Altered mRNA and lncRNA expression profiles in the striated muscle complex of anorectal malformation rats.

BACKGROUND: Striated muscle complex (SMC) dysplasia has been confirmed to contribute to postoperative defecation dysfunction of patients with anorectal malformations (ARMs). To date, the potential molecular mechanisms of SMC dysplasia underlying the development of ARMs have not been clearly explained. This study examined the expression profiles of mRNAs and lncRNAs in the malformed SMC of ARM rats using RNA sequencing (RNA-seq). METHODS: A rat model of ARMs was established by the intragastric administration of 1% ethylene thiourea (ETU) on an embryonic day 10 (E10). The rats were subjected to euthanasia and the SMC samples were collected on E19. The expression of mRNAs and lncRNAs was analyzed by RNA-seq on the Illumina HiSeq2500 platform. qRT-PCR was used to confirm the results of RNA-seq. RESULTS: Compared with the levels in control rats, 1408 mRNAs and 472 lncRNAs were differentially expressed in the SMC of E19 ARM rats. GO and KEGG pathway analyses showed that the top enriched GO terms were mainly related to muscle development and the enriched pathways were associated with muscle and synaptic development. Protein-protein interaction network analysis was also performed using the STRING database. The network map revealed the interaction between the WNT3 protein and NTRK1, NTF4, MUSK, and BMP5 proteins. Finally, the qRT-PCR results further confirmed the RNA-seq data. CONCLUSION: Our findings indicate the involvement of these dysregulated mRNAs and lncRNAs in the pathogenesis of SMC dysplasia in ARMs, providing a theoretical foundation for developing interventions to improve postoperative defecation function.

A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle.

In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.

Striated-for-smooth muscle replacement in the developing mouse esophagus.

The esophagus is a muscular tube which transports swallowed content from the oral cavity and the pharynx to the stomach. Early in mouse development, an entire layer of the esophagus, the muscularis externa, consists of differentiated smooth muscle cells. Starting shortly after mid-gestation till about two weeks after birth, the muscularis externa almost entirely consists of striated muscle. This proximal-to-distal replacement of smooth muscle by the striated muscle depends on a number of factors. To identify the nature of the hypothetical "proximal" (mainly striated muscle originating) and "distal" (mainly smooth muscle originating) signals that govern the striated-for-smooth muscle replacement, we compared the esophagus of Myf5:MyoD null fetuses completely lacking striated muscle to the normal control using cDNA microarray analysis, followed by a comprehensive database search. Here we provide an insight into the nature of "proximal" and "distal" signals that govern the striated-for-smooth muscle replacement in the esophagus.

The regulatory pathway from genes directly activated by maternal factors to muscle structural genes in ascidian embryos.

Striated muscle cells in the tail of ascidian tadpole larvae differentiate cell-autonomously. Although several key regulatory factors have been identified, the genetic regulatory pathway is not fully understood; comprehensive understanding of the regulatory pathway is essential for accurate modeling in order to deduce principles for gene regulatory network dynamics, and for comparative analysis on how ascidians have evolved the cell-autonomous gene regulatory mechanism. Here, we reveal regulatory interactions among three key regulatory factors, Zic-r.b, Tbx6-r.b and Mrf, and elucidate the mechanism by which these factors activate muscle structural genes. We reveal a cross-regulatory circuit among these regulatory factors, which maintains the expression of Tbx6-r.b and Mrf during gastrulation. Although these two factors combinatorially activate muscle structural genes in late-stage embryos, muscle structural genes are activated mainly by Tbx6-r.b before gastrulation. Time points when expression of muscle structural genes become first detectable are strongly correlated with the degree of Tbx6-r.b occupancy. Thus, the genetic pathway, starting with Tbx6-r.b and Zic-r.b, which are activated by maternal factors, and ending with expression of muscle structural genes, has been revealed.

Cullin-3-RING ubiquitin ligase activity is required for striated muscle function in mice.

