Atacama Clear for Complex 3D Imaging of Organs.

3D reconstructive imaging is a powerful strategy to interrogate the global architecture of tissues. We developed Atacama Clear (ATC), a novel method that increases 3D imaging signal-to-noise ratios (SNRs) while simultaneously increasing the capacity of tissue to be cleared. ATC potentiated the clearing capacity of all tested chemical reagents currently used for optical clearing by an average of 68%, and more than doubled SNRs. This increased imaging efficacy enabled multiplex interrogation of tough fibrous tissue and specimens that naturally exhibit high levels of background noise, including the heart, kidney, and human biopsies. Indeed, ATC facilitated visualization of previously undocumented adjacent nephron segments that exhibit notoriously high autofluorescence, elements of the cardiac conduction system, and the distinct human glomerular tissue layers, at single cell resolution. Moreover, ATC was validated to be compatible with fluorescent reporter proteins in murine, zebrafish, and 3D stem cell model systems. These data establish ATC for 3D imaging studies of challenging tissue types.

SARS-CoV-2 infection causes dopaminergic neuron senescence.

COVID-19 patients commonly present with signs of central nervous system and/or peripheral nervous system dysfunction. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively susceptible and permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 infection of DA neurons triggers an inflammatory and cellular senescence response. High-throughput screening in hPSC-derived DA neurons identified several FDA-approved drugs that can rescue the cellular senescence phenotype by preventing SARS-CoV-2 infection. We also identified the inflammatory and cellular senescence signature and low levels of SARS-CoV-2 transcripts in human substantia nigra tissue of COVID-19 patients. Furthermore, we observed reduced numbers of neuromelanin+ and tyrosine-hydroxylase (TH)+ DA neurons and fibers in a cohort of severe COVID-19 patients. Our findings demonstrate that hPSC-derived DA neurons are susceptible to SARS-CoV-2, identify candidate neuroprotective drugs for COVID-19 patients, and suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.

A pancreatic cancer organoid platform identifies an inhibitor specific to mutant KRAS.

KRAS mutations, mainly G12D and G12V, are found in more than 90% of pancreatic ductal adenocarcinoma (PDAC) cases. The success of drugs targeting KRASG12C suggests the potential for drugs specifically targeting these alternative PDAC-associated KRAS mutations. Here, we report a high-throughput drug-screening platform using a series of isogenic murine pancreatic organoids that are wild type (WT) or contain common PDAC driver mutations, representing both classical and basal PDAC phenotypes. We screened over 6,000 compounds and identified perhexiline maleate, which can inhibit the growth and induce cell death of pancreatic organoids carrying the KrasG12D mutation both in vitro and in vivo and primary human PDAC organoids. scRNA-seq analysis suggests that the cholesterol synthesis pathway is upregulated specifically in the KRAS mutant organoids, including the key cholesterol synthesis regulator SREBP2. Perhexiline maleate decreases SREBP2 expression levels and reverses the KRAS mutant-induced upregulation of the cholesterol synthesis pathway.

Personalized medicine in the dish to prevent calcium leak associated with short-coupled polymorphic ventricular tachycardia in patient-derived cardiomyocytes.

BACKGROUND: Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca2+ handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro. METHODS: Previously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs. RESULTS: The voltage-dependent Ca2+ channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca2+ in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control. CONCLUSIONS: By testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca2+ release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.

Isogenic human trophectoderm cells demonstrate the role of NDUFA4 and associated variants in ZIKV infection.

