Urokinase-Type Plasminogen Activator Receptor (uPAR) in Inflammation and Disease: A Unique Inflammatory Pathway Activator.

The urokinase-type plasminogen activator receptor (uPAR) is a unique protease binding receptor, now recognized as a key regulator of inflammation. Initially, uPA/uPAR was considered thrombolytic (clot-dissolving); however, recent studies have demonstrated its predominant immunomodulatory functions in inflammation and cancer. The uPA/uPAR complex has a multifaceted central role in both normal physiological and also pathological responses. uPAR is expressed as a glycophosphatidylinositol (GPI)-linked receptor interacting with vitronectin, integrins, G protein-coupled receptors, and growth factor receptors within a large lipid raft. Through protein-to-protein interactions, cell surface uPAR modulates intracellular signaling, altering cellular adhesion and migration. The uPA/uPAR also modifies extracellular activity, activating plasminogen to form plasmin, which breaks down fibrin, dissolving clots and activating matrix metalloproteinases that lyse connective tissue, allowing immune and cancer cell invasion and releasing growth factors. uPAR is now recognized as a biomarker for inflammatory diseases and cancer; uPAR and soluble uPAR fragments (suPAR) are increased in viral sepsis (COVID-19), inflammatory bowel disease, and metastasis. Here, we provide a comprehensive overview of the structure, function, and current studies examining uPAR and suPAR as diagnostic markers and therapeutic targets. Understanding uPAR is central to developing diagnostic markers and the ongoing development of antibody, small-molecule, nanogel, and virus-derived immune-modulating treatments that target uPAR.

The Transcription Factor Mohawk Facilitates Skeletal Muscle Repair via Modulation of the Inflammatory Environment.

Efficient repair of skeletal muscle relies upon the precise coordination of cells between the satellite cell niche and innate immune cells that are recruited to the site of injury. The expression of pro-inflammatory cytokines and chemokines such as TNFα, IFNγ, CXCL1, and CCL2, by muscle and tissue resident immune cells recruits neutrophils and M1 macrophages to the injury and activates satellite cells. These signal cascades lead to highly integrated temporal and spatial control of muscle repair. Despite the therapeutic potential of these factors for improving tissue regeneration after traumatic and chronic injuries, their transcriptional regulation is not well understood. The transcription factor Mohawk (Mkx) functions as a repressor of myogenic differentiation and regulates fiber type specification. Embryonically, Mkx is expressed in all progenitor cells of the musculoskeletal system and is expressed in human and mouse myeloid lineage cells. An analysis of mice deficient for Mkx revealed a delay in postnatal muscle repair characterized by impaired clearance of necrotic fibers and smaller newly regenerated fibers. Further, there was a delay in the expression of inflammatory signals such as Ccl2, Ifnγ, and Tgfß. This was coupled with impaired recruitment of pro-inflammatory macrophages to the site of muscle damage. These studies demonstrate that Mkx plays a critical role in adult skeletal muscle repair that is mediated through the initial activation of the inflammatory response.

Pharmacotherapeutic Approaches to Treatment of Muscular Dystrophies.

Muscular dystrophies are a heterogeneous group of genetic muscle-wasting disorders that are subdivided based on the region of the body impacted by muscle weakness as well as the functional activity of the underlying genetic mutations. A common feature of the pathophysiology of muscular dystrophies is chronic inflammation associated with the replacement of muscle mass with fibrotic scarring. With the progression of these disorders, many patients suffer cardiomyopathies with fibrosis of the cardiac tissue. Anti-inflammatory glucocorticoids represent the standard of care for Duchenne muscular dystrophy, the most common muscular dystrophy worldwide; however, long-term exposure to glucocorticoids results in highly adverse side effects, limiting their use. Thus, it is important to develop new pharmacotherapeutic approaches to limit inflammation and fibrosis to reduce muscle damage and promote repair. Here, we examine the pathophysiology, genetic background, and emerging therapeutic strategies for muscular dystrophies.

Single cell analysis reveals satellite cell heterogeneity for proinflammatory chemokine expression.

Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood. Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence. Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2 h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury. Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.

