and mouse tracheas in vivo ( Figure 1E and S1D , E ). In comparison, the MCC transcription factor FOXJ1 increased 7.5-fold during transcription activation and remained elevated throughout differentiation ( Figure 1E – F ). These results indicate that DNA damage occurs during the centriole amplification stage of MCC differentiation. Given this robust DNA damage during MCC differentiation, we tested whether exogenous damage could induce differentiation by treating cells with the DNA damaging drug etoposide, either during the transcription activation stage or centriole amplification stage. Etoposide treatment during either stage did not induce differentiation and instead decreased the number of MCCs ( Figure 1H – J ). Furthermore, etoposide treatment generated γH2AX foci that were larger and fewer than those observed in MCCs ( Figure 1H ). Together this demonstrates that MCCs show physiologic DNA damage during differentiation that is unique from exogenous damage. To verify that DNA damage is dependent on the MCC differentiation program, we used the auxin inducible degron (AID) system to degrade the earliest MCC transcription factor, GEMC1. GEMC1 is upstream of FOXJ1, thus GEMC1 loss completely prevents transcription activation and differentiation of MCCs 7 – 9 . To temporally control GEMC1 function, we knocked in an AIDRuby tag at the endogenous GEMC1 locus in mice expressing the E3 ligase adaptor OsTIR1. GEMC1 depletion with IAA from the onset of differentiation resulted in loss of MCCs ( Figure S1F ), which is consistent with GEMC1 KO models 7 – 9 . Moreover, GEMC1 depletion abolished DNA damage ( Figure S1F ), suggesting that DNA damage is downstream of transcription activation and specific to MCC differentiation. DNA damage scales with centriole number Given that DNA damage correlates with the timing of centriole amplification, we then asked whether the amount of DNA damage relates to centriole number. Different multiciliated tissues have varying numbers of cilia and thus centrioles so we compared the amount of DNA damage across these MCC types relative to stem cells. This analysis revealed that nuclear γH2AX intensity was strongest in trachea MCCs (average 250-300 centrioles per cell 10 , 11 ). Brain ventricle MCCs, which make an average of 40-100 centrioles 11 – 14 , also showed DNA damage during centriole amplification, but it was decreased relative to trachea MCCs ( Figure 1E , 2A , B ). Therefore, both trachea and brain MCCs show DNA damage during differentiation, and at the tissue level, MCCs with more centrioles have more DNA damage. Figure 2. DNA damage scales with centriole number. Open in a new tab ( A ) Primary mouse brain ventricle ependymal cells fixed at differentiation days 7-10 and stained for the MCC transcription factor (FOXJ1, cyan), DNA damage (γH2AX, magenta), centrioles (CenGFP, green), and DNA (DAPI, blue). MCCs were grouped into differentiation stages based on FOXJ1 and CenGFP staining. ( B ) Fold-change in γH2AX nuclear intensities for trachea and brain ventricle MCCs compared to neighboring stem cells. Each dot represents the nuclear average from a field of view, and 3 biological replicates are represented. Welch’s t-test was used to determine P-values. ( C ) Primary mouse trachea epithelia cells fixed at differentiation day 3 and stained for the centriole protein (PLK4, green), DNA damage (γH2AX, magenta), and DNA (DAPI, blue). ( D, E ) Linear correlation plot of the fold-change in cytoplasmic centriole protein intensity (PLK4 in ( D ) and cytoplasmic deuterosome protein intensity DEUP1 in ( E )) versus fold-change in γH2AX nuclear intensity. Each dot represents a single cell, and 3 biological replicates are represented. Simple linear regression was used to calculate R-squared and P-values. All graphs show mean ± SD. See also Figure S2 . To further test whether DNA damage scales with centriole amplification, the quantity of DNA damage and centriole protein levels were assessed in individual trachea MCCs. The cytoplasmic protein intensity of two centriole proteins (PLK4 and PCNT) and the deuterosome protein (DEUP1) were quantified relative to nuclear γH2AX intensity. Centriole and deuterosome protein intensities positively correlated with γH2AX intensity ( Figure 2C – E , S2A ). In summary, DNA damage increases with centriole number in tissues and centriole protein abundance in individual MCCs. DNA damage sites co-localize with repair kinases and RNA-DNA hybrids DNA damage is a