Myomesin is part of an integrity pathway that responds to sarcomere damage and disease.

The structure and function of the sarcomere of striated muscle is well studied but the steps of sarcomere assembly and maintenance remain under-characterized. With the aid of chaperones and factors of the protein quality control system, muscle proteins can be folded and assembled into the contractile apparatus of the sarcomere. When sarcomere assembly is incomplete or the sarcomere becomes damaged, suites of chaperones and maintenance factors respond to repair the sarcomere. Here we show evidence of the importance of the M-line proteins, specifically myomesin, in the monitoring of sarcomere assembly and integrity in previously characterized zebrafish muscle mutants. We show that myomesin is one of the last proteins to be incorporated into the assembling sarcomere, and that in skeletal muscle, its incorporation requires connections with both titin and myosin. In diseased zebrafish sarcomeres, myomesin1a shows an early increase of gene expression, hours before chaperones respond to damaged muscle. We found that myomesin expression is also more specific to sarcomere damage than muscle creatine kinase, and our results and others support the use of myomesin assays as an early, specific, method of detecting muscle damage

<i>Still Heart</i> Encodes a Structural HMT, SMYD1b, with Chaperone-Like Function during Fast Muscle Sarcomere Assembly

<div><p>The vertebrate sarcomere is a complex and highly organized contractile structure whose assembly and function requires the coordination of hundreds of proteins. Proteins require proper folding and incorporation into the sarcomere by assembly factors, and they must also be maintained and replaced due to the constant physical stress of muscle contraction. Zebrafish mutants affecting muscle assembly and maintenance have proven to be an ideal tool for identification and analysis of factors necessary for these processes. The <i>still heart</i> mutant was identified due to motility defects and a nonfunctional heart. The cognate gene for the mutant was shown to be <i>smyd1b</i> and the <i>still heart</i> mutation results in an early nonsense codon. SMYD1 mutants show a lack of heart looping and chamber definition due to a lack of expression of heart morphogenesis factors <i>gata4</i>, <i>gata5</i> and <i>hand2</i>. On a cellular level, fast muscle fibers in homozygous mutants do not form mature sarcomeres due to the lack of fast muscle myosin incorporation by SMYD1b when sarcomeres are first being assembled (19hpf), supporting SMYD1b as an assembly protein during sarcomere formation.</p></div

Wnt signaling and polarity in freshwater sponges

Abstract Background The Wnt signaling pathway is uniquely metazoan and used in many processes during development, including the formation of polarity and body axes. In sponges, one of the earliest diverging animal groups, Wnt pathway genes have diverse expression patterns in different groups including along the anterior-posterior axis of two sponge larvae, and in the osculum and ostia of others. We studied the function of Wnt signaling and body polarity formation through expression, knockdown, and larval manipulation in several freshwater sponge species. Results Sponge Wnts fall into sponge-specific and sponge-class specific subfamilies of Wnt proteins. Notably Wnt genes were not found in transcriptomes of the glass sponge Aphrocallistes vastus. Wnt and its signaling genes were expressed in archaeocytes of the mesohyl throughout developing freshwater sponges. Osculum formation was enhanced by GSK3 knockdown, and Wnt antagonists inhibited both osculum development and regeneration. Using dye tracking we found that the posterior poles of freshwater sponge larvae give rise to tissue that will form the osculum following metamorphosis. Conclusions Together the data indicate that while components of canonical Wnt signaling may be used in development and maintenance of osculum tissue, it is likely that Wnt signaling itself occurs between individual cells rather than whole tissues or structures in freshwater sponges

SMYD1b is co-regulated with other myosin chaperones HSP90a1 and UNC45b.