Control of protein homeostasis is an essential cellular process that, when perturbed, can result in the deregulation or toxic accumulation of proteins. Owing to constant mechanical stress, striated muscle proteins are particularly prone to wear and tear and require several protein quality-control mechanisms to coordinate protein turnover and removal of damaged proteins. Kelch-like proteins, substrate adapters for the Cullin-3 (Cul3)-RING ligase (CRL3) complex, are emerging as critical regulators of striated muscle development and function, highlighting the importance of Cul3-mediated proteostasis in muscle function. To explore the role of Cul3-mediated proteostasis in striated muscle, here we deleted Cul3 specifically in either skeletal muscle (SkM-Cul3 KO) or cardiomyocytes (CM-Cul3 KO) of mice. The loss of Cul3 caused neonatal lethality and dramatic alterations in the proteome, which were unique to each striated muscle type. Many of the proteins whose expression was significantly changed in the SkM-Cul3 KO were components of the extracellular matrix and sarcomere, whereas proteins altered in the CM-Cul3 KO were involved in metabolism. These findings highlight the requirement for striated muscle-specific CRL3 activity and indicate how the CRL3 complex can control different nodes of protein interaction networks in different types of striated muscle. Further identification of Cul3 substrates, and how these substrates are targeted, may reveal therapeutic targets and treatment regimens for striated muscle diseases.

Spatiotemporal expression of Wnt3a during striated muscle complex development in rat embryos with ethylenethiourea-induced anorectal malformations.

Numerous patients with anorectal malformations (ARMs) continue to experience fecal incontinence and constipation following surgical procedures. One of the most important factors that influences defecation is the striated muscle complex (SMC). Wnt signaling regulates the expression of myogenic regulatory factors, which serve an important role in muscle development. Therefore, the present study aimed to investigate the expression pattern of Wnt3a protein (by immunohistochemistry and western blot analysis) and mRNA [by reverse transcription­quantitative polymerase chain reaction (RT-qPCR)] in the SMC of ARM model rats that were exposed to ethylenethiourea. Immunostaining revealed that the expression of Wnt3a exhibits space­ and time­dependent changes in the developing SMC of ARM model rat embryos. Immunohistochemistry demonstrated that on embryonic day 17 (E17), Wnt3a­positive cells were observed in the SMC in normal embryos, and expression levels gradually increased as the rat embryos developed. Similar changes in Wnt3a protein expression were detected in ARM model rat embryos; however, the expression of Wnt3a was significantly reduced compared with the normal rat embryos. Western blotting and RT­qPCR also revealed lower expression levels of Wnt3a protein and mRNA, respectively, in the SMC of ARMs model rat embryos compared with normal rat embryos. These data revealed that the expression of Wnt3a in ARM embryos was notably reduced, indicating a potential role for Wnt3a in the maldevelopment of the SMC in patients with ARMs.

Embracing change: striated-for-smooth muscle replacement in esophagus development.

The esophagus functions to transport food from the oropharyngeal region to the stomach via waves of peristalsis and transient relaxation of the lower esophageal sphincter. The gastrointestinal tract, including the esophagus, is ensheathed by the muscularis externa (ME). However, while the ME of the gastrointestinal tract distal to the esophagus is exclusively smooth muscle, the esophageal ME of many vertebrate species comprises a variable amount of striated muscle. The esophageal ME is initially composed only of smooth muscle, but its developmental maturation involves proximal-to-distal replacement of smooth muscle with striated muscle. This fascinating phenomenon raises two important questions: what is the developmental origin of the striated muscle precursor cells, and what are the cellular and morphogenetic mechanisms underlying the process? Studies addressing these questions have provided controversial answers. In this review, we discuss the development of ideas in this area and recent work that has shed light on these issues. A working model has emerged that should permit deeper understanding of the role of ME development and maturation in esophageal disorders and in the functional and evolutionary underpinnings of the variable degree of esophageal striated myogenesis in vertebrate species.

The Chromatin Remodeling Complex Chd4/NuRD Controls Striated Muscle Identity and Metabolic Homeostasis.

Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life.

A Cranial Mesoderm Origin for Esophagus Striated Muscles.