Population-based genome-wide association studies (GWAS) normally require a large sample size, which can be labor intensive and costly. Recently, we reported a human induced pluripotent stem cell (hiPSC) array-based GWAS method, identifying NDUFA4 as a host factor for Zika virus (ZIKV) infection. In this study, we extended our analysis to trophectoderm cells, which constitute one of the major routes of mother-to-fetus transmission of ZIKV during pregnancy. We differentiated hiPSCs from various donors into trophectoderm cells. We then infected cells carrying loss of function mutations in NDUFA4, harboring risk versus non-risk alleles of SNPs (rs917172 and rs12386620) or having deletions in the NDUFA4 cis-regulatory region with ZIKV. We found that loss/reduction of NDUFA4 suppressed ZIKV infection in trophectoderm cells. This study validated our published hiPSC array-based system as a useful platform for GWAS and confirmed the role of NDUFA4 as a susceptibility locus for ZIKV in disease-relevant trophectoderm cells.

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

A targetable pathway to eliminate TRA-1-60+/TRA-1-81+ chemoresistant cancer cells.

Chemoresistance is a primary cause of treatment failure in pancreatic cancer. Identifying cell surface markers specifically expressed in chemoresistant cancer cells (CCCs) could facilitate targeted therapies to overcome chemoresistance. We performed an antibody-based screen and found that TRA-1-60 and TRA-1-81, two 'stemness' cell surface markers, are highly enriched in CCCs. Furthermore, TRA-1-60+/TRA-1-81+ cells are chemoresistant compared to TRA-1-60-/TRA-1-81- cells. Transcriptome profiling identified UGT1A10, shown to be both necessary and sufficient to maintain TRA-1-60/TRA-1-81 expression and chemoresistance. From a high-content chemical screen, we identified Cymarin, which downregulates UGT1A10, eliminates TRA-1-60/TRA-1-81 expression, and increases chemosensitivity both in vitro and in vivo. Finally, TRA-1-60/TRA-1-81 expression is highly specific in primary cancer tissue and positively correlated with chemoresistance and short survival, which highlights their potentiality for targeted therapy. Therefore, we discovered a novel CCC surface marker regulated by a pathway that promotes chemoresistance, as well as a leading drug candidate to target this pathway.

Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells.

While Mek1/2 and Gsk3ß inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

An evolutionarily conserved pacemaker role for HCN ion channels in smooth muscle.

Although hyperpolarization-activated cation (HCN) ion channels are well established to underlie cardiac pacemaker activity, their role in smooth muscle organs remains controversial. HCN-expressing cells are localized to renal pelvic smooth muscle (RPSM) pacemaker tissues of the murine upper urinary tract and HCN channel conductance is required for peristalsis. To date, however, the Ih pacemaker current conducted by HCN channels has never been detected in these cells, raising questions on the identity of RPSM pacemakers. Indeed, the RPSM pacemaker mechanisms of the unique multicalyceal upper urinary tract exhibited by humans remains unknown. Here, we developed immunopanning purification protocols and demonstrate that 96% of isolated HCN+ cells exhibit Ih . Single-molecule STORM to whole-tissue imaging showed HCN+ cells express single HCN channels on their plasma membrane and integrate into the muscular syncytium. By contrast, PDGFR-α+ cells exhibiting the morphology of ICC gut pacemakers were shown to be vascular mural cells. Translational studies in the homologous human and porcine multicalyceal upper urinary tracts showed that contractions and pacemaker depolarizations originate in proximal calyceal RPSM. Critically, HCN+ cells were shown to integrate into calyceal RPSM pacemaker tissues, and HCN channel block abolished electrical pacemaker activity and peristalsis of the multicalyceal upper urinary tract. Cumulatively, these studies demonstrate that HCN ion channels play a broad, evolutionarily conserved pacemaker role in both cardiac and smooth muscle organs and have implications for channelopathies as putative aetiologies of smooth muscle disorders. KEY POINTS: Pacemakers trigger contractions of involuntary muscles. Hyperpolarization-activated cation (HCN) ion channels underpin cardiac pacemaker activity, but their role in smooth muscle organs remains controversial. Renal pelvic smooth muscle (RPSM) pacemakers trigger contractions that propel waste away from the kidney. HCN+ cells localize to murine RPSM pacemaker tissue and HCN channel conductance is required for peristalsis. The HCN (Ih ) current has never been detected in RPSM cells, raising doubt whether HCN+ cells are bona fide pacemakers. Moreover, the pacemaker mechanisms of the unique multicalyceal RPSM of higher order mammals remains unknown. In total, 97% of purified HCN+ RPSM cells exhibit Ih . HCN+ cells integrate into the RPSM musculature, and pacemaker tissue peristalsis is dependent on HCN channels. Translational studies in human and swine demonstrate HCN channels are conserved in the multicalyceal RPSM and that HCN channels underlie pacemaker activity that drives peristalsis. These studies provide insight into putative channelopathies that can underlie smooth muscle dysfunction.