normally harmful process that cells rapidly repair 5 . Our data demonstrate that MCCs show physiologic DNA damage, so we next explored the composition of these DNA damage sites. We first examined the localization of the three main DDR kinases ATR, ATM, and DNA-PK, which are activated by phosphorylation 15 . Phospho-antibodies were used to indicate the activity of ATR, ATM, or DNA-PK, and the nuclear intensity was analyzed at the single cell level. pATM and pDNA-PK showed a 3-fold intensity increase in cells undergoing centriole amplification compared to stem cells ( Figure 3A – D , S3A ). After activation, the DDR kinases phosphorylate downstream substrates, so substrate phosphorylation was analyzed. An antibody recognizing the phospho-motif of the DDR substrates also increased during centriole amplification and remained slightly elevated during the ciliation stage ( Figure S3C , D ). This suggests that DDR kinases are activating downstream effectors involved in DNA damage signaling. Finally, foci of pATM, pDNA-PK, and phosphorylated DDR substrates colocalized with γH2AX foci ( Figure S3E – G ). In contrast, pATR was only mildly increased in MCCs and did not colocalize with γH2AX foci ( Figure S3H , I ). Together this suggests that DNA damage sites are recruiting specific DDR machinery for signaling and repair. Figure 3. DNA damage sites co-localize with repair kinases and RNA-DNA hybrids. Open in a new tab ( A ) Primary mouse trachea epithelia cells fixed at differentiation day 5 and stained for centrioles (Centrin, green), deuterosomes (DEUP1, red), phosphorylated-ATM (pATM, magenta), and DNA (DAPI, blue). MCCs were grouped into differentiation stages based on Centrin and DEUP1 staining. Antibody conflicts at the transcription activation stage precluded pATM staining. See also Figure S3A . ( B ) Fold-change in pATM nuclear intensity compared to neighboring stem cells. ( C ) Primary mouse trachea epithelia cells fixed at differentiation day 5 and stained for the MCC transcription factor (FOXJ1, cyan), phosphorylated-DNA-PK (pDNAPK, magenta), microtubules, centrioles, and cilia (β-tubulin, green), and DNA (DAPI, blue). MCCs were grouped into differentiation stages based on FOXJ1 and β-tubulin staining. See also Figure S3B . ( D ) Fold-change in pDNA-PK nuclear intensity compared to neighboring stem cells. ( E ) Primary mouse trachea epithelia cells fixed at differentiation day 5 and stained for the RNA-DNA hybrid/R-loop marker (S9.6, gray), DNA damage (γH2AX, magenta), microtubules, centrioles, and cilia (β-tubulin, green), and DNA (DAPI, blue). Arrows show examples of colocalization between γH2AX and R-loop foci. MCCs were grouped into differentiation stages based on γH2AX and β-tubulin staining. ( F ) Fold-change in S9.6 nuclear intensity compared to neighboring stem cells. For B , D , and F , each dot represents the nuclear average from a field of view, and 3 biological replicates are represented. Paired t-test was used to determine P-values. All graphs show mean ± SD. Some common sources of DNA damage include replication stress, transcription stress, and oxidative stress. The pseudo cell cycle state of MCCs permits centriole duplication without DNA replication 3 , however, replication stress can occur at dysfunctional telomeres in brain MCC progenitors with GEMC1 gain of function 13 . Therefore, we tested whether the DNA damage observed during centriole amplification occurs at telomeres. DNA FISH with a telomere probe combined with γH2AX immunofluorescence revealed that most DNA damage sites were distinct from telomeres ( Figure S3J , K ), suggesting that telomeric replication stress is not the primary cause of DNA damage in differentiating trachea MCCs. We next explored whether the unique transcriptome of MCCs requiring robust expression of many centriole and cilia genes could create a transcriptional burden contributing to DNA damage. R-loops are RNA-DNA structures that form in regions of heavy transcription and occur when a nascent RNA hybridizes to its DNA template displacing the non-template DNA strand 16 . R-loops are typically transient structures but if left unresolved cause DNA damage 16 . We therefore asked whether R-loops were present during MCC differentiation. To test this, MCCs were stained with the S9.6 antibody which recognizes R-loops 17 . In neighboring stem cells, R-loops could be visualized exclusively in the nucleolus ( Figure 3E , arrows); however, in MCCs many R-loops were also