Jennifer A. Lawson

Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration

Muscle regeneration requires the coordinated interaction of multiple cell types. Satellite cells have been implicated as the primary stem cell responsible for regenerating muscle, yet the necessity of these cells for regeneration has not been tested. Connective tissue fibroblasts also are likely to play a role in regeneration, as connective tissue fibrosis is a hallmark of regenerating muscle. However, the lack of molecular markers for these fibroblasts has precluded an investigation of their role. Using Tcf4, a newly identified fibroblast marker, and Pax7, a satellite cell marker, we found that after injury satellite cells and fibroblasts rapidly proliferate in close proximity to one another. To test the role of satellite cells and fibroblasts in muscle regeneration in vivo, we created Pax7CreERT2 and Tcf4CreERT2 mice and crossed these to R26RDTA mice to genetically ablate satellite cells and fibroblasts. Ablation of satellite cells resulted in a complete loss of regenerated muscle, as well as misregulation of fibroblasts and a dramatic increase in connective tissue. Ablation of fibroblasts altered the dynamics of satellite cells, leading to premature satellite cell differentiation, depletion of the early pool of satellite cells, and smaller regenerated myofibers. Thus, we provide direct, genetic evidence that satellite cells are required for muscle regeneration and also identify resident fibroblasts as a novel and vital component of the niche regulating satellite cell expansion during regeneration. Furthermore, we demonstrate that reciprocal interactions between fibroblasts and satellite cells contribute significantly to efficient, effective muscle regeneration.

Connective tissue fibroblasts and Tcf4 regulate myogenesis

Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4GFPCre mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis.

Muscle stem cells contribute to myofibres in sedentary adult mice

Skeletal muscle is essential for mobility, stability, and whole body metabolism, and muscle loss, for instance during sarcopenia, has profound consequences. Satellite cells (muscle stem cells) have been hypothesized, but not yet demonstrated, to contribute to muscle homeostasis and a decline in their contribution to myofiber homeostasis to play a part in sarcopenia. To test their role in muscle maintenance, we genetically labeled and ablated satellite cells in adult sedentary mice. We demonstrate via genetic lineage experiments that even in the absence of injury, satellite cells contribute to myofibers in all adult muscles, although the extent and timing differs. However, genetic ablation experiments showed that satellite cells are not globally required to maintain myofiber cross-sectional area of uninjured adult muscle.

Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias

The diaphragm is an essential mammalian skeletal muscle, and defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDHs), a common and often lethal birth defect. The diaphragm is derived from multiple embryonic sources, but how these give rise to the diaphragm is unknown, and, despite the identification of many CDH-associated genes, the etiology of CDH is incompletely understood. Using mouse genetics, we show that the pleuroperitoneal folds (PPFs), which are transient embryonic structures, are the source of the diaphragms muscle connective tissue and regulate muscle development, and we show that the striking migration of PPF cells controls diaphragm morphogenesis. Furthermore, Gata4 mosaic mutations in PPF-derived muscle connective tissue fibroblasts result in the development of localized amuscular regions that are biomechanically weaker and more compliant, leading to CDH. Thus, the PPFs and muscle connective tissue are critical for diaphragm development, and mutations in PPF-derived fibroblasts are a source of CDH.

Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome

Nucleoporins are essential components of the nuclear pore complex (NPC). Only a few diseases have been attributed to NPC dysfunction. Steroid-resistant nephrotic syndrome (SRNS), a frequent cause of chronic kidney disease, is caused by dysfunction of glomerular podocytes. Here we identify in eight families with SRNS mutations in NUP93, its interaction partner NUP205 or XPO5 (encoding exportin 5) as hitherto unrecognized monogenic causes of SRNS. NUP93 mutations caused disrupted NPC assembly. NUP93 knockdown reduced the presence of NUP205 in the NPC, and, reciprocally, a NUP205 alteration abrogated NUP93 interaction. We demonstrate that NUP93 and exportin 5 interact with the signaling protein SMAD4 and that NUP93 mutations abrogated interaction with SMAD4. Notably, NUP93 mutations interfered with BMP7-induced SMAD transcriptional reporter activity. We hereby demonstrate that mutations of NUP genes cause a distinct renal disease and identify aberrant SMAD signaling as a new disease mechanism of SRNS, opening a potential new avenue for treatment.