Myxomavirus Serp-1 Protein Ameliorates Inflammation in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is an X-linked disease afflicting 1 in 3500 males that is characterized by muscle weakness and wasting during early childhood, and loss of ambulation and death by early adulthood. Chronic inflammation due to myofiber instability leads to fibrosis, which is a primary cause of loss of ambulation and cardiorespiratory insufficiency. Current standard of care focuses on reducing inflammation with corticosteroids, which have serious adverse effects. It is imperative to identify alternate immunosuppressants as treatments to reduce fibrosis and mortality. Serp-1, a Myxoma virus-derived 55 kDa secreted glycoprotein, has proven efficacy in a range of animal models of acute inflammation, and its safety and efficacy has been shown in a clinical trial. In this initial study, we examined whether pegylated Serp-1 (PEGSerp-1) treatment would ameliorate chronic inflammation in a mouse model for Duchenne muscular dystrophy. Our data revealed a significant reduction in diaphragm fibrosis and increased myofiber diameter, and significantly decreased pro-inflammatory M1 macrophage infiltration. The M2a macrophage and overall T cell populations showed no change. These data demonstrate that treatment with this new class of poxvirus-derived immune-modulating serpin has potential as a therapeutic approach designed to ameliorate DMD pathology and facilitate muscle regeneration.

Anatomical and histological analyses reveal that tail repair is coupled with regrowth in wild-caught, juvenile American alligators (Alligator mississippiensis).

Reptiles are the only amniotes that maintain the capacity to regenerate appendages. This study presents the first anatomical and histological evidence of tail repair with regrowth in an archosaur, the American alligator. The regrown alligator tails constituted approximately 6-18% of the total body length and were morphologically distinct from original tail segments. Gross dissection, radiographs, and magnetic resonance imaging revealed that caudal vertebrae were replaced by a ventrally-positioned, unsegmented endoskeleton. This contrasts with lepidosaurs, where the regenerated tail is radially organized around a central endoskeleton. Furthermore, the regrown alligator tail lacked skeletal muscle and instead consisted of fibrous connective tissue composed of type I and type III collagen fibers. The overproduction of connective tissue shares features with mammalian wound healing or fibrosis. The lack of skeletal muscle contrasts with lizards, but shares similarities with regenerated tails in the tuatara and regenerated limbs in Xenopus adult frogs, which have a cartilaginous endoskeleton surrounded by connective tissue, but lack skeletal muscle. Overall, this study of wild-caught, juvenile American alligator tails identifies a distinct pattern of wound repair in mammals while exhibiting features in common with regeneration in lepidosaurs and amphibia.

Transcriptome changes during the initiation and progression of Duchenne muscular dystrophy in Caenorhabditis elegans.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by nonsense and missense mutations in the dystrophin gene, leading to instability of the sarcolemma and skeletal muscle necrosis and atrophy. Resulting changes in muscle-specific gene expression that take place in dystrophin's absence remain largely uncharacterized, as they are potentially obscured by the chronic inflammation elicited by muscle damage in humans. Caenorhabditis elegans possess a mild inflammatory response that is not active in the muscle, and lack a satellite cell equivalent. This allows for the characterization of the transcriptome rearrangements affecting disease progression independently of inflammation and regeneration. In effort to better understand these dynamics, we have isolated and sequenced body muscle-specific transcriptomes from C. elegans lacking functional dystrophin at distinct stages of disease progression. We have identified an upregulation of genes involved in mitochondrial function early in disease progression, and an upregulation of genes related to muscle repair in later stages. Our results suggest that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract muscle degradation caused by loss of dystrophin. We have also developed a temperature-based screening method for synthetic paralysis that can be used to rapidly identify genetic partners of dystrophin. Our results allow for the comprehensive identification of transcriptome changes that potentially serve as independent drivers of disease progression and may in turn allow for the identification of new therapeutic targets for the treatment of DMD.

Poly(N-isopropylacrylamide)-based dual-crosslinking biohybrid injectable hydrogels for vascularization.

