Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Regulation of bone development, growth, and remodeling traditionally has been thought to depend on endocrine and autocrine/paracrine modulators. Recently, however, brain-derived signals have emerged as key regulators of bone metabolism, although their mechanisms of action have been poorly understood. We reveal the existence of an ancient parathyroid hormone (Pth)4 in zebrafish that was secondarily lost in the eutherian mammals' lineage, including humans, and that is specifically expressed in neurons of the hypothalamus and appears to be a central neural regulator of bone development and mineral homeostasis. Transgenic fish lines enabled mapping of axonal projections leading from the hypothalamus to the brainstem and spinal cord. Targeted laser ablation demonstrated an essential role for of pth4-expressing neurons in larval bone mineralization. Moreover, we show that Runx2 is a direct regulator of pth4 expression and that Pth4 can activate cAMP signaling mediated by Pth receptors. Finally, gain-of-function experiments show that Pth4 can alter calcium/phosphorus levels and affect expression of genes involved in phosphate homeostasis. Based on our discovery and characterization of Pth4, we propose a model for evolution of bone homeostasis in the context of the vertebrate transition from an aquatic to a terrestrial lifestyle.-Suarez-Bregua, P., Torres-Nuñez, E., Saxena, A., Guerreiro, P., Braasch, I., Prober, D. A., Moran, P., Cerda-Reverter, J. M., Du, S. J., Adrio, F., Power, D. M., Canario, A. V. M., Postlethwait, J. H., Bronner, M E., Cañestro, C., Rotllant, J. Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Comparative proteomic analyses of Asian cotton ovules with attached fibers in the early stages of fiber elongation process.

BACKGROUND: Plenty of proteomic studies were performed to characterize the allotetraploid upland cotton fiber elongation process, whereas little is known about the elongating diploid cotton fiber proteome. METHODS: In this study, we used a two-dimensional electrophoresis-based comparative proteomic approach to profile dynamic proteomes of diploid Asian cotton ovules with attached fibers in the early stages of fiber elongation process. One-way ANOVA and Student-Newman-Keuls test were used to find the differentially displayed protein (DDP) spots. RESULTS: A total of 55 protein spots were found having different abundance ranging from 1 to 9 days post-anthesis (DPA) in a two-day interval. These 55 DDP spots were all successfully identified using high-resolution mass spectrometric analyses. Gene ontology analyses revealed that proteoforms involved in energy/carbohydrate metabolism, redox homeostasis, and protein metabolism are the most abundant. In addition, orthologues of the 13 DDP spots were also found in differential proteome of allotetraploid elongating cotton fibers, suggesting their possible essential roles in fiber elongation process. CONCLUSIONS: Our results not only revealed the dynamic proteome change of diploid Asian cotton fiber and ovule during early stages of fiber elongation process but also provided valuable resource for future studies on the molecular mechanism how the polyploidization improves the trait of fiber length.

GhMCS1, the Cotton Orthologue of Human GRIM-19, Is a Subunit of Mitochondrial Complex I and Associated with Cotton Fibre Growth.

GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a subunit of mitochondrial respiratory complex I in mammalian systems, and it has been demonstrated to be a multifunctional protein involved in the cell cycle, cell motility and innate immunity. However, little is known about the molecular functions of its homologues in plants. Here, we characterised GhMCS1, an orthologue of human GRIM-19 from cotton (Gossypium hirsutum L.), and found that it was essential for maintaining complex integrity and mitochondrial function in cotton. GhMCS1 was detected in various cotton tissues, with high levels expressed in developing fibres and flowers and lower levels in leaves, roots and ovules. In fibres at different developmental stages, GhMCS1 expression peaked at 5-15 days post anthesis (dpa) and then decreased at 20 dpa and diminished at 25 dpa. By Western blot analysis, GhMCS1 was observed to be localised to the mitochondria of cotton leaves and to colocalise with complex I. In Arabidopsis, GhMCS1 overexpression enhanced the assembly of complex I and thus respiratory activity, whereas the GhMCS1 homologue (At1g04630) knockdown mutants showed significantly decreased respiratory activities. Furthermore, the mutants presented with some phenotypic changes, such as smaller whole-plant architecture, poorly developed seeds and fewer trichomes. More importantly, in the cotton fibres, both the GhMCS1 transcript and protein levels were correlated with respiratory activity and fibre developmental phase. Our results suggest that GhMCS1, a functional ortholog of the human GRIM-19, is an essential subunit of mitochondrial complex I and is involved in cotton fibre development. The present data may deepen our knowledge on the potential roles of mitochondria in fibre morphogenesis.

