Sufu- and Spop-mediated regulation of Gli2 is essential for the control of mammalian cochlear hair cell differentiation.

Development of mammalian auditory epithelium, the organ of Corti, requires precise control of both cell cycle withdrawal and differentiation. Sensory progenitors (prosensory cells) in the cochlear apex exit the cell cycle first but differentiate last. Sonic hedgehog (Shh) signaling is required for the spatiotemporal regulation of prosensory cell differentiation, but the underlying mechanisms remain unclear. Here, we show that suppressor of fused (Sufu), a negative regulator of Shh signaling, is essential for controlling the timing and progression of hair cell (HC) differentiation. Removal of Sufu leads to abnormal Atoh1 expression and a severe delay of HC differentiation due to elevated Gli2 mRNA expression. Later in development, HC differentiation defects are restored in the Sufu mutant by the action of speckle-type PDZ protein (Spop), which promotes Gli2 protein degradation. Deletion of both Sufu and Spop results in robust Gli2 activation, exacerbating HC differentiation defects. We further demonstrate that Gli2 inhibits HC differentiation through maintaining the progenitor state of Sox2+ prosensory cells. Along the basal-apical axis of the developing cochlea, the Sox2 expression level is higher in the progenitor cells than in differentiating cells and is down-regulated from base to apex as differentiation proceeds. The dynamic spatiotemporal change of Sox2 expression levels is controlled by Shh signaling through Gli2. Together, our results reveal key functions of Gli2 in sustaining the progenitor state, thereby preventing HC differentiation and in turn governing the basal-apical progression of HC differentiation in the cochlea.

SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

Direct Interaction of Sox10 With Cadherin-19 Mediates Early Sacral Neural Crest Cell Migration: Implications for Enteric Nervous System Development Defects.

BACKGROUND AND AIMS: The enteric nervous system, which regulates many gastrointestinal functions, is derived from neural crest cells (NCCs). Defective NCC migration during embryonic development may lead to enteric neuropathies such as Hirschsprung's disease (hindgut aganglionosis). Sox10 is known to be essential for cell migration but downstream molecular events regulating early NCC migration have not been fully elucidated. This study aimed to determine how Sox10 regulates migration of sacral NCCs toward the hindgut using Dominant megacolon mice, an animal model of Hirschsprung's disease with a Sox10 mutation. METHODS: We used the following: time-lapse live cell imaging to determine the migration defects of mutant sacral NCCs; genome-wide microarrays, site-directed mutagenesis, and whole embryo culture to identify Sox10 targets; and liquid chromatography and tandem mass spectrometry to ascertain downstream effectors of Sox10. RESULTS: Sacral NCCs exhibited retarded migration to the distal hindgut in Sox10-null embryos with simultaneous down-regulated expression of cadherin-19 (Cdh19). Sox10 was found to bind directly to the Cdh19 promoter. Cdh19 knockdown resulted in retarded sacral NCC migration in vitro and ex vivo, whereas re-expression of Cdh19 partially rescued the retarded migration of mutant sacral NCCs in vitro. Cdh19 formed cadherin-catenin complexes, which then bound to filamentous actin of the cytoskeleton during cell migration. CONCLUSIONS: Cdh19 is a direct target of Sox10 during early sacral NCC migration toward the hindgut and forms cadherin-catenin complexes which interact with the cytoskeleton in migrating cells. Elucidation of this novel molecular pathway helps to provide insights into the pathogenesis of enteric nervous system developmental defects.

Notch signalling regulates epibranchial placode patterning and segregation.

Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2+/Irx5+ rostral domain and a Sox2+/Fgf3+/Etv5+ caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.

The Hong Kong Principles for assessing researchers: Fostering research integrity.

For knowledge to benefit research and society, it must be trustworthy. Trustworthy research is robust, rigorous, and transparent at all stages of design, execution, and reporting. Assessment of researchers still rarely includes considerations related to trustworthiness, rigor, and transparency. We have developed the Hong Kong Principles (HKPs) as part of the 6th World Conference on Research Integrity with a specific focus on the need to drive research improvement through ensuring that researchers are explicitly recognized and rewarded for behaviors that strengthen research integrity. We present five principles: responsible research practices; transparent reporting; open science (open research); valuing a diversity of types of research; and recognizing all contributions to research and scholarly activity. For each principle, we provide a rationale for its inclusion and provide examples where these principles are already being adopted.

Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization.

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

BACKGROUND & AIMS: Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. METHODS: We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. RESULTS: We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. CONCLUSIONS: We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

Epigenetic dysregulation of leukaemic HOX code in MLL-rearranged leukaemia mouse model.

HOX genes are frequently dysregulated in human leukaemia with the gene rearrangement between mixed lineage leukaemia (MLL) and partner genes. The resultant MLL fusion proteins are known to mediate leukaemia through disruption of the normal epigenetic regulation at the target gene loci. To elucidate the pathogenic role of MLL fusion proteins in HOX dysregulation in leukaemia, we generated a novel haematopoietic lineage-specific Mll-Een knock-in mouse model using a Cre-mediated inversion strategy. The Mll(Een) (/+) invertor mice developed acute myeloid leukaemia, with organomegaly of the spleen, liver and mesenteric lymph nodes caused by infiltration of blast cells. Using Mll-Een-expressing leukaemic cell lines derived from bone marrow of Mll(Een) (/+) mutant mice, we showed that induction of Hox genes in leukaemic cells was associated with hypomethylated promoter regions and an aberrant active chromatin state at the Hox loci. Knock-down of Prmt1 was insufficient to reverse the active chromatin status and the hypomethylated Hox loci, suggesting that Prmt1-mediated histone arginine methylation was only partially involved in the maintenance of Hox expression in leukaemic cells. Furthermore, in vivo analysis of bone marrow cells of Mll(Een) (/+) mice revealed a Hox expression profile similar to that of wild-type haematopoietic stem cells. The leukaemic Hox profile was highly correlated with aberrant hypomethylation of Hox promoters in the mutant mice, which highlights the importance of DNA methylation in leukaemogenic mechanisms induced by MLL fusion proteins. Our results point to the involvement of dynamic epigenetic regulations in the maintenance of the stem cell-like HOX code that initiates leukaemic stem cells in MLL-rearranged leukaemia. This provides insights for the development of alternative strategies for leukaemia treatment.

The role of SOX10 during enteric nervous system development.

The SOX10 transcription factor is a characteristic marker for migratory multipotent neural crest (NC) progenitors as well as several of their differentiated derivatives. The involvement of SOX10 in Waardenburg-Hirschsprung disease (pigmentation defects, deafness and intestinal aganglionosis) and studies of mutant animal models have contributed significantly to the understanding of its function in neural crest cells (NCC) in general and in the melanocytes and enteric nervous system (ENS) in particular. Cell-based studies have further demonstrated the important roles of this transcription factor in maintaining the NC progenitor cell number and in determining glial cell fate. Phenotypic variability observed among patients presenting with SOX10 mutations is in agreement with molecular genetics and animal model studies, which revealed that SOX10 cooperates with different partner factors; a number of genetic modifiers of SOX10 have been identified. This study reviews the expression, regulation, and function of SOX10 in normal development of the ENS and in disease conditions, as well as the genetic and molecular interactions of SOX10 with other ENS genes/factors. We also discuss future research areas. Further understanding of SOX10 function will benefit from genomic and cell biological studies that integrate the cell-intrinsic molecular mechanisms and the interactions of the enteric NCC with the niche environment.

Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development.

ATP-dependent SWI/SNF chromatin remodeling complexes alter the structure of chromatin at specific loci and facilitate tissue-specific gene regulation during development. Several SWI/SNF subunits are required for cardiogenesis. However, the function and mechanisms of SWI/SNF in mediating cardiac progenitor cell (CPC) differentiation during cardiogenesis are not well understood. Our studies of the SWI/SNF chromatin remodeling complex identified that BAF250a, a regulatory subunit of the SWI/SNF, plays a key role in CPC differentiation. BAF250a ablation in mouse second heart field (SHF) led to trabeculation defects in the right ventricle, ventricular septal defect, persistent truncus arteriosus, reduced myocardial proliferation, and embryonic lethality around E13. Using an embryonic stem cell culture system that models the formation and differentiation of SHF CPCs in vivo, we have shown that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. Moreover, BAF250a selectively regulates the expression of key cardiac factors Mef2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunoprecipitation and DNase I digestion assays indicate that BAF250a regulates gene expression by binding selectively to its target gene promoters and recruiting Brg1, the catalytic subunit of SWI/SNF, to modulate chromatin accessibility. Our results thus identify BAF250a-mediated chromatin remodeling as an essential epigenetic mechanism mediating CPC differentiation.

