The IgCAM BT-IgSF (IgSF11) is essential for connexin43-mediated astrocyte-astrocyte coupling in mice.

The type I transmembrane protein BT-IgSF is predominantly localized in the brain and testes. It belongs to the CAR subgroup of Ig cell adhesion proteins, that are hypothesized to regulate connexin expression or localization. Here, we studied the putative link between BT-IgSF and connexins in astrocytes, ependymal cells and neurons of the mouse. Global knockout of BT-IgSF caused an increase in the clustering of connexin43 (Gja1), but not of connexin30 (Gjb6), on astrocytes and ependymal cells. Additionally, knockout animals displayed reduced expression levels of connexin43 protein in the cortex and hippocampus. Importantly, analysis of biocytin spread in hippocampal or cortical slices from mature mice of either sex revealed a decrease in astrocytic cell-cell coupling in the absence of BT-IgSF. Blocking either protein biosynthesis or proteolysis showed that the lysosomal pathway increased connexin43 degradation in astrocytes. Localization of connexin43 in subcellular compartments was not impaired in astrocytes of BT-IgSF mutants. In contrast to connexin43 the localization and expression of connexin36 (Gjd2) on neurons was not affected by the absence of BT-IgSF. Overall, our data indicate that the IgCAM BT-IgSF is essential for correct gap junction-mediated astrocyte-to-astrocyte cell communication.Significance Statement Astrocytes regulate a variety of physiological processes in the developing and adult brain that are essential for proper brain function. Astrocytes form extensive networks in the brain and communicate via gap junctions. Disruptions of gap junction coupling are found in several diseases such as neurodegeneration or epilepsy. Here, we demonstrate that the cell adhesion protein BT-IgSF is essential for gap junction mediated coupling between astrocytes in the cortex and hippocampus.

Regulation of developmental tempo in different vertebrate species.

In this issue, Lazaro et al.1 use iPSC-derived presomitic mesoderm cells to analyze the oscillatory expression of somitic clock genes. Comparison of a wide range of species, including mouse, rabbit, cattle, rhinoceros, human, and marmoset, demonstrates an excellent correlation between biochemical reaction speed and the tempo of the clock.

Prox2 and Runx3 vagal sensory neurons regulate esophageal motility.

Vagal sensory neurons monitor mechanical and chemical stimuli in the gastrointestinal tract. Major efforts are underway to assign physiological functions to the many distinct subtypes of vagal sensory neurons. Here, we use genetically guided anatomical tracing, optogenetics, and electrophysiology to identify and characterize vagal sensory neuron subtypes expressing Prox2 and Runx3 in mice. We show that three of these neuronal subtypes innervate the esophagus and stomach in regionalized patterns, where they form intraganglionic laminar endings. Electrophysiological analysis revealed that they are low-threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis in freely behaving mice. Our work defines the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders.

c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy.

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.

Visualizing MyoD Oscillations in Muscle Stem Cells.

The bHLH transcription factor MyoD is a master regulator of myogenic differentiation, and its sustained expression in fibroblasts suffices to differentiate them into muscle cells. MyoD expression oscillates in activated muscle stem cells of developing, postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. The oscillatory period is around 3 h and thus much shorter than the cell cycle or circadian rhythm. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed when stem cells undergo myogenic differentiation. The oscillatory expression of MyoD is driven by the oscillatory expression of the bHLH transcription factor Hes1 that periodically represses MyoD. Ablation of the Hes1 oscillator interferes with stable MyoD oscillations and leads to prolonged periods of sustained MyoD expression. This interferes with the maintenance of activated muscle stem cells and impairs muscle growth and repair. Thus, oscillations of MyoD and Hes1 control the balance between the proliferation and differentiation of muscle stem cells. Here, we describe time-lapse imaging methods using luciferase reporters, which can monitor dynamic MyoD gene expression in myogenic cells.

Molecular identity of proprioceptor subtypes innervating different muscle groups in mice.

The precise execution of coordinated movements depends on proprioception, the sense of body position in space. However, the molecular underpinnings of proprioceptive neuron subtype identities are not fully understood. Here we used a single-cell transcriptomic approach to define mouse proprioceptor subtypes according to the identity of the muscle they innervate. We identified and validated molecular signatures associated with proprioceptors innervating back (Tox, Epha3), abdominal (C1ql2), and hindlimb (Gabrg1, Efna5) muscles. We also found that proprioceptor muscle identity precedes acquisition of receptor character and comprise programs controlling wiring specificity. These findings indicate that muscle-type identity is a fundamental aspect of proprioceptor subtype differentiation that is acquired during early development and includes molecular programs involved in the control of muscle target specificity.