present throughout the nucleus ( Figure 3E , dashed circles). These R-loops formed specifically during the centriole amplification stage of MCC differentiation and colocalized with γH2AX foci ( Figure 3E , F ). To confirm the specificity of the S9.6 antibody for R-loops, nuclear extracts were treated with RNase H to digest the RNA strand of the RNA-DNA hybrid. Using a dot blot, loss of S9.6 signal was observed with RNase H treatment ( Figure S3L ). Together, this suggests that during MCC differentiation, DNA damage sites recruit active repair kinases and contain RNA-DNA hybrid structures. DNA damage response kinases are required for centriole amplification During a canonical cell cycle, DNA damage triggers the DDR pathway during which the DDR kinases phosphorylate substrates ultimately activating a checkpoint that allows time for DNA repair before the cell cycle proceeds. Since MCCs undergo a pseudo cell cycle to amplify centrioles 3 , 4 , and the DDR kinases are active during centriole amplification, we asked whether the activity of these kinases was required for MCC differentiation. To address this, kinase inhibitors to ATR, ATM, or DNA-PK were used. In many epithelial cell types, drug efflux pumps reduce effective drug concentration, especially for kinase inhibitors 18 . To overcome this in MCCs, all drug experiments were performed in the presence of verapamil, a drug pump inhibitor 19 . Kinase inhibitor concentrations were optimized with cell viability assays in proliferating mouse epithelial cells, dose response curves in MCCs, and validation with phospho-antibody staining ( Figure S4A – F ) (see methods ). We treated MCCs at the onset of differentiation with inhibitors to ATR, ATM, or DNA-PK and examined the number of MCCs one week later. Treatment with any of the kinase inhibitors reduced the number of MCCs ( Figure 4A , B ) suggesting that the DNA damage response pathway is required for MCC differentiation. We observed a slight decrease in the total number of cells with inhibitor treatment ( Figure S4G ). However, this was not enough to account for the decrease in MCCs, ruling out cell death as the cause for MCC loss. ATM and DNA-PK inhibition had the most significant decrease in the number of MCCs ( Figure 4A – B ), and the largest increase in kinase phosphorylation that indicates activity during differentiation ( Figure 3A – D ). Figure 4. DNA damage response kinases are required for centriole amplification. Open in a new tab ( A ) Primary mouse trachea epithelia cells fixed at differentiation day 7 and stained for the MCC transcription factor (FOXJ1, cyan), centrioles (γTub, gray), microtubules, centrioles, and cilia (β-tubulin, green), and DNA (DAPI, blue). Cells were treated with DMSO plus verapamil or kinase inhibitors plus verapamil from the onset of differentiation to fixation (differentiation days 0-7). ( B ) Percent of FOXJ1+ multiciliated cells with kinase inhibitor treatment. Each dot represents a field of view, and 3 biological replicates are represented. Mann-Whitney test was used to determine P-values. ( C ) Schematic broadly depicting differentiation day timing in culture relative to stages of MCC differentiation. ( D ) Quantitation of time course experiments where primary mouse trachea epithelia cells were treated with DMSO plus verapamil or kinase inhibitors plus verapamil from the onset of differentiation and fixed every day for 7 days and again at 14 days. Graph shows the percentage of FOXJ1+ cells throughout the time course. Each dot represents a field of view, and 3 biological replicates are represented. ( E-G ) Quantitation of time course experiments where primary mouse trachea epithelia cells were treated with DMSO plus verapamil or kinase inhibitors plus verapamil from the onset of differentiation and fixed every day for 7 days. Graphs show the percent of cells in early centriole amplification ( E ), late centriole amplification ( F ), and ciliation ( G ) stages. PCNT, CEP164, and β-tubulin were used to stage cells. Each dot represents a field of view, and 3 biological replicates are represented. ( H ) Fold-change in γH2AX nuclear intensity compared to neighboring stem cells for DMSO and kinase inhibitor treated cells at each stage of differentiation. Each dot represents the nuclear average from a field of view, and 3 biological replicates are represented. B and H show mean ± SD. D-G show mean ± SEM. See also Figure S4 . Given that DNA damage occurs with centriole amplification, we