SWI/SNF in cardiac progenitor cell differentiation.

Cardiogenesis requires proper specification, proliferation, and differentiation of cardiac progenitor cells (CPCs). The differentiation of CPCs to specific cardiac cell types is likely guided by a comprehensive network comprised of cardiac transcription factors and epigenetic complexes. In this review, we describe how the ATP-dependent chromatin remodeling SWI/SNF complexes work synergistically with transcription and epigenetic factors to direct specific cardiac gene expression during CPC differentiation. Furthermore, we discuss how SWI/SNF may prime chromatin for cardiac gene expression at a genome-wide level. A detailed understanding of SWI/SNF-mediated CPC differentiation will provide important insight into the etiology of cardica defects and help design novel therapies for heart disease.

Hoxb3 negatively regulates Hoxb1 expression in mouse hindbrain patterning.

The spatial regulation of combinatorial expression of Hox genes is critical for determining hindbrain rhombomere (r) identities. To address the cross-regulatory relationship between Hox genes in hindbrain neuronal specification, we have generated a gain-of-function transgenic mouse mutant Hoxb3(Tg) using the Hoxb2 r4-specific enhancer element. Interestingly, in r4 of the Hoxb3(Tg) mutant where Hoxb3 was ectopically expressed, the expression of Hoxb1 was specifically abolished. The hindbrain neuronal defects of the Hoxb3(Tg) mutant mice were similar to those of Hoxb1(-/-) mutants. Therefore, we hypothesized that Hoxb3 could directly suppress Hoxb1 expression. We first identified a novel Hoxb3 binding site S3 on the Hoxb1 locus and confirmed protein binding to this site by EMSA, and by in vivo ChIP analysis using P19 cells and hindbrain tissues from the Hoxb3(Tg) mutant. We further showed that Hoxb3 could suppress Hoxb1 transcriptional activity by chick in ovo luciferase reporter assay. Moreover, in E10.5 wildtype caudal hindbrain, where Hoxb1 is not expressed, we showed by in vivo ChIP that Hoxb3 was consistently bound to the S3 site on the Hoxb1 gene. This study reveals a novel negative regulatory mechanism by which Hoxb3 as a posterior gene serves to restrict Hoxb1 expression in r4 by direct transcriptional repression to maintain the rhombomere identity.

Cerebellar defects in Pdss2 conditional knockout mice during embryonic development and in adulthood.

PDSS2 is a gene that encodes one of the two subunits of trans-prenyl diphosphate synthase that is essential for ubiquinone biosynthesis. It is known that mutations in PDSS2 can cause primary ubiquinone deficiency in humans and a similar disease in mice. Cerebellum is the most often affected organ in ubiquinone deficiency, and cerebellar atrophy has been diagnosed in many infants with this disease. In this study, two Pdss2 conditional knockout mouse lines directed by Pax2-cre and Pcp2-cre were generated to investigate the effect of ubiquinone deficiency on cerebellum during embryonic development and in adulthood, respectively. The Pdss2(f/-); Pax2-cre mouse recapitulates some symptoms of ubiquinone deficiency in infants, including severe cerebellum hypoplasia and lipid accumulation in skeletal muscles at birth. During early cerebellum development (E12.5-14.5), Pdss2 knockout initially causes the delay of radial glial cell growth and neuron progenitor migration, so the growth of mutant cerebellum is retarded. During later development (E15.5-P0), increased ectopic apoptosis of neuroblasts and impaired cell proliferation result in the progression of cerebellum hypoplasia in the mutant. Thus, the mutant cerebellum contains fewer neurons at birth, and the cells are disorganized. The developmental defect of mutant cerebellum does not result from reduced Fgf8 expression before E12.5. Electron microscopy reveals mitochondrial defects and increased autophagic-like vacuolization that may arise in response to abnormal mitochondria in the mutant cerebellum. Nevertheless, the mutant mice die soon after birth probably due to cleft palate and micrognathia, which may result from Pdss2 knockout caused by ectopic Pax2-cre expression in the first branchial arch. On the other hand, the Pdss2(f/-); Pcp2-cre mouse is healthy at birth but gradually loses cerebellar Purkinje cells and develops ataxia-like symptoms at 9.5 months; thus this conditional knockout mouse may serve as a model for ubiquinone deficiency in adult patients. In conclusion, this study provides two mouse models of Pdss2 based ubiquinone deficiency. During cerebellum development, Pdss2 knockout results in severe cerebellum hypoplasia by impairing cell migration and eliciting ectopic apoptosis, whereas Pdss2 knockout in Purkinje cells at postnatal stages leads to the development of cerebellar ataxia.

