Muscle injury induces a transient senescence-like state that is required for myofiber growth during muscle regeneration.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by the cell cycle inhibitors Cdkn2a, Cdkn1a, and Trp53. Senescent cells are implicated in chronic diseases and tissue repair through their increased secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). Here, we use spatial transcriptomics and single-cell RNA sequencing (scRNAseq) to demonstrate that cells displaying senescent characteristics are "transiently" present within regenerating skeletal muscle and within the muscles of D2-mdx mice, a model of Muscular Dystrophy. Following injury, multiple cell types including macrophages and fibrog-adipogenic progenitors (FAPs) upregulate senescent features such as senescence pathway genes, SASP factors, and senescence-associated beta-gal (SA-ß-gal) activity. Importantly, when these cells were removed with ABT-263, a senolytic compound, satellite cells are reduced, and muscle fibers were impaired in growth and myonuclear accretion. These results highlight that an "acute" senescent phenotype facilitates regeneration similar to skin and neonatal myocardium.

Loss of dystrophin expression in skeletal muscle is associated with senescence of macrophages and endothelial cells.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21, and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence-associated secretory phenotype, which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne muscular dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling, and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-wk-old) and D2-mdx (4-wk-old and 8-wk-old) mice, two mouse models of DMD, that cells displaying canonical markers of senescence are found within the skeletal muscle. Eight-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation, which were mostly Cdkn1a-positive macrophages, whereas in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wild-type (WT) muscle, which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes [Cdkn1a (p21), Cdkn2a (p16INK4A), and Trp53 (p53)], which correlated with the quantity of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.

Three Optimized Methods for In Situ Quantification of Progenitor Cell Proliferation in Embryonic Kidneys Using BrdU, EdU, and PCNA.

BACKGROUND: Nephron progenitor cells derived from the metanephric mesenchyme undergo a complex balance of self-renewal and differentiation throughout kidney development to give rise to the mature nephron. Cell proliferation is an important index of progenitor population dynamics. However, accurate and reproducible in situ quantification of cell proliferation within progenitor populations can be technically difficult to achieve due to the complexity and harsh tissue treatment required of certain protocols. OBJECTIVE: To optimize and compare the performance of the 3 most accurate S phase-specific labeling methods used for in situ detection and quantification of nephron progenitor and ureteric bud cell proliferation in the developing kidney, namely, 5-bromo-2'-deoxyuridine (BrdU), 5-ethynyl-2'-deoxyuridine (EdU), and proliferating cell nuclear antigen (PCNA). METHODS: Protocols for BrdU, EdU, and PCNA were optimized for fluorescence labeling on paraformaldehyde-fixed, paraffin-embedded mouse kidney tissue sections, with co-labeling of nephron progenitor cells and ureteric bud with Six2 and E-cadherin antibodies, respectively. Image processing and analysis, including quantification of proliferating cells, were carried out using free ImageJ software. RESULTS: All 3 methods detect similar ratios of nephron progenitor and ureteric bud proliferating cells. The BrdU staining protocol is the lengthiest and most complex protocol to perform, requires tissue denaturation, and is most subject to interexperimental signal variability. In contrast, bound PCNA and EdU protocols are relatively more straightforward, consistently yield clear results, and far more easily lend themselves to co-staining; however, the bound PCNA protocol requires substantive additional postexperimental analysis to distinguish the punctate nuclear PCNA staining pattern characteristic of proliferating cells. CONCLUSIONS: All 3 markers exhibit distinct advantages and disadvantages in quantifying cell proliferation in kidney progenitor populations, with EdU and PCNA protocols being favored due to greater technical ease and reproducibility of results associated with these methods.