SMER28 Attenuates PI3K/mTOR Signaling by Direct Inhibition of PI3K p110 Delta.

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here, we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and, consequently, growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction in phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3Kδ and to a lesser extent p110γ. The biological relevance of our observations was underscored by SMER28 interfering with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor tyrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited. Lastly, in B cell lymphoma cells, which predominantly depend on tonic signaling through PI3Kδ, apoptosis upon SMER28 treatment is profound in comparison to non-hematopoietic cells. This indicates SMER28 as a possible drug candidate for the treatment of diseases that derive from aberrant PI3Kδ activity.

Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting.

BACKGROUND: Sepsis and inflammation can cause intensive care unit-acquired weakness (ICUAW). Increased interleukin-6 (IL-6) plasma levels are a risk factor for ICUAW. IL-6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT-pathway, but its role in sepsis-induced muscle wasting is uncertain. In a clinical observational study, we found that the IL-6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT-pathway activation. We tested the hypothesis that the IL-6/gp130-pathway mediates ICUAW muscle atrophy. METHODS: We sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture-operated (CLP) and sham-operated wildtype (WT) mice. The effects of the IL-6/gp130/JAK2/STAT3-pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h. RESULTS: Analyses of differentially expressed genes in RNAseq (≥2-log2-fold change, P < 0.01) revealed an activation of IL-6-signalling and JAK/STAT-signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL-6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three-fold, P < 0.01) and Socs3 mRNA expression (3.1-fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL-6-induced STAT3 phosphorylation (-30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (- 29.0%; P < 0.01) or STAT3 inhibition (-38.7%; P < 0.05) decreased IL-6-induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL-6. CLP-operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st-cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st-cKO mice (WT: -22.3%, P < 0.001, cKO: -13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin-1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin-1 were unaltered between CLP-treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (-22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin-1-mRNA (-81.3%, P < 0.05) and Trim63/MuRF1-mRNA expression (-77.6%, P < 0.05) and protein content. CONCLUSIONS: IL-6 via the gp130/JAK2/STAT3-pathway mediates sepsis-induced muscle atrophy possibly contributing to ICUAW.

A medullary centre for lapping in mice.

It has long been known that orofacial movements for feeding can be triggered, coordinated, and often rhythmically organized at the level of the brainstem, without input from higher centers. We uncover two nuclei that can organize the movements for ingesting fluids in mice. These neuronal groups, IRtPhox2b and Peri5Atoh1, are marked by expression of the pan-autonomic homeobox gene Phox2b and are located, respectively, in the intermediate reticular formation of the medulla and around the motor nucleus of the trigeminal nerve. They are premotor to all jaw-opening and tongue muscles. Stimulation of either, in awake animals, opens the jaw, while IRtPhox2b alone also protracts the tongue. Moreover, stationary stimulation of IRtPhox2b entrains a rhythmic alternation of tongue protraction and retraction, synchronized with jaw opening and closing, that mimics lapping. Finally, fiber photometric recordings show that IRtPhox2b is active during volitional lapping. Our study identifies one of the subcortical nuclei underpinning a stereotyped feeding behavior.

An oscillatory network controlling self-renewal of skeletal muscle stem cells.

The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. Muscle stem cells can proliferate, they can generate differentiating cells, or they self-renew to produce new stem cells. Notch signaling plays a crucial role in this process. Recent studies revealed that expression of the Notch effector HES1 oscillates in activated muscle stem cells. The oscillatory expression of HES1 periodically represses transcription from the genes encoding the myogenic transcription factor MYOD and the Notch ligand DLL1, thereby driving MYOD and DLL1 oscillations. This oscillatory network allows muscle progenitor cells and activated muscle stem cells to remain in a proliferative and 'undecided' state, in which they can either differentiate or self-renew. When HES1 is downregulated, MYOD oscillations become unstable and are replaced by sustained expression, which drives the cells into terminal differentiation. During development and regeneration, proliferating stem cells contact each other and the stability of the oscillatory expression depends on regular DLL1 inputs provided by neighboring cells. In such communities of cells that receive and provide Notch signals, the appropriate timing of DLL1 inputs is important, as sustained DLL1 cannot replace oscillatory DLL1. Thus, in cell communities, DLL1 oscillations ensure the appropriate balance between self-renewal and differentiation. In summary, oscillations in myogenic cells are an important example of dynamic gene expression determining cell fate.