next wanted to temporally resolve when the DDR kinases ATM and DNA-PK function during the stages of differentiation. Cells were treated with ATM or DNA-PK inhibitor from the onset of differentiation, and cells were fixed every day for one week and then at two weeks. ATM and DNA-PK activity were not required at early timepoints (days 0-2) for transcription activation but were required for centriole amplification and ciliation (days 3-14) ( Figure 4C , D ). We next examined how kinase inhibition affected centrioles and cilia. ATM or DNA-PK inhibition delayed and reduced the number of cells in early centriole amplification, late centriole amplification, and ciliation stages ( Figure 4D – G , see methods for staging). This suggests that ATM and DNA-PK activity are dispensable for transcription activation but required for robust initiation and completion of centriole amplification and ciliation. Finally, we asked whether ATM and DNA-PK activity were required for DNA damage in MCCs. Examining the initial γH2AX intensity profile revealed no change in γH2AX intensity during transcription activation, consistent with the timing of kinase function ( Figure 4H ). The few cells that did undergo centriole amplification or ciliation with kinase inhibition showed nuclear γH2AX at similar levels to control cells ( Figure 4H , S4H ). However, these centrioles were immature, fewer, and clumped together leading to cilia that were short, sparse, and disorganized compared to controls ( Figure S4H , I ). This suggests that compensatory DDR pathways may promote the MCC program but fail to assemble normal centrioles and cilia. Altogether, this demonstrates that DNA damage and the DDR kinases are required for the centriole amplification and ciliation stages of MCC differentiation. A fundamental problem in cell biology is how centrioles are duplicated precisely once during a canonical cell cycle to prevent centriole overduplication, mitotic errors, and aneuploidy 20 . Here, we use the MCC system to study how canonical cell cycle counting mechanisms are bypassed to permit centriole overduplication required for MCC differentiation. We discovered that MCCs display large quantities of endogenous DNA damage during centriole amplification. Further, DDR kinase activity is required for MCC differentiation. The purpose of this widespread DNA damage and DDR pathway activation could be multifaceted. One possibility is that DNA damage is a byproduct of the MCC differentiation program and this damage must be repaired for proper development. In this context, DDR kinases are activated and required to repair DNA damage and prevent genomic instability. In this scenario, the MCC system will be a useful model to understand how cells tolerate robust physiologic DNA damage during development. Alternatively, but not mutually exclusive, DNA damage could activate DDR signaling which triggers a checkpoint holding cells in a pseudo S-phase that permits time for centriole amplification. In support of this model, the amount of DNA damage scales with the number of centrioles. Further, in brain MCCs, the p53/p21 pathway is activated in cycling progenitors that exit the cell cycle to differentiate 13 , and p21 is highly expressed in trachea MCCs during centriole amplification 3 . Given that DNA damage in canonical cycling cells delays the cell cycle until DNA damage is repaired, it is possible that MCCs repurpose the DDR pathway to reprogram the cell cycle and create a state conducive to centriole amplification. We observe RNA-DNA hybrids at damage sites suggesting that MCCs experience transcription stress that coincides with DNA damage. Therefore, DNA damage should occur at or near heavily transcribed genes, such as centriole or cilia genes. Further studies in MCCs are needed to confirm the location of DNA breaks and rule out other sources of DNA damage. For example, replication stress is a common cause of DNA damage. Given that centriole amplification is typically coupled to DNA replication, the unique cell cycle state of MCCs could lend itself to replication-induced damage. EdU incorporation to monitor DNA replication has been observed in MCCs when their pseudo cell cycle is perturbed 3 , 4 , 13 , 21 . However, EdU incorporation is not observed during normal MCC differentiation ( Figure S4K ; 3 ), and our data show that DNA damage is not at telomeres arguing against replication-associated damage. However, GEMC1 is a pioneer transcription factor in MCCs