Injectable hydrogels provide a powerful and non-invasive approach for numerous applications in cell transplantation, growth factor delivery, tissue regeneration and so forth. The properties of injectable hydrogels should be well-tuned for specific applications, where their overall design should ensure biocompatibility, non-toxicity, robust mechanical properties, and most importantly the ability to promote vascularization and integration with the host tissue/organ. Among these criteria, vascularization remains a key design element in the development of functional therapeutic hydrogels for successful translation into clinical settings. To that end, there is still a critical need for the development of the next generation of injectable hydrogels with precisely tuned biophysical and biochemical properties which could simultaneously promote tissue vascularization. In this work, we developed a temperature responsive, dual-crosslinking, biohybrid hydrogels, modified with a vasculogenic peptide for applications in regenerative medicine, specifically tissue vascularization. The synthesized hydrogels consisted of poly(N-isopropylacrylamide)-based copolymer, functionalized gelation and angiogenic VEGF-mimetic QK peptide with enhanced shear-thinning and injectability properties. QK peptide is a VEGF-mimetic vasculogenic peptide which binds to VEGF receptors and activates intercellular pathway for vascularization. Apart from the presence of QK peptide, the mechanical properties of the hydrogels were precisely tuned by altering the polymer concentration, enabling successful assembly and endothelial cell network formation. Extended in vitro studies demonstrated successful encapsulation and homogeneous distribution of endothelial cells within the three-dimensional (3D) environment of the hydrogel matrix with significantly enhanced vascularization in presence of the QK peptide as early as 3 days of culture. A small, preliminary in vivo study in mice showed a trend of increased blood vessel formation in hydrogels that incorporated the QK peptide. Overall, our study presents the design and characterization of injectable, dual-crosslinking and vasculogenic hydrogels with controlled properties which could be utilized for numerous applications in regenerative medicine, minimally invasive cell and drug delivery as well as fundamental studies on tissue vascularization and angiogenesis. STATEMENT OF SIGNIFICANCE: In this work, we synthesized a new class of temperature responsive, dual-crosslinking, biohybrid injectable hydrogels with enhanced vascularization properties for broad applications in regenerative medicine and minimally invasive cell/drug delivery. The developed hydrogels properly accommodated 3D culture, assembly and network formation of endothelial cells, as evidenced by in vitro and in vivo studies.

Molecular analysis of muscle progenitor cells on extracellular matrix coatings and hydrogels.

Development of an ex vivo culture system to expand satellite cells, the resident muscle stem cell population, will be necessary for the development of their use as therapeutics. The loss of the niche environment is often cited as the reason that culture results in both the loss of myogenic potential and low re-engraftment rates of these cells. Studies have shown that culture of satellite cells on more elastic substrates maintained their quiescence and potential and increased re-engraftment, but there was limited proliferation. We examined whether substrates composed of extracellular matrix proteins, as either coatings or hydrogels, could support expansion of this population whilst maintaining the potency of these cells. The collagen based hydrogels were very pliant and our studies demonstrate that stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, and the ECM composition was not significantly important. Our data further indicates that culture on highly elastic substrates allowed satellite cells to down-regulate myogenic specific transcription factors, resulting in an expression profile similar to a Galert state. These satellite cells could be subsequently cultured on Matrigel and induced to differentiate. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for studies of satellite cell proliferation and differentiation on alternative substrates. STATEMENT OF SIGNIFICANCE: Although biomaterials and muscle stem cell-based therapeutics for muscle loss due to trauma and disease have been studied intensely for the last decade, significant challenges remain; satellite cells lose their myogenic potential after in vitro culture, and do not re-engraft efficiently when delivered to skeletal muscle. We cultured adult mouse derived satellite cells and C2C12 cells on biomimetic, extracellular matrix based, hydrogels and coated plates and carried out a detailed analysis of proliferation, differentiation, and gene expression. These studies were designed to allow for the examination of the roles of both matrix composition and elasticity. The data demonstrated that satellite cells were most affected by elasticity. Examining the expression of lineage and cell cycle specific genes provided important insight into the behavior of both cell types, and point to fundamental differences that affect the interpretation of studies aimed at understanding the in vitro requirements of muscle progenitor cells. Understanding how satellite cells respond to various biochemical and biophysical cues at the molecular level will inform future efforts to design therapeutics for muscle regeneration.

Identification of satellite cells from anole lizard skeletal muscle and demonstration of expanded musculoskeletal potential.

The lizards are evolutionarily the closest vertebrates to humans that demonstrate the ability to regenerate entire appendages containing cartilage, muscle, skin, and nervous tissue. We previously isolated PAX7-positive cells from muscle of the green anole lizard, Anolis carolinensis, that can differentiate into multinucleated myotubes and express the muscle structural protein, myosin heavy chain. Studying gene expression in these satellite/progenitor cell populations from A. carolinensis can provide insight into the mechanisms regulating tissue regeneration. We generated a transcriptome from proliferating lizard myoprogenitor cells and compared them to transcriptomes from the mouse and human tissues from the ENCODE project using XGSA, a statistical method for cross-species gene set analysis. These analyses determined that the lizard progenitor cell transcriptome was most similar to mammalian satellite cells. Further examination of specific GO categories of genes demonstrated that among genes with the highest level of expression in lizard satellite cells were an increased number of genetic regulators of chondrogenesis, as compared to mouse satellite cells. In micromass culture, lizard PAX7-positive cells formed Alcian blue and collagen 2a1 positive nodules, without the addition of exogenous morphogens, unlike their mouse counterparts. Subsequent quantitative RT-PCR confirmed up-regulation of expression of chondrogenic regulatory genes in lizard cells, including bmp2, sox9, runx2, and cartilage specific structural genes, aggrecan and collagen 2a1. Taken together, these data suggest that tail regeneration in lizards involves significant alterations in gene regulation with expanded musculoskeletal potency.