The esophagus links the oral cavity to the stomach and facilitates the transfer of bolus. Using genetic tracing and mouse mutants, we demonstrate that esophagus striated muscles (ESMs) are not derived from somites but are of cranial origin. Tbx1 and Isl1 act as key regulators of ESMs, which we now identify as a third derivative of cardiopharyngeal mesoderm that contributes to second heart field derivatives and head muscles. Isl1-derived ESM progenitors colonize the mouse esophagus in an anterior-posterior direction but are absent in the developing chick esophagus, thus providing evolutionary insight into the lack of ESMs in avians. Strikingly, different from other myogenic regions, in which embryonic myogenesis establishes a scaffold for fetal fiber formation, ESMs are established directly by fetal myofibers. We propose that ESM progenitors use smooth muscle as a scaffold, thereby bypassing the embryonic program. These findings have important implications in understanding esophageal dysfunctions, including dysphagia, and congenital disorders, such as DiGeorge syndrome.

A thioredoxin fold protein Sh3bgr regulates Enah and is necessary for proper sarcomere formation.

The sh3bgr (SH3 domain binding glutamate-rich) gene encodes a small protein containing a thioredoxin-like fold, SH3 binding domain, and glutamate-rich domain. Originally, it was suggested that increased expression of Sh3bgr may cause the cardiac phenotypes in Down's syndrome. However, it was recently reported that the overexpression of Sh3bgr did not cause any disease phenotypes in mice. In this study, we have discovered that Sh3bgr is critical for sarcomere formation in striated muscle tissues and also for heart development. Sh3bgr is strongly expressed in the developing somites and heart in Xenopus. Morpholino mediated-knockdown of sh3bgr caused severe malformation of heart tissue and disrupted segmentation of the somites. Further analysis revealed that Sh3bgr specifically localized to the Z-line in mature sarcomeres and that knockdown of Sh3bgr completely disrupted sarcomere formation in the somites. Moreover, overexpression of Sh3bgr resulted in abnormally discontinues thick firmaments in the somitic sarcomeres. We suggest that Sh3bgr does its function at least partly by regulating localization of Enah for the sarcomere formation. In addition, we provide the data supporting Sh3bgr is also necessary for proper heart development in part by affecting the Enah protein level.

Loss of Prox1 in striated muscle causes slow to fast skeletal muscle fiber conversion and dilated cardiomyopathy.

Correct regulation of troponin and myosin contractile protein gene isoforms is a critical determinant of cardiac and skeletal striated muscle development and function, with misexpression frequently associated with impaired contractility or disease. Here we reveal a novel requirement for Prospero-related homeobox factor 1 (Prox1) during mouse heart development in the direct transcriptional repression of the fast-twitch skeletal muscle genes troponin T3, troponin I2, and myosin light chain 1. A proportion of cardiac-specific Prox1 knockout mice survive beyond birth with hearts characterized by marked overexpression of fast-twitch genes and postnatal development of a fatal dilated cardiomyopathy. Through conditional knockout of Prox1 from skeletal muscle, we demonstrate a conserved requirement for Prox1 in the repression of troponin T3, troponin I2, and myosin light chain 1 between cardiac and slow-twitch skeletal muscle and establish Prox1 ablation as sufficient to cause a switch from a slow- to fast-twitch muscle phenotype. Our study identifies conserved roles for Prox1 between cardiac and skeletal muscle, specifically implicated in slow-twitch fiber-type specification, function, and cardiomyopathic disease.

Post-transcriptional regulation of myotube elongation and myogenesis by Hoi Polloi.

Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression.

Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion.

Striated muscles that enable mouth opening and swallowing during feeding are essential for efficient energy acquisition, and are likely to have played a fundamental role in the success of early jawed vertebrates. The developmental origins and genetic requirements of these muscles are uncertain. Here, we determine by indelible lineage tracing in mouse that fibres of sternohyoid muscle (SHM), which is essential for mouth opening during feeding, and oesophageal striated muscle (OSM), which is crucial for voluntary swallowing, arise from Pax3-expressing somite cells. In vivo Kaede lineage tracing in zebrafish reveals the migratory route of cells from the anteriormost somites to OSM and SHM destinations. Expression of pax3b, a zebrafish duplicate of Pax3, is restricted to the hypaxial region of anterior somites that generate migratory muscle precursors (MMPs), suggesting that Pax3b plays a role in generating OSM and SHM. Indeed, loss of pax3b function led to defective MMP migration and OSM formation, disorganised SHM differentiation, and inefficient ingestion and swallowing of microspheres. Together, our data demonstrate Pax3-expressing somite cells as a source of OSM and SHM fibres, and highlight a conserved role of Pax3 genes in the genesis of these feeding muscles of vertebrates.