A multi-organoid platform identifies CIART as a key factor for SARS-CoV-2 infection.

COVID-19 is a systemic disease involving multiple organs. We previously established a platform to derive organoids and cells from human pluripotent stem cells to model SARS-CoV-2 infection and perform drug screens1,2. This provided insight into cellular tropism and the host response, yet the molecular mechanisms regulating SARS-CoV-2 infection remain poorly defined. Here we systematically examined changes in transcript profiles caused by SARS-CoV-2 infection at different multiplicities of infection for lung airway organoids, lung alveolar organoids and cardiomyocytes, and identified several genes that are generally implicated in controlling SARS-CoV-2 infection, including CIART, the circadian-associated repressor of transcription. Lung airway organoids, lung alveolar organoids and cardiomyocytes derived from isogenic CIART-/- human pluripotent stem cells were significantly resistant to SARS-CoV-2 infection, independently of viral entry. Single-cell RNA-sequencing analysis further validated the decreased levels of SARS-CoV-2 infection in ciliated-like cells of lung airway organoids. CUT&RUN, ATAC-seq and RNA-sequencing analyses showed that CIART controls SARS-CoV-2 infection at least in part through the regulation of NR4A1, a gene also identified from the multi-organoid analysis. Finally, transcriptional profiling and pharmacological inhibition led to the discovery that the Retinoid X Receptor pathway regulates SARS-CoV-2 infection downstream of CIART and NR4A1. The multi-organoid platform identified the role of circadian-clock regulation in SARS-CoV-2 infection, which provides potential therapeutic targets for protection against COVID-19 across organ systems.

Development of a New Methodology for Dearomative Borylation of Coumarins and Chromenes and Its Applications to Synthesize Boron-Containing Retinoids.

Dearomative borylation of coumarins and chromenes via conjugate addition represents a relatively unexplored and challenging task. To address this issue, herein, we report a new and general copper (I) catalyzed dearomative borylation process to synthesize boron-containing oxacycles. In this report, the borylation of coumarins, chromones, and chromenes comprising functional groups, such as esters, nitriles, carbonyls, and amides, has been achieved. In addition, the method generates different classes of potential boron-based retinoids, including the ones with oxadiazole and anthocyanin motifs. The borylated oxacycles can serve as suitable intermediates to generate a library of compounds.

A human iPSC-array-based GWAS identifies a virus susceptibility locus in the NDUFA4 gene and functional variants.

Population-based studies to identify disease-associated risk alleles typically require samples from a large number of individuals. Here, we report a human-induced pluripotent stem cell (hiPSC)-based screening strategy to link human genetics with viral infectivity. A genome-wide association study (GWAS) identified a cluster of single-nucleotide polymorphisms (SNPs) in a cis-regulatory region of the NDUFA4 gene, which was associated with susceptibility to Zika virus (ZIKV) infection. Loss of NDUFA4 led to decreased sensitivity to ZIKV, dengue virus, and SARS-CoV-2 infection. Isogenic hiPSC lines carrying non-risk alleles of SNPs or deletion of the cis-regulatory region lower sensitivity to viral infection. Mechanistic studies indicated that loss/reduction of NDUFA4 causes mitochondrial stress, which leads to the leakage of mtDNA and thereby upregulation of type I interferon signaling. This study provides proof-of-principle for the application of iPSC arrays in GWAS and identifies NDUFA4 as a previously unknown susceptibility locus for viral infection.