Evolutionary patterns of metazoan microRNAs reveal targeting principles in the let-7 and miR-10 families.

MicroRNAs (miRNAs) regulate gene output by targeting degenerate elements in mRNAs and have undergone drastic expansions in higher metazoan genomes. The evolutionary advantage of maintaining copies of highly similar miRNAs is not well understood, nor is it clear what unique functions, if any, miRNA family members possess. Here, we study evolutionary patterns of metazoan miRNAs, focusing on the targeting preferences of the let-7 and miR-10 families. These studies reveal hotspots for sequence evolution with implications for targeting and secondary structure. High-throughput screening for functional targets reveals that each miRNA represses sites with distinct features and regulates a large number of genes with cooperative function in regulatory networks. Unexpectedly, given the high degree of similarity, single-nucleotide changes grant miRNA family members with distinct targeting preferences. Together, our data suggest complex functional relationships among miRNA duplications, novel expression patterns, sequence change, and the acquisition of new targets.

Notch signaling represses GATA4-induced expression of genes involved in steroid biosynthesis.

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway.

Comparative RNA-Seq analysis reveals pervasive tissue-specific alternative polyadenylation in Caenorhabditis elegans intestine and muscles.

BACKGROUND: Tissue-specific RNA plasticity broadly impacts the development, tissue identity and adaptability of all organisms, but changes in composition, expression levels and its impact on gene regulation in different somatic tissues are largely unknown. Here we developed a new method, polyA-tagging and sequencing (PAT-Seq) to isolate high-quality tissue-specific mRNA from Caenorhabditis elegans intestine, pharynx and body muscle tissues and study changes in their tissue-specific transcriptomes and 3'UTRomes. RESULTS: We have identified thousands of novel genes and isoforms differentially expressed between these three tissues. The intestine transcriptome is expansive, expressing over 30% of C. elegans mRNAs, while muscle transcriptomes are smaller but contain characteristic unique gene signatures. Active promoter regions in all three tissues reveal both known and novel enriched tissue-specific elements, along with putative transcription factors, suggesting novel tissue-specific modes of transcription initiation. We have precisely mapped approximately 20,000 tissue-specific polyadenylation sites and discovered that about 30% of transcripts in somatic cells use alternative polyadenylation in a tissue-specific manner, with their 3'UTR isoforms significantly enriched with microRNA targets. CONCLUSIONS: For the first time, PAT-Seq allowed us to directly study tissue specific gene expression changes in an in vivo setting and compare these changes between three somatic tissues from the same organism at single-base resolution within the same experiment. We pinpoint precise tissue-specific transcriptome rearrangements and for the first time link tissue-specific alternative polyadenylation to miRNA regulation, suggesting novel and unexplored tissue-specific post-transcriptional regulatory networks in somatic cells.

Numb-deficient satellite cells have regeneration and proliferation defects.

The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.

Regulation of mesenchymal-to-epithelial transition by PARAXIS during somitogenesis.

BACKGROUND: Dynamic alterations in cell shape, migration, and adhesion play a central role in tissue morphogenesis during embryonic development and congenital disease. The mesenchymal-to-epithelial transition that occurs during vertebrate somitogenesis is required for proper patterning of the axial musculoskeletal system. Somitic MET is initiated in the presomitic mesoderm by PARAXIS-dependent changes in cell adhesion, cell polarity, and the composition of the extracellular matrix. However, the target genes downstream of the transcription factor PARAXIS remain poorly described. RESULTS: A genome-wide comparison of gene expression in the anterior presomitic mesoderm and newly formed somites of Paraxis(-/-) embryos resulted in a set of deregulated genes enriched for factors associated with extracellular matrix and cytoskeletal organization and cell-cell and cell-ECM adhesion. The greatest change in expression was seen in fibroblast activation protein alpha (Fap), encoding a dipeptidyl peptidase capable of increasing fibronectin and collagen fiber organization in extracellular matrix. Further, downstream genes in the Wnt and Notch signaling pathways were downregulated, predicting that PARAXIS participates in positive feedback loops in both pathways. CONCLUSIONS: These data demonstrate that PARAXIS initiates and stabilizes somite epithelialization by integrating signals from multiple pathways to control the reorganization of the ECM, cytoskeleton, and adhesion junctions during MET.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality.