CONTEXTE: Les cellules progénitrices de néphrons dérivées du mésenchyme métanéphrique subissent une séquence complexe d'auto-régénération et de différenciation tout au long du développement du rein pour donner naissance aux néphrons matures. La prolifération cellulaire est un indice de la dynamique des populations de cellules progénitrices. La quantification in situ précise et reproductible de la prolifération cellulaire au sein de populations de cellules progénitrices peut cependant s'avérer techniquement difficile à réaliser en raison de la complexité et de la sévérité du traitement tissulaire requis par certains protocoles. OBJECTIF: Optimiser et comparer la performance des trois plus précises méthodes de marquage spécifiques à la phase S pour détecter et quantifier in situ la prolifération des cellules progénitrices de néphrons et de bourgeons urétéraux dans le rein en développement, à savoir la 5-bromo-2'-désoxyuridine (BrdU), la 5-éthynyl-2'-désoxyuridine (EdU), et l'antigène nucléaire de prolifération cellulaire (PCNA). MÉTHODOLOGIE: Les protocoles pour BrdU, EdU et PCNA ont été optimisés pour le marquage fluorescent de coupes de tissus rénaux de souris, fixés au paraformaldéhyde et enchassés dans la paraffine, avec co-marquage des cellules progénitrices de néphrons et de bourgeons urétéraux avec les anticorps Six2 et E-cadhérine, respectivement. Le traitement et l'analyse des images, y compris la quantification des cellules en prolifération, ont été réalisés à l'aide du logiciel gratuit ImageJ. RÉSULTATS: Les trois méthodes ont détecté des ratios similaires de cellules progénitrices de néphrons et de bourgeons urétéraux en prolifération. Le protocole de coloration BrdU est le plus long et le plus complexe à effectuer. Il requiert la dénaturation des tissus et il est le plus sujet à la variabilité du signal inter-expériences. En revanche, les protocoles de liaison de PCNA et d'EdU sont relativement plus simples, donnent systématiquement des résultats clairs et se prêtent beaucoup plus facilement à la coloration conjointe. Toutefois, le protocole de liaison de PCNA requiert une analyse supplémentaire approfondie post-expérience pour distinguer le schéma de coloration ponctuée du noyau caractéristique des cellules en prolifération. CONCLUSION: Les trois méthodes ont montré des avantages et des inconvénients distincts pour la quantification de la prolifération cellulaire des populations de cellules progénitrices du rein. Les protocoles avec EdU et PCNA sont favorisés en raison de leur simplicité technique et de la reproductibilité des résultats obtenus.

SOX4 regulates gonad morphogenesis and promotes male germ cell differentiation in mice.

The group C SOX transcription factors SOX4, -11 and -12 play important and mutually overlapping roles in development of a number of organs. Here, we examined the role of SoxC genes during gonadal development in mice. All three genes were expressed in developing gonads of both sexes, predominantly in somatic cells, with Sox4 being most strongly expressed. Sox4 deficiency resulted in elongation of both ovaries and testes, and an increased number of testis cords. While female germ cells entered meiosis normally, male germ cells showed reduced levels of differentiation markers Nanos2 and Dnmt3l and increased levels of pluripotency genes Cripto and Nanog, suggesting that SOX4 may normally act to restrict the pluripotency period of male germ cells and ensure their proper differentiation. Finally, our data reveal that SOX4 (and, to a lesser extent, SOX11 and -12) repressed transcription of the sex-determining gene Sox9 via an upstream testis-specific enhancer core (TESCO) element in fetal gonads, raising the possibility that SOXC proteins may function as transcriptional repressors in a context-dependent manner.

Estimation of total glomerular number using an integrated disector method in embryonic and postnatal kidneys.

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are a polymorphic group of clinical disorders comprising the major cause of renal failure in children. Included within CAKUT is a wide spectrum of developmental malformations ranging from renal agenesis, renal hypoplasia and renal dysplasia (maldifferentiation of renal tissue), each characterized by varying deficits in nephron number. First presented in the Brenner Hypothesis, low congenital nephron endowment is becoming recognized as an antecedent cause of adult-onset hypertension, a leading cause of coronary heart disease, stroke, and renal failure in North America. Genetic mouse models of impaired nephrogenesis and nephron endowment provide a critical framework for understanding the origins of human kidney disease. Current methods to quantitate nephron number include (i) acid maceration (ii) estimation of nephron number from a small number of tissue sections (iii) imaging modalities such as MRI and (iv) the gold standard physical disector/fractionator method. Despite its accuracy, the physical disector/fractionator method is rarely employed because it is labour-intensive, time-consuming and costly to perform. Consequently, less rigourous methods of nephron estimation are routinely employed by many laboratories. Here we present an updated, digitized version of the physical disector/fractionator method using free open source Fiji software, which we have termed the integrated disector method. This updated version of the gold standard modality accurately, rapidly and cost-effectively quantitates nephron number in embryonic and post-natal mouse kidneys, and can be easily adapted for stereological measurements in other organ systems.