and belongs to the geminin family known to regulate the cell cycle and DNA replication 22 . Our data demonstrate that GEMC1 is required for DNA damage in MCCs, and DNA damage has been observed with GEMC1 overexpression in brain MCCs 13 . Therefore, even though we do not detect DNA replication by EdU incorporation in MCCs, we cannot rule out a replication-like damage eluding EdU detection perhaps driven by GEMC1. DNA damage has also been observed during massive genome remodeling events, such as the maternal to zygotic transition and neuronal activation 23 – 25 . Because MCCs must rewire their genome for robust expression of centriole and cilia genes, genome remodeling could contribute to DNA damage during differentiation. Finally, MCCs line an epithelial surface where they are exposed to environmental insults that promote DNA damage. Therefore, a combination of factors could contribute to the DNA damage observed during MCC differentiation. Given that we observe heterogeneity in γH2AX foci number and size throughout differentiation, it will be interesting to determine if different foci correlate with different levels of transcription, different genomic regions, or different sources of DNA damage. Our data show that ATM and DNA-PK inhibition show the strongest MCC phenotypes. However, it remains to be determined whether they have redundant or distinct roles during MCC differentiation. One possibility is that DNA-PK is important for the repair of lesions at transcriptionally active sites, whereas ATM slows the cell cycle. Consistent with this idea, DNA-PK is not required for p53-dependent cell cycle arrest in mouse fibroblasts 26 – 29 . At activation-induced neuronal and hormone genes, the DNA-PK pathway is required to repair DNA breaks 30 , 31 . Furthermore, the phenotypes of ATM and DNA-PK loss are distinct. ATM knockout mice are sterile and have increased tumorigenesis, whereas DNA-PK knockout mice exhibit severe combined immunodeficiency 32 – 34 . However, ATM and DNA-PK double knockout mice are embryonic lethal 35 , 36 indicating synthetic lethality. Moreover, in other systems, DNA-PK and ATM function redundantly to activate p53 and arrest the cell cycle 37 , 38 . Whether the DDR is a cause and/or consequence of the MCC differentiation program and the precise roles of the DDR kinases during MCC differentiation remain important questions for uncovering why the DDR is critical for MCC differentiation. Hydrocephaly, a phenotype associated with MCC loss, has been reported in knockout mouse models lacking genes involved in the DDR pathway, including RAD50, NEK1, CDKN1B, CDKN2A, TRP53BP2, SLX4, GNL1, CASP3 39 – 47 . Additionally, a large-scale proteomic analysis of mammalian MCCs identified a protein cluster associated with double-strand breaks and DDR 48 . While hydrocephaly has not been specifically reported in the DDR kinase knockout animal models 32 – 34 , 49 , 50 , DDR knockout mouse models typically do not have as severe phenotypes as drug inhibition or kinase dead mouse models due to the compensatory nature of DDR pathways 15 . Future experiments examining MCCs in DDR kinase knockout, kinase dead, and acute kinase depletion models alongside p53, p21, and cell cycle perturbations will be important for understanding the contribution of each DDR kinase and its repair pathway to MCC differentiation. Altogether our work identifies DNA damage as a prerequisite for centriole amplification in MCCs. Identifying how massive DNA damage is not harmful to MCCs and instead a physiologic component of the differentiation trajectory will illuminate how specialized cells utilize the DDR to maintain control over potentially pathological processes. Resource availability Lead Contact: Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Chad Pearson ( [email protected] ). Materials Availability: The mouse line generated in this study has been deposited to The Jackson Laboratory Repository and is available under JAX Stock # 040910. STAR methods Experimental model and Study participant details Mice were housed and cared for in AAALAC-accredited facilities. All animal experiments were performed according to the Johns Hopkins University Institute Animal Care and Use Committee (MO21M300) or the University of Colorado Institute Animal Care and Use Committee (01490). Strains were maintained on a standard chow diet, and a mixture of male and female C57BL/6J mice were used for experiments. No differences in sexes were observed. Mice aged 