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.

Characterization of the DNA-binding properties of the Mohawk homeobox transcription factor.

The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.

Somitogenesis in the anole lizard and alligator reveals evolutionary convergence and divergence in the amniote segmentation clock.

The axial skeleton is a defining feature of vertebrates and is patterned during somitogenesis. Cyclically expressed members of the notch and other signaling pathways, described as the 'segmentation clock', regulate the formation of somite boundaries. Comparisons among vertebrate model systems have revealed fundamental shifts in the regulation of expression among critical genes in the notch pathway. However, insights into the evolution of these expression differences have been limited by the lack of information from non-avian reptiles. We analyzed the segmentation clock of the first Lepidosaurian reptile sequenced, the green anole lizard, Anolis carolinensis, for comparison with avian and mammalian models. Using genomic sequence, RNA-Seq transcriptomic data, and in situ hybridization analysis of somite-stage embryos, we carried out comparative analyses of key genes and found that the anole segmentation clock displays features common to both amniote and anamniote vertebrates. Shared features with anamniotes, represented by Xenopus laevis and Danio rerio, include an absence of lunatic fringe (lfng) expression within the presomitic mesoderm (PSM), a hes6a gradient in the PSM not observed in the chicken or mouse, and EGF repeat structure of the divergent notch ligand, dll3. The anole and mouse share cycling expression of dll1 ligand in the PSM. To gain insight from an Archosaurian reptile, we analysed LFNG and DLL1 expressions in the American alligator. LFNG expression was absent in the alligator PSM, like the anole but unlike the chicken. In contrast, DLL1 expression does not cycle in the PSM of the alligator, similar to the chicken but unlike the anole. Thus, our analysis yields novel insights into features of the segmentation clock that are evolutionarily basal to amniotes versus those that are specific to mammals, Lepidosaurian reptiles, or Archosaurian reptiles.

Scoliosis and segmentation defects of the vertebrae.

The vertebral column derives from somites, which are transient paired segments of mesoderm that surround the neural tube in the early embryo. Somites are formed by a genetic mechanism that is regulated by cyclical expression of genes in the Notch, Wnt, and fibroblast growth factor (FGF) signaling pathways. These oscillators together with signaling gradients within the presomitic mesoderm help to set somitic boundaries and rostral-caudal polarity that are essential for the precise patterning of the vertebral column. Disruption of this mechanism has been identified as the cause of severe segmentation defects of the vertebrae in humans. These segmentation defects are part of a spectrum of spinal disorders affecting the skeletal elements and musculature of the spine, resulting in curvatures such as scoliosis, kyphosis, and lordosis. While the etiology of most disorders with spinal curvatures is still unknown, genetic and developmental studies of somitogenesis and patterning of the axial skeleton and musculature are yielding insights into the causes of these diseases.

Mutations in the Notch pathway alter the patterning of multifidus.

Clinical studies have suggested that defects in the epaxial muscles, particularly multifidus, may contribute to the etiology of idiopathic scoliosis. While the epaxial muscles and the vertebrae derive from the same embryonic segmentation process, the mechanisms that pattern the multisegmental back muscles are still unclear. The process of segmentation is regulated by the Notch signaling pathway, and mutations in the modulators delta-like 3 (Dll3) and lunatic fringe (Lfng) are genetic models for spinal disorders such as scoliosis. Osteological defects have been characterized in these genetic models, but myological phenotypes have not previously been studied. We analyzed the multifidus muscle in the mouse (Mus musculus) and observed intriguing changes in the cranio-caudal borders of multifidus in Dll3 and Lfng models. Statistical analysis did not find a significant association between the majority of the multifidus anomalies and the vertebral defects, suggesting a previously unappreciated role for Notch signaling in patterning epaxial muscle groups. These findings indicate an additional mechanism by which DLL3 and LFNG may play a role in the etiology of human idiopathic scoliosis.