Analysis of the sacral neural crest cell contribution to the hindgut enteric nervous system in the mouse embryo.

BACKGROUND & AIMS: The majority of the enteric nervous system is derived from the vagal neural crest, with a second contribution, which is restricted to the post-umbilical gut, originating from the sacral neural crest. In mammals, although sacral neural crest cells (NCCs) have been shown to enter the hindgut, information on their development and role remains scant. Our aim was to determine the migratory routes of sacral NCCs to the hindgut, their timing and site of entry into the gut, and their migratory behaviors and differentiation within the hindgut. METHODS: We used in situ cell labeling, whole embryo culture, immunofluorescence, organotypic culture, and time-lapse live-cell imaging in mouse embryos. RESULTS: Sacral NCCs emigrated from the neural tube at embryonic day 9.5, accumulated bilateral to the hindgut to form prospective pelvic ganglia at embryonic day 11.5, and from there entered the distal hindgut through its ventrolateral side at embryonic day 13.5. They then migrated along nerve fibers extending from the pelvic ganglia toward the proximal hindgut, intermingling with rostrocaudally migrating vagal NCCs to differentiate into neurons and glia. In organotypic culture, genetically labeled sacral and vagal NCCs displayed different capabilities of entering the hindgut, implying differences in their intrinsic migratory properties. Time-lapse live-cell imaging on explants ex vivo showed that sacral NCCs migrated along nerve fibers and exhibited different migratory behaviors from vagal NCCs. CONCLUSIONS: Murine sacral NCCs are a distinct group of cells that migrate along defined pathways from neural tube to hindgut. They exhibit discrete migratory behaviors within the gut mesenchyme and contribute neurons and glial cells to the hindgut enteric nervous system.

MQuad enables clonal substructure discovery using single cell mitochondrial variants.

Mitochondrial mutations are increasingly recognised as informative endogenous genetic markers that can be used to reconstruct cellular clonal structure using single-cell RNA or DNA sequencing data. However, identifying informative mtDNA variants in noisy and sparse single-cell sequencing data is still challenging with few computation methods available. Here we present an open source computational tool MQuad that accurately calls clonally informative mtDNA variants in a population of single cells, and an analysis suite for complete clonality inference, based on single cell RNA, DNA or ATAC sequencing data. Through a variety of simulated and experimental single cell sequencing data, we showed that MQuad can identify mitochondrial variants with both high sensitivity and specificity, outperforming existing methods by a large extent. Furthermore, we demonstrate its wide applicability in different single cell sequencing protocols, particularly in complementing single-nucleotide and copy-number variations to extract finer clonal resolution.

seRNA PAM controls skeletal muscle satellite cell proliferation and aging through trans regulation of Timp2 expression synergistically with Ddx5.

Muscle satellite cells (SCs) are responsible for muscle homeostasis and regeneration and lncRNAs play important roles in regulating SC activities. Here, in this study, we identify PAM (Pax7 Associated Muscle lncRNA) that is induced in activated/proliferating SCs upon injury to promote SC proliferation as myoblast cells. PAM is generated from a myoblast-specific super-enhancer (SE); as a seRNA it binds with a number of target genomic loci predominantly in trans. Further studies demonstrate that it interacts with Ddx5 to tether PAM SE to its inter-chromosomal targets Timp2 and Vim to activate the gene expression. Lastly, we show that PAM expression is increased in aging SCs, which leads to enhanced inter-chromosomal interaction and target genes upregulation. Altogether, our findings identify PAM as a previously unknown lncRNA that regulates both SC proliferation and aging through its trans gene regulatory activity.