2-8 months were used for experiments. No differences were observed among mice from different ages. Methods details Generation of GEMC1-AID mice GEMC1-AID mice were created using CRISPR-Cas9 genome editing. A double stranded DNA donor template targeting the last exon of GEMC1 and containing mAID tagged with mRuby was microinjected along with preassembled crRNA + tracrRNA + Cas9 ribonucleoprotein complexes into B6SJLF1/J mouse embryos at the one-cell stage and transplanted into pseudo pregnant females. Microinjection and transplantation were performed by the Johns Hopkins Transgenic Core Facility. Offspring were screened by PCR and sequencing, and confirmed knock-ins were outcrossed to C57BL6/J mice for two generations. The GEMC1 strain was deposited to The Jackson Laboratory Repository and is available under JAX Stock # 040910. GEMC1-AID-mRuby heterozygous mice were then outcrossed to Rosa26-OsTIRF74G 51 mice and incrossed to generate homozygous lines. Primary mouse tracheal epithelia cell culture Mouse trachea epithelia cultures were prepared as described previously 52 . Briefly, tracheas were harvested from mice ranging from 2-8 months of age, digested overnight in pronase solution, after which stem cells were harvested and plated on transwell filters. Stem cells were allowed to proliferate for 5-7 days in mTEC Plus media before moving to an air-liquid interface with NuSerum media in the basal chamber to trigger differentiation. The day at which cells were moved to the air-liquid interface was called differentiation day 0. For experiments with DDR kinase inhibitors, verapamil and kinase inhibitor were added to the basal chamber on day 0. DMSO controls were also treated with verapamil. For experiments with etoposide, verapamil was not added. Drugs and concentrations are listed in the Key Resources Table . Key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-FOXJ1, clone 2A5 ThermoFisher Scientific Cat # 14-9965-82 Rabbit polyclonal phosphorylated histone H2A.X (Ser139) (γH2AX) Cell Signaling Technology Cat # 2577 Mouse monoclonal anti-53BP1, clone BP13 Milllipore Sigma Cat # MAB3802 Guinea pig polyclonal anti-β-Tubulin Geneva Antibody Facility Cat # AA344 Goat polyclonal anti-γ-Tubulin Holland Lab N/A Rabbit polyclonal anti-PLK4 Holland Lab N/A Rabbit polyclonal anti-DEUP1 Holland Lab N/A Mouse monoclonal anti-phosphorylated-ATM (Ser1981) Milllipore Sigma Cat # 05-740-25UG Rabbit polyclonal anti-phosphorylated-DNA-PKcs (Ser2056) Abcam Cat # ab18192 Rabbit polyclonal anti-phosphorylated-ATR (Ser428) Cell Signaling Technology Cat # 2853 Rabbit monoclonal phosphorylated-ATM/ATR substrate motif [(pS/pT) QG] Cell Signaling Technology Cat # 6966 Mouse monoclonal anti-DNA-RNA hybrid, clone S9.6 Milllipore Sigma Cat # MABE1095 Mouse monoclonal anti-PCNT BD Transduction Laboratories Cat # 611815 Rabbit polyclonal anti-CEP164 Milllipore Sigma Cat # ABE2621 Chemicals, peptides, and recombinant proteins KU-60019 ATM inhibitor Selleck Chemicals Cat # S1570 NU7441 DNA-PK inhibitor Selleck Chemicals Cat # S2638 VE-822 ATR inhibitor Selleck Chemicals Cat # S7102 Verapamil HCl Selleck Chemicals Cat # S4202 Etoposide Selleck Chemicals Cat # S1225 5-Ph-IAA Selleck Chemicals Cat # E8177 MTT Milllipore Sigma Cat # 475989-1GM Experimental models: Cell lines mouse: IMCD3 cells Laboratory of Andrew Holland N/A Experimental models: Organisms/strains Mouse: C57BL/6J JAX Stock # 000664 Mouse: GEMC1-AID-Ruby Created for this study; deposited at JAX Stock # 040910 Software and algorithms ImageJ Schindelin et al. 49 https://imagej.nih.gov/ij/ Illustrator Adobe https://www.adobe.com GraphPad Prism 10 GraphPad https://www.graphpad.com Open in a new tab Differentiation and staging of MCCs Stem cells were allowed to proliferate into a polarized epithelial monolayer before moving to an air-liquid interface to induce differentiation. This allowed for a consistent number of differentiating MCC between experiments. Due to verapamil treatment, we observed slightly reduced total MCC numbers compared to untreated cultures 18 . In our experiments, around 25-30% of cells were FOXJ1+ after 7 days at an air-liquid interface, and this increased to around 35% of FOXJ1+ cells after 14 days at an air-liquid interface. The non-MCCs remained as stem/basal cells or differentiated into secretory cells if cultured at an air-liquid interface for multiple weeks. The following immunofluorescence markers were used to stage fixed MCCs. To determine FOXJ1+ cells, the nuclei were segmented and thresholded using a semi-automated ImageJ macro. For the transcription activation stage, the nuclei were FOXJ1+ but the cytoplasm did not show centriole markers (labeled with PCNT, CEP164, Centrin, DEUP1) nor cytoplasmic microtubule networks (labeled with β-tubulin). For the centriole amplification stage, the nuclei were FOXJ1+ and the cytoplasm showed centriole markers (labeled with PCNT, CEP164, Centrin, DEUP1) and diffuse cytoplasmic microtubule networks (labeled with β-tubulin). We found that cytoplasmic microtubule networks correlated with the presence of centriole markers ( Figure S1B ), and therefore staged cells in centriole amplification based on the presence of cytoplasmic microtubule staining. We can further classify centriole amplification into early, mid, and late stages. During early centriole amplification, PCNT forms a cloud-type arrangement within the cytoplasm, cytoplasmic microtubules are starting to surround the cloud, and CEP164 has not yet accumulated within this cloud. During mid centriole amplification, the PCNT cloud becomes larger, and cytoplasmic microtubules increasingly surround the cloud, but CEP164 has not yet strongly accumulated within this cloud. During late centriole amplification, the PCNT cloud and cytoplasmic microtubules increase in intensity and CEP164 strongly accumulates within this cloud. For the ciliation stage, the nuclei were FOXJ1+ and some centriole markers persisted such as CEP164 and Centrin, but the diffuse cytoplasmic microtubule staining was lost and tubulin staining transitioned to labeling distinct cilia ( Figure S1B ). Primary mouse brain ependymal cell culture Ependymal cells were prepared as described previously 53 . Briefly, brains were harvested from P0-P3 pups, the telecephalons were isolated, enzymatically digested with DNase and papain, and progenitor cells were grown to confluence for 4-5 days. Cells were then plated at high density on coverslips and grown in serum-free media to promote differentiation. The day at which cells were moved to serum-free media was called differentiation day 0. Immunofluorescence Cells were fixed in either 4% paraformaldehyde for 15 minutes at room temperature or ice cold 100% methanol for 10 minutes at −20°C. Paraformaldehyde fixed cells were quenched for 5 minutes with glycine. Cells were then washed twice with PBS and permeabilized for 10 minutes in 0.1% Triton-X in PBS. Cells were blocked for 1-2 hours in block buffer (10% normal donkey serum, 0.1% Triton-X in PBS). Primary antibodies were diluted in block buffer and incubated overnight at room temperature. Cells were washed with 0.1% Triton-X in PBS and then incubated with secondary antibody for 1-2 hours at room temperature. Cells were washed again with 0.1% Triton-X in PBS and then mounted. Primary antibodies are listed in the Key Resources Table . Confocal microscopy Slides were imaged on either a Zeiss Axio Observer 7 inverted microscope with Slidebook 2023 software (3i—Intelligent, Imaging Innovations, Inc.), CSU-W1 (Yokogawa) T1 Super-Resolution SoRa Spinning Disk, and Prime 95B CMOS camera (Teledyne Photometrics) with a 63x/1.40 NA plan-apochromat oil immersion objective or a Nikon A1R confocal system with Nikon Elements software a 60x/1.40 NA. Images were processed in FIJI 54 . All images presented in figures are max projections. Comet assay The alkaline Comet assay 55 was modified for MCCs. Cells were harvested either on day 0 (stem cells) or day 5 (MCCs) by trypsinization and resuspended in ice cold PBS. 1500 cells were added to 1% agarose in a 1:2 ratio and 100uL of the cell/agarose mixture was quickly pipetted onto an agarose-coated slide. The mixture solidified for 5 minutes and then was submerged in cold lysis solution (1.2M NaCl, 100mM Na 2 EDTA, 0.1% sodium lauryl sarcosinate, 0.26M NaOH, pH >13) overnight at 4°C. Slides were then submerged in cold rinse solution (0.03M NaOH, 2mM Na 2 EDTA, pH >12.3) for 1 hour, replacing with fresh rinse solution every 20 minutes. Slides were moved to an electrophoresis chamber with fresh rinse solution just covering the top of the agarose mixture and run at a voltage of 0.6V/cm for 25 minutes. Slides were then neutralized in distilled water followed by staining with 2.5ug/mL propidium iodide for 20 minutes. Cells were rinsed in distilled water and imaged on a Nikon Ti Eclipse widefield microscope with a 40x/0.75NA air objective and Teledyne Photometrics Kinetix 22 camera. Images were analyzed in FIJI using line scans across the comet to measure intensity values. Dot blot Primary mouse trachea epithelia cells were cultured on filters as described above and differentiated at an air-liquid interface for 5 days before harvesting by trypsinization. 