Les anomalies congénitales du rein et des voies urinaires (Congenital Anomalies of the Kidney and Urinary Tract, CAKUT) désignent un groupe polymorphe d'entités cliniques qui constitue la cause la plus fréquente d'insuffisance rénale chez l'enfant. Le CAKUT comprend aussi un grand nombre de malformations développementales, dont le syndrome de Potter, l'hypoplasie rénale, ainsi que la dysplasie rénale (maldifférentiation des tissus rénaux), toutes caractérisées par un déficit de néphrons. On reconnaît de plus en plus une masse néphronique congénitale réduite, d'abord présentée dans l'hypothèse de Brenner, comme une cause de l'hypertension chez l'adulte, de coronaropathie, d'AVC, et d'insuffisance rénale en Amérique du Nord. Les modèles génétiques de souris comportant une détérioration de la fonction rénale et de la masse néphronique fournissent un cadre pour permettre la compréhension de l'origine des néphropathies chez l'humain. Les méthodes actuelles de quantification des néphrons comprennent (i) la macération acide (ii) l'estimation du nombre de néphrons à partir d'une petite quantité de tissus sectionnés (iii) les modes d'imagerie tels que l'IRM et (iv) la technique de référence du disecteur et fractionnement. Malgré sa précision, cette dernière méthode n'est employée que rarement, puisqu'elle requiert main-d'œuvre, temps et argent. Par conséquent, plusieurs laboratoires emploient systématiquement des méthodes moins rigoureuses d'estimation du nombre de néphrons. Nous présentons ici une version mise à jour et numérisée de la technique du disecteur et fractionnement, que nous appelons la technique intégrée du disecteur, en utilisant Fiji, un logiciel ouvert et gratuit. Cette version mise à jour de la modalité de référence permet de quantifier les néphrons de manière précise, rapide et rentable dans les reins de souris à l'état embryonnaire ou postnatal, et peut aisément être adaptée aux mesures stéréologiques d'autres organes.

The transcription factor Sry-related HMG box-4 (SOX4) is required for normal renal development in vivo.

BACKGROUND: The DNA-binding transcription factor Wilms' Tumor Suppressor-1 (WT1) plays an essential role in nephron progenitor differentiation during renal development. We previously used Wt1 chromatin-immunoprecipitation coupled to microarray (ChIP-chip) to identify novel Wt1 target genes that may regulate nephrogenesis in vivo. We discovered that all three members of the SoxC subfamily, namely, Sox4, Sox11, and Sox12, are bound by Wt1 in mouse embryonic kidneys in vivo. SoxC genes play master roles in determining neuronal and mesenchymal progenitor cell fate in a multitude of developmental processes, but their function in the developing kidney is largely unknown. RESULTS: Here we show that all three SoxC genes are expressed in the nephrogenic lineages during renal development. Conditional ablation of Sox4 in nephron progenitors and their cellular descendants (Sox4(nephron-) mice) results in a significant reduction in nephron endowment. By postnatal day (P)7, Sox4(nephron-) renal corpuscles exhibit reduced numbers of Wt1+ podocytes together with loss of expression of the slit diaphragm protein nephrin. Sox4(nephron-) mice develop early-onset proteinacious glomerular injury within 2 weeks of birth progressing to end-stage renal failure within 5-9 months. CONCLUSIONS: Collectively, our results demonstrate an essential requirement of Sox4 for normal renal development in vivo.

Inositol polyphosphate 4-phosphatase B as a regulator of bone mass in mice and humans.

Osteoporosis is a multifactorial genetic disease characterized by reduction of bone mass due to dysregulation of osteoclast differentiation or maturation. Herein, we identified a regulator of osteoclastogenesis, the murine homolog of inositol polyphosphate 4-phosphatase type IIα (Inpp4bα). Expression of Inpp4bα is detected from early osteoclast differentiation to activation stage. Targeted expression of native Inpp4bα ex vivo repressed whereas phosphatase-inactive Inpp4bα stimulated osteoclast differentiation. Inpp4bα acts on intracellular calcium level that modulates NFATc1 nuclear translocation and activation. In vivo mice deficient in Inpp4b displayed increased osteoclast differentiation rate and potential resulting in decreased bone mass and osteoporosis. Importantly, INPP4B in human was identified as a susceptibility locus for osteoporosis. This study defined Inpp4b as a major modulator of the osteoclast differentiation and as a gene linked to variability of bone mineral density in mice and humans.