External gill motility and striated muscle presence in the embryos of anuran amphibians.

Anuran external gills were assessed for motility and striated muscle content in 16 species from seven families. Motility of three kinds was observed. Pulsatory movements related to heart beat rhythm were common. In embryos developing to a late stage before hatching, movements of the whole embryo were frequent, with gills rearranging as a consequence. The only clearly active movement, presumably muscle driven, was 'gill flicking', a posterior movement of the entire gill into the body either on one side only, or both together, followed by a return to the normal spread-out position. Some species may actively spread their gills when hanging from the water surface film, but we did not observe this. In some species, active gill movement developed over time, but we were not able to follow all species over such a developmental sequence. The relationship between active motility and muscle content was good in most cases. Observations on late stage embryos of the aromobatid Mannophryne trinitatis are presented for the first time. In one species, we noted spread external gills being used to adhere hatchlings to a surface.

Cytoskeletal heart-enriched actin-associated protein (CHAP) is expressed in striated and smooth muscle cells in chick and mouse during embryonic and adult stages.

We recently identified a new Z-disc protein, CHAP (Cytoskeletal Heart-enriched Actin-associated Protein), which is expressed in striated muscle and plays an important role during embryonic muscle development in mouse and zebrafish. Here, we confirm and further extend these findings by (i) the identification and characterization of the CHAP orthologue in chick and (ii) providing a detailed analysis of CHAP expression in mouse during embryonic and adult stages. Chick CHAP contains a PDZ domain and a nuclear localization signal, resembling the human and mouse CHAPa. CHAP is expressed in the developing heart and somites, as well as muscle precursors of the limb buds in mouse and chick embryos. CHAP expression in heart and skeletal muscle is maintained in adult mice, both in slow and fast muscle fibers. Moreover, besides expression in striated muscle, we demonstrate that CHAP is expressed in smooth muscle cells of aorta, carotid and coronary arteries in adult mice, but not during embryonic development.

The development of satellite cells and their niche in striated muscle complex of anorectal malformations rat embryos.

BACKGROUND: It has been demonstrated that different degrees of pelvic floor muscle (PFM) maldevelop in anorectal malformations (ARMs); yet the development of satellite cells, the myogenic stem cells responsible for muscle growth, repair, and maintenance remains elusive during the embryogenesis of PFM. Striated muscle complex (SMC) is one of the most important components of PFM. The objective of this study was to observe the development pattern of satellite cells and their niche of SMC and investigate its possible role in PFM dysplasia in ARMs. METHODS: Immunohistochemistry, cell culture, transmission electron microscopy (TEM), real-time quantitative PCR, and Western blot were performed to trace the dynamic development pattern of satellite cells during the morphogenesis of PFM in ethylenethiourea (ETU)-induced ARMs rat embryos. RESULTS: In ARMs rat embryos, earlier presentation and higher number of Pax7-expressing cell were observed in SMC. The expression of Pax7 and vimentin were up-regulated, while the expression of myogenin, vWF, and neurofilament were down-regulated. Ultrastructure analysis of SMC was characterized by increased amount of nuclear heterochromatin of satellite cell nuclei, thickened basal lamina, widened gap between satellite cell and myofiber, and disarrangement of muscle fibers. The satellite cells demonstrated abnormal differentiation after they were isolated and cultured in vitro. CONCLUSIONS: Our results suggest that premature origination of satellite cell from myogenic progenitor or precursor may result in the depletion of myogenic precursor and cessation of muscle growth; intrinsic defect in satellite cell structure, and extrinsic impairment of microenvironment compromised the myogenic competence of satellite cell, which might contribute substantially to the hypoplastic SMC in ARMs.

Hox genes regulate muscle founder cell pattern autonomously and regulate morphogenesis through motor neurons.