Synthesis of a boron-containing amidoxime reagent and its application to synthesize functionalized oxadiazole and quinazolinone derivatives.

Herein, we report the design, synthesis and application of a borylated amidoxime reagent for the direct synthesis of functionalized oxadiazole and quinazolinone derivatives. This reagent exhibits broad synthetic utility to obtain a variety of biologically relevant drug-like molecules. It can be easily prepared at large scale from relatively inexpensive reagents, and can undergo facile transformations to obtain target compounds. The developed amidoxime reagent was synthesized from 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile and hydroxyl amine hydrochloride using N,N-diisopropylethylamine as a base in ethanol under reflux conditions. Overall advantages include a metal-free route to boronated oxadiazoles, quinazolinone derivatives, and restriction of the multistep sequences. Importantly, the boron-rich pharmacophore derived compounds were obtained through an efficient and inexpensive strategy.

Boron Chemicals in Drug Discovery and Development: Synthesis and Medicinal Perspective.

A standard goal of medicinal chemists has been to discover efficient and potent drug candidates with specific enzyme-inhibitor abilities. In this regard, boron-based bioactive compounds have provided amphiphilic properties to facilitate interaction with protein targets. Indeed, the spectrum of boron-based entities as drug candidates against many diseases has grown tremendously since the first clinically tested boron-based drug, Velcade. In this review, we collectively represent the current boron-containing drug candidates, boron-containing retinoids, benzoxaboroles, aminoboronic acid, carboranes, and BODIPY, for the treatment of different human diseases.In addition, we also describe the synthesis, key structure-activity relationship, and associated biological activities, such as antimicrobial, antituberculosis, antitumor, antiparasitic, antiprotozoal, anti-inflammatory, antifolate, antidepressant, antiallergic, anesthetic, and anti-Alzheimer's agents, as well as proteasome and lipogenic inhibitors. This compilation could be very useful in the exploration of novel boron-derived compounds against different diseases, with promising efficacy and lesser side effects.

A dual SHOX2:GFP; MYH6:mCherry knockin hESC reporter line for derivation of human SAN-like cells.

The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and limitations in robust in vitro derivation methods. We developed a dual SHOX2:GFP; MYH6:mCherry knockin human embryonic stem cell (hESC) reporter line, which allows the identification and purification of SAN-like cells. Using this line, we performed several rounds of chemical screens and developed an efficient strategy to generate and purify hESC-derived SAN-like cells (hESC-SAN). The derived hESC-SAN cells display molecular and electrophysiological characteristics of bona fide nodal cells, which allowed exploration of their transcriptional profile at single-cell level. In sum, our dual reporter system facilitated an effective strategy for deriving human SAN-like cells, which can potentially be used for future disease modeling and drug discovery.

SARS-CoV-2 Infection Induces Ferroptosis of Sinoatrial Node Pacemaker Cells.