<p>In-situ hybridization staining of wild type and <i>still heart</i> embryos shows that <i>hsp90a1</i> expression is normal in <i>still heart</i> mutants, when compared to wild type embryos at 19hpf (A&B). However, the expression of <i>hsp90a1</i> and <i>unc45b</i> dramatically increases in <i>still heart</i> mutants at 24hpf (C-F & K) and 48hpf throughout the somites (G-J & M). Additionally, <i>smyd1b</i> expression increases significantly when UNC45b is absent in <i>steif</i> mutants (N), supporting co-regulation of these three genes. (O) A time course of <i>smyd1b</i> expression during muscle formation reveals that <i>smyd1b</i> is expressed early at 10hpf when <i>unc45b</i> is expressed and increases in its expression as muscle development progresses. Due to the rapid development of <i>unc45b</i> staining in the somites of the embryo in panel J, the head while present, has no background staining and is not clear in this focal plane. (qPCR: n = 3, 30 embryos each time/phenotype. Error bars are standard deviation.).</p

SMYD1b is required for fast myosin incorporation during sarcomere assembly.

<p>At 48hpf, fast myosin (F310—green) and actin (phalloidin—red) staining is visible in the premyofibrils in wt zebrafish tails (A&B), while absent from the premyofibrils in <i>sth</i> fast muscle tissue (C&D). Slow muscle (F59) develops normally in both wild type and <i>still heart</i> zebrafish at 48hpf (E&F, G&H). At 19hpf, in wild type embryos, fast myosin (F310) is beginning to be incorporated into the maturing myofibril (I) and overlaps (white arrowheads) with the developing actin (phalloidin) (J) fibers in trunk muscle (K, merge, K’ inset, white arrowhead). Fast myosin is not incorporated into the maturing premyofibril (L), although actin fibers are still present (M&N, N’ inset).</p

<i>Still heart</i>, a <i>smyd1b</i> mutant, has defects in heart and fast skeletal muscle tissue.

<p>A lateral view of 48hpf wild type (A) and <i>still heart</i> (<i>sth</i>) mutants (B), which have pericardial edema, small eyes, malformed head and reduced motility. Black arrowheads highlight the pericardial edema in sth mutants and the absence of edema in wild type. White arrowheads indicate blood pooling in the mutant and the absence of pooling in wild type. (C&D) <i>Sth</i> mutant hearts are underdeveloped and do not beat. (E-G) Examination of lateral myofibers at 5dpf under DIC microscopy revealed striations, indicative of fully formed sarcomeres, are visible in the myofibers of wild type muscle (E, black arrowheads), while absent in the fast muscle of <i>still heart</i> mutants (F); striations are present in <i>sth</i> slow muscle (G, black arrowhead) but are disturbed by nuclei and fluid-filled spaces (G, white arrowhead). Sequencing of <i>smyd1b</i> cDNA from wild type embryos (H) and <i>sth</i> mutant embryos (I) revealed a 9 nucleotide insertion between exon 1 and 2 in the <i>smyd1b</i> mRNA, creating an in-frame stop codon (I, underlined sequence). The insertion is the first 9 nucleotides of intron one as sequenced from wild type smyd1b genomic sequence (J). This is a result of a transition mutation in the splice donor site of intron 1 (I, green letter in sequence, J, outlined letter in sequence). This results in a premature truncation of the SMYD1b protein after exon 1 (K).</p

Additional file 7: of Wnt signaling and polarity in freshwater sponges

Fluorescent in situ hybridization and antibody images showing separate channels for images shown in Fig. 2. A) wntB, B) wntA/wntC, C) wntA/wntB and D) β-catenin antibody. (TIFF 6439 kb

Additional file 11: of Wnt signaling and polarity in freshwater sponges

Accession numbers of previously published sequences used for phylogenetic analysis in Fig.1 and Additional files 3 and 5. (PDF 64 kb

Additional file 2: of Wnt signaling and polarity in freshwater sponges

Mafft alignment of sponge Wnt protein sequences prepared in Boxshade ( http://www.ch.embnet.org/software/BOX_form.html ). Conserved cysteine residues are marked with a red asterisk, and the conserved RWNC motif is indicated with a green bracket. (PDF 9686 kb

Additional file 3: of Wnt signaling and polarity in freshwater sponges

Raw phylogenetic trees used to create the consensus tree presented in Fig. 1. A) PhyML tree with support values from 1000 bootstrap replicates. B) RAxML tree showing bootstrap support from 100 replicates. C) IQ-TREE with support values from 1000 SH-aLRT replicates/aBayes/1000 ultrafast bootstrap replicates. (PDF 820 kb