The differentiation of myoblasts to form functional muscle fibers is a consequence of interactions between the mesoderm and ectoderm. The authors examine the role of segment identity in directing these interactions by studying the role of Hox genes in patterning adult muscles in Drosophila. Using the 'four-winged fly' to remove Ultrabithorax function in the developing adult, the authors alter the identity of the ectoderm of the third thoracic segment towards the second and show that this is sufficient to inductively alter most properties of the mesoderm-myoblast number, molecular diversity, and migration pattern-to that of the second thoracic segment. Not all aspects of myogenesis are determined by the segment identity of the ectoderm. The autonomous identity of the mesoderm is important for choosing muscle founder cells in the correct segmental pattern. The authors show this by removal of the function of Antennapedia, the Hox gene expressed in the mesoderm of the third thoracic segment. This results in the transformation of founder cells to a second-thoracic pattern. The authors also report a role for the nervous system in later aspects of muscle morphogenesis by specifically altering Ultrabithorax gene expression in motor neurons. Thus, ectoderm and mesoderm segment identities collaborate to direct muscle differentiation by affecting distinct aspects of the process.

MADD-2, a homolog of the Opitz syndrome protein MID1, regulates guidance to the midline through UNC-40 in Caenorhabditis elegans.

The body muscles of Caenorhabditis elegans extend plasma membrane extensions called muscle arms to the midline motor axons to form the postsynaptic membrane of the neuromuscular junction. Through a screen for muscle arm development defective (Madd) mutants, we previously discovered that the UNC-40/DCC guidance receptor directs muscle arm extension through the Rho-GEF UNC-73. Here, we describe a gene identified through our mutant screen called madd-2, and show that it functions in an UNC-40 pathway. MADD-2 is a C1-TRIM protein and a homolog of human MID1, mutations in which cause Opitz Syndrome. We demonstrate that MADD-2 functions cell autonomously to direct muscle and axon extensions to the ventral midline of worms. Our results suggest that MADD-2 may enhance UNC-40 pathway activity by facilitating an interaction between UNC-40 and UNC-73. The analogous phenotypes that result from MADD-2 and MID1 mutations suggest that C1-TRIM proteins may have a conserved biological role in midline-oriented developmental events.

Apoptosis during the development of pelvic floor muscle in anorectal malformation rats.

PURPOSE: Fecal incontinence and constipation still remain as major postoperative complications after procedures for anorectal malformations (ARM). The striated muscle complex (SMC) is one of the most important factors that influence defecation. Previous studies have demonstrated different degrees of the muscle complex dysplasia dependent on the complexity of ARM. To explore the mechanisms of maldevelopment of SMC in ARM, apoptosis was investigated during pelvic floor muscle development in rat embryos with ARM. METHODS: Anorectal malformations in rat embryos were induced by treating pregnant rats with ethylenethiourea on the 10th embryonic day (E10). Normal and ARM rat embryos from E16 to E21 were serial-sectioned transversely or sagittally, and SMCs were dissected and snap frozen. TdT mediated dUTP Nick Ending Labeling (TUNEL) staining and DNA ladder analysis were performed to identify apoptosis and expression of Bax/Bcl-2 were confirmed with immunohistochemical staining and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis. RESULTS: Hypoplastic and disordered SMC sling shifted cephalad, ventrally, and converged inferior to the rectourethral fistula and infiltrated connective tissue in ARM embryos. In the normal group, TUNEL-positive cells became evident on E17; sporadic positive staining was mainly localized in 2 areas as follows: the junction area between SMC and bulbocarvernosus muscle and posterior to the rectum where bilateral SMC converged. In the ARM group, massive positive staining of nuclei was observed from E16 to E21 and was mainly distributed in the dorsal part of the SMC. Electrophoresis of DNA samples yielded a "ladder" pattern of migration both in normal and the ARM group from E17 to E21, the ladders were stronger in the ARM group. In both groups, the expression of Bax/Bcl-2 was detectable on E17, the immunoreactivity increased on E19 and E21. Compared with the normal group, the expression of Bax was increased, whereas Bcl-2 was declined in the ARM group. Significant upregulation of Bax messenger RNA (mRNA) levels and downregulation of Bcl-2 mRNA levels were observed in ARM embryos. CONCLUSIONS: In the current study, abnormal apoptosis and disturbed expression of Bax/Bcl-2 were identified during SMC development in ARM embryos. It is suggested that precocious, excessive, and dislocated apoptosis might be a fundamental pathogenesis for the maldeveloped SMC in ARM rats. The temporospatial expressions of Bax/Bcl-2 indicate they may have an important role in the regulation of apoptosis of SMC.