Transiently Active Wnt/β-Catenin Signaling Is Not Required but Must Be Silenced for Stem Cell Function during Muscle Regeneration

Summary Adult muscle’s exceptional capacity for regeneration is mediated by muscle stem cells, termed satellite cells. As with many stem cells, Wnt/β-catenin signaling has been proposed to be critical in satellite cells during regeneration. Using new genetic reagents, we explicitly test in vivo whether Wnt/β-catenin signaling is necessary and sufficient within satellite cells and their derivatives for regeneration. We find that signaling is transiently active in transit-amplifying myoblasts, but is not required for regeneration or satellite cell self-renewal. Instead, downregulation of transiently activated β-catenin is important to limit the regenerative response, as continuous regeneration is deleterious. Wnt/β-catenin activation in adult satellite cells may simply be a vestige of their developmental lineage, in which β-catenin signaling is critical for fetal myogenesis. In the adult, surprisingly, we show that it is not activation but rather silencing of Wnt/β-catenin signaling that is important for muscle regeneration.

Whole exome sequencing identifies causative mutations in the majority of consanguineous or familial cases with childhood-onset increased renal echogenicity

Chronically increased echogenicity on renal ultrasound is a sensitive early finding of chronic kidney disease that can be detected before manifestation of other symptoms. Increased echogenicity, however, is not specific for a certain etiology of chronic kidney disease. Here, we performed whole exome sequencing in 79 consanguineous or familial cases of suspected nephronophthisis in order to determine the underlying molecular disease cause. In 50 cases, there was a causative mutation in a known monogenic disease gene. In 32 of these cases whole exome sequencing confirmed the diagnosis of a nephronophthisis-related ciliopathy. In 8 cases it revealed the diagnosis of a renal tubulopathy. The remaining 10 cases were identified as Alport syndrome (4), autosomal-recessive polycystic kidney disease (2), congenital anomalies of the kidney and urinary tract (3), and APECED syndrome (1). In 5 families, in whom mutations in known monogenic genes were excluded, we applied homozygosity mapping for variant filtering, and identified 5 novel candidate genes (RBM48, FAM186B, PIAS1, INCENP, and RCOR1) for renal ciliopathies. Thus, whole exome sequencing allows the detection of the causative mutation in 2/3 of affected individuals, thereby presenting the etiologic diagnosis and allows identification of novel candidate genes.

Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis or Nephrocalcinosis

BACKGROUND AND OBJECTIVES Nephrolithiasis is a prevalent condition that affects 10%-15% of adults in their lifetime. It is associated with high morbidity due to colicky pain, the necessity for surgical intervention, and sometimes progression to CKD. In recent years, multiple monogenic causes of nephrolithiasis and nephrocalcinosis have been identified. However, the prevalence of each monogenic gene in a pediatric renal stone cohort has not yet been extensively studied. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS To determine the percentage of cases that can be explained molecularly by mutations in one of 30 known nephrolithiasis/nephrocalcinosis genes, we conducted a high-throughput exon sequencing analysis in an international cohort of 143 individuals <18 years of age, with nephrolithiasis (n=123) or isolated nephrocalcinosis (n=20). Over 7 months, all eligible individuals at three renal stone clinics in the United States and Europe were approached for study participation. RESULTS We detected likely causative mutations in 14 of 30 analyzed genes, leading to a molecular diagnosis in 16.8% (24 of 143) of affected individuals; 12 of the 27 detected mutations were not previously described as disease causing (44.4%). We observed that in our cohort all individuals with infantile manifestation of nephrolithiasis or nephrocalcinosis had causative mutations in recessive rather than dominant monogenic genes. In individuals who manifested later in life, causative mutations in dominant genes were more frequent. CONCLUSIONS We present the first exclusively pediatric cohort examined for monogenic causes of nephrolithiasis/nephrocalcinosis, and suggest that important therapeutic and preventative measures may result from mutational analysis in individuals with early manifestation of nephrolithiasis or nephrocalcinosis.

Evolutionarily conserved morphogenetic movements at the vertebrate head–trunk interface coordinate the transport and assembly of hypopharyngeal structures

The vertebrate head–trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today. Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarily conserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head–trunk interface.

IFT81 , encoding an IFT-B core protein, as a very rare cause of a ciliopathy phenotype

Background Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. Methods We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. Results Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. Conclusions This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development.