BACKGROUND: Increasing evidence suggests that cardiac arrhythmias are frequent clinical features of coronavirus disease 2019 (COVID-19). Sinus node damage may lead to bradycardia. However, it is challenging to explore human sinoatrial node (SAN) pathophysiology due to difficulty in isolating and culturing human SAN cells. Embryonic stem cells (ESCs) can be a source to derive human SAN-like pacemaker cells for disease modeling. METHODS: We used both a hamster model and human ESC (hESC)-derived SAN-like pacemaker cells to explore the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the pacemaker cells of the heart. In the hamster model, quantitative real-time polymerase chain reaction and immunostaining were used to detect viral RNA and protein, respectively. We then created a dual knock-in SHOX2:GFP;MYH6:mCherry hESC reporter line to establish a highly efficient strategy to derive functional human SAN-like pacemaker cells, which was further characterized by single-cell RNA sequencing. Following exposure to SARS-CoV-2, quantitative real-time polymerase chain reaction, immunostaining, and RNA sequencing were used to confirm infection and determine the host response of hESC-SAN-like pacemaker cells. Finally, a high content chemical screen was performed to identify drugs that can inhibit SARS-CoV-2 infection, and block SARS-CoV-2-induced ferroptosis. RESULTS: Viral RNA and spike protein were detected in SAN cells in the hearts of infected hamsters. We established an efficient strategy to derive from hESCs functional human SAN-like pacemaker cells, which express pacemaker markers and display SAN-like action potentials. Furthermore, SARS-CoV-2 infection causes dysfunction of human SAN-like pacemaker cells and induces ferroptosis. Two drug candidates, deferoxamine and imatinib, were identified from the high content screen, able to block SARS-CoV-2 infection and infection-associated ferroptosis. CONCLUSIONS: Using a hamster model, we showed that primary pacemaker cells in the heart can be infected by SARS-CoV-2. Infection of hESC-derived functional SAN-like pacemaker cells demonstrates ferroptosis as a potential mechanism for causing cardiac arrhythmias in patients with COVID-19. Finally, we identified candidate drugs that can protect the SAN cells from SARS-CoV-2 infection.

Stage-specific regulation of DNA methylation by TET enzymes during human cardiac differentiation.

Changes in DNA methylation are associated with normal cardiogenesis, whereas altered methylation patterns can occur in congenital heart disease. Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA demethylation. Here, we characterize stage-specific methylation dynamics and the function of TETs during human cardiomyocyte differentiation. Human embryonic stem cells (hESCs) in which all three TET genes are inactivated fail to generate cardiomyocytes (CMs), with altered mesoderm patterning and defective cardiac progenitor specification. Genome-wide methylation analysis shows TET knockout causes promoter hypermethylation of genes encoding WNT inhibitors, leading to hyperactivated WNT signaling and defects in cardiac mesoderm patterning. TET activity is also needed to maintain hypomethylated status and expression of NKX2-5 for subsequent cardiac progenitor specification. Finally, loss of TETs causes a set of cardiac structural genes to fail to be demethylated at the cardiac progenitor stage. Our data demonstrate key roles for TET proteins in controlling methylation dynamics at sequential steps during human cardiac development.

An airway organoid-based screen identifies a role for the HIF1α-glycolysis axis in SARS-CoV-2 infection.

It is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we derive airway organoids from human pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 infection. Using this platform, we perform a high content screen and identify GW6471, which blocks SARS-CoV-2 infection. GW6471 can also block infection of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 infection at least in part by inhibiting hypoxia inducible factor 1 subunit alpha (HIF1α), which is further validated by chemical inhibitor and genetic perturbation targeting HIF1α. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1α-glycolysis axis in mediating SARS-CoV-2 infection of human airway epithelium.

Sirt1 promotes tissue regeneration in zebrafish through regulating the mitochondrial unfolded protein response.

The mitochondrial unfolded protein response (UPRmt) is an organellar stress signaling pathway that functions to detect and restore disruption of mitochondrial proteostasis. The UPRmt is involved in a wide range of physiological and disease conditions, including aging, stem cell maintenance, innate immunity, neurodegeneration, and cancer. Here we report that the UPRmt is integral to zebrafish fin regeneration. Taking advantage of a novel zebrafish UPRmt reporter, we observed that UPRmt activation occurs in regenerating fin tissue shortly after injury. Through chemical and genetic approaches, we discovered that the Sirt1-UPRmt pathway, best known for its role in promoting lifespan extension, is crucial for fin regeneration. The metabolism of NAD+ is an important contributor to Sirt1 activity in this context. We propose that Sirt1 activation induces mitochondrial biogenesis in injured fin tissue, which leads to UPRmt activation and promotes tissue regeneration.