1.5 x 10 6 cells were lysed for nuclear fractionation and genomic DNA/RNA extraction as previously described 56 . Genomic extracts were incubated at 37°C for 2 hours either with or without addition of RNase H. 100ng of untreated and RNase H treated DNA was spotted onto two separate, positively charged nylon membranes and allowed to dry for 10 minutes before UV crosslinking. One membrane was then stained with methylene blue for 2 hours, washed with water, and imaged with white light. The second membrane was blocked for 1 hour in Tris-buffered saline with 0.05% Tween-20 and 5% milk, incubated with S9.6 primary antibody overnight, washed, incubated with IRDye 680RD goat anti-mouse for 2 hours, washed and imaged on a LI-COR Odyssey Scanner. Cell growth and viability assays DDR kinase inhibitor concentrations were optimized for MCCs. First, to measure endpoint growth and viability with prolonged drug treatment, thiazolyl blue tetrazolium bromide (MTT) assays were performed as described previously 57 . Immortalized mouse epithelial (IMCD3) cells were used for growth and viability assays due to the large number of cells needed. Briefly, IMCD3 cells were plated in triplicate for each condition, inhibitors were added the following day, and cells were left to proliferate. After 5 days, cell viability was assayed with MTT solution and absorbance was measured at 570 nm. Drug titration curves were generated and the highest concentration that did not produce a decrease in cell viability was used for dose response curves in MCCs. For dose response curves, MCCs were treated with a range of drug concentrations from day 0 through day 7, then cells were fixed and analyzed for markers of differentiation and cell viability. The optimal drug concentration was determined by selecting the highest concentration that decreased differentiation but not cell viability/density. The effectiveness of these drug concentrations was validated by loss of immunofluorescence signal when staining with antibodies to the phosphorylated/active form of each DDR kinase. Quantification and statistical analysis A minimum of three biological replicates were performed for each experiment. Statistical tests are indicated in figure legends. GraphPad Prism 10 was used for statistical analysis. For the fold-change intensity graphs, each point on the graph represents the average of all the cells at that stage from a single field of view, typically 300-400 cells. Values were normalized to the average stem cell intensity for that field of view. FIJI was used to manually draw an ROI around each nucleus. A minimum of three biological replicates were performed for each experiment, although some fields of view did not have cells at a particular stage thereby reducing the number of replicates for that stage. For the graphs displaying % of cells with a particular marker (i.e. FOXJ1+, centriole, cilia) each point on the graph represents a field of view encompassing 300-400 cells. A semi-automated macro was used to segment nuclei and count the total number of cells per field of view. Supplementary Material 1 NIHMS2157284-supplement-1.pdf (19.1MB, pdf) Highlights: Multiciliated cells show endogenous DNA damage during differentiation The quantity of DNA damage scales with centriole number DNA damage sites contain R-loops DNA damage response kinases are required for differentiation Acknowledgments We are grateful to Carolyn Ott and Alex Stemm-Wolf for critical reading of the manuscript, and members of the Holland and Pearson labs for helpful discussions. We thank Margaret Strong and Craig Zikan for help with mouse husbandry. This work was funded by the Damon Runyon Cancer Research Foundation (CEJ is a Merck Fellow DRG-2478-22); a Hartwell Foundation Fellowship to CEJ; R01GM133897, R01GM114119, R01CA266199 to AJH; R35GM140813 to CGP. Abbreviations: MCCs Multiciliated cells DDR DNA damage response Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Declaration of interests The authors declare no competing interests. Declaration of generative AI and AI-assisted technologies No AI technologies in any form were used in the preparation of this manuscript. 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