Met and Cxcr4 cooperate to protect skeletal muscle stem cells against inflammation-induced damage during regeneration.

Acute skeletal muscle injury is followed by an inflammatory response, removal of damaged tissue, and the generation of new muscle fibers by resident muscle stem cells, a process well characterized in murine injury models. Inflammatory cells are needed to remove the debris at the site of injury and provide signals that are beneficial for repair. However, they also release chemokines, reactive oxygen species, as well as enzymes for clearance of damaged cells and fibers, which muscle stem cells have to withstand in order to regenerate the muscle. We show here that MET and CXCR4 cooperate to protect muscle stem cells against the adverse environment encountered during muscle repair. This powerful cyto-protective role was revealed by the genetic ablation of Met and Cxcr4 in muscle stem cells of mice, which resulted in severe apoptosis during early stages of regeneration. TNFα neutralizing antibodies rescued the apoptosis, indicating that TNFα provides crucial cell-death signals during muscle repair that are counteracted by MET and CXCR4. We conclude that muscle stem cells require MET and CXCR4 to protect them against the harsh inflammatory environment encountered in an acute muscle injury.

Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells.

Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice. Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities.

Technologies for profiling the impact of genomic variants on transcription factor binding.

Transcription factors (TFs) bind DNA in a sequence-specific manner and thereby regulate target gene expression. TF binding and its regulatory activity is highly context dependent, and is not only determined by specific cell types or differentiation stages but also relies on other regulatory mechanisms, such as DNA and chromatin modifications. Interactions between TFs and their DNA binding sites are critical mediators of phenotypic variation and play important roles in the onset of disease. A continuously growing number of studies therefore attempts to elucidate TF:DNA interactions to gain knowledge about regulatory mechanisms and disease-causing variants. Here we summarize how TF-binding characteristics and the impact of variants can be investigated, how bioinformatic tools can be used to analyze and predict TF:DNA binding, and what additional information can be obtained from the TF protein structure.

Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells.

Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. This revealed distinct nuclear subtypes unrelated to fiber type diversity, previously unknown subtypes as well as the expected ones at the neuromuscular and myotendinous junctions. In fibers of the Mdx dystrophy mouse model, distinct subtypes emerged, among them nuclei expressing a repair signature that were also abundant in the muscle of dystrophy patients, and a nuclear population associated with necrotic fibers. Finally, modifications of our approach revealed the compartmentalization in the rare and specialized muscle spindle. Our data identifies nuclear compartments of the myofiber and defines a molecular roadmap for their functional analyses; the data can be freely explored on the MyoExplorer server ( https://shiny.mdc-berlin.de/MyoExplorer/ ).

SIX1 and SIX4 homeoproteins regulate PAX7+ progenitor cell properties during fetal epaxial myogenesis.

Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.

EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake.

Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

Self-Organizing 3D Human Trunk Neuromuscular Organoids.

Neuromuscular networks assemble during early human embryonic development and are essential for the control of body movement. Previous neuromuscular junction modeling efforts using human pluripotent stem cells (hPSCs) generated either spinal cord neurons or skeletal muscles in monolayer culture. Here, we use hPSC-derived axial stem cells, the building blocks of the posterior body, to simultaneously generate spinal cord neurons and skeletal muscle cells that self-organize to generate human neuromuscular organoids (NMOs) that can be maintained in 3D for several months. Single-cell RNA-sequencing of individual organoids revealed reproducibility across experiments and enabled the tracking of the neural and mesodermal differentiation trajectories as organoids developed and matured. NMOs contain functional neuromuscular junctions supported by terminal Schwann cells. They contract and develop central pattern generator-like neuronal circuits. Finally, we successfully use NMOs to recapitulate key aspects of myasthenia gravis pathology, thus highlighting the significant potential of NMOs for modeling neuromuscular diseases in the future.

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7.

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.