Exercise training activates neuregulin 1/ErbB signaling and promotes cardiac repair in a rat myocardial infarction model.

AIMS: Exercise training (ET) has a cardioprotective effect and can alter the molecular response to myocardial infarction (MI). The Neuregulin 1 (NRG1)/ErbB signaling plays a critical role in cardiac repair and regeneration in the failing heart. We sought to investigate whether ET following MI could activate the NRG1/ErbB signaling and promote cardiac repair and regeneration. MAIN METHODS: Male Sprague-Dawley rats were used to establish the MI model. Exercise-trained animals were subjected to four weeks of exercise (16m/min, 50min/d, 5d/wk) following the surgery. AG1478 was used as an inhibitor of ErbB (1mg/kg body weight, administered i.v. every other day during the process of training). NRG1/ErbB signaling activation, cardiomyocyte (CM) proliferation and apoptosis were evaluated. KEY FINDINGS: In the exercise-trained rats, NRG1 expression was up-regulated and ErbB/PI3K/Akt signaling was activated compared with the MI group. In addition, ET preserved heart function accompanied with increased numbers of BrdU(+) CMs, PCNA(+) CMs and c-kit(+) cells, and reduced apoptosis level in the MI rats. In contrast, blocking ErbB signaling by AG1478 attenuated the ET-induced cardiac repair and regeneration. SIGNIFICANCE: ET up-regulates NRG1 expression and activates ErbB2, ErbB4 and PI3K/Akt signal transduction to promote cardiac repair through endogenous regeneration. Activation of ErbB may be an underlying mechanism for the ET-induced cardiac repair and regeneration following MI.

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

The duplicated paired box protein 7 (pax7) genes differentially transcribed during Japanese flounder (Paralichthys olivaceus) embryogenesis.

PAX are important regulators of developmental processes. PAX7 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest (NC), and skeletal muscle. Here, we identified six spliced isoforms of pax7a and one pax7b and characterized their expression patterns. All of flounder Pax7a-1, Pax7a-2, Pax7a-3, and Pax7b contain a conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). However, the PD of Pax7a-4 and the HD of Pax7a-5 are not intact, and there is no HD in Pax7a-4 and Pax7a-6. pax7a and pax7b show distinct spatiotemporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that the expression patterns of pax7a and pax7b are overlapping but distinguishable in the dorsal central nervous system. pax7a is expressed in most part of the brain and the neural tube, while pax7b is expressed exclusively in the diencephalon and the midbrain. In addition, pax7a is also expressed in the cranial NC and the trunk NC. RT-PCR results show that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated pax7 genes. The duplicated pax7 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis.

A transgenic zebrafish model for monitoring xbp1 splicing and endoplasmic reticulum stress in vivo.

Accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) triggers ER stress that initiates unfolded protein response (UPR). XBP1 is a transcription factor that mediates one of the key signaling pathways of UPR to cope with ER stress through regulating gene expression. Activation of XBP1 involves an unconventional mRNA splicing catalyzed by IRE1 endonuclease that removes an internal 26 nucleotides from xbp1 mRNA transcripts in the cytoplasm. Researchers have taken advantage of this unique activation mechanism to monitor XBP1 activation, thereby UPR, in cell culture and transgenic models. Here we report a Tg(ef1α:xbp1δ-gfp) transgenic zebrafish line to monitor XBP1 activation using GFP as a reporter especially in zebrafish oocytes and developing embryos. The Tg(ef1α:xbp1δ-gfp) transgene was constructed using part of the zebrafish xbp1 cDNA containing the splicing element. ER stress induced splicing results in the cDNA encoding a GFP-tagged partial XBP1 without the transactivation activation domain (XBP1Δ-GFP). The results showed that xbp1 transcripts mainly exist as the spliced active isoform in unfertilized oocytes and zebrafish embryos prior to zygotic gene activation at 3 hours post fertilization. A strong GFP expression was observed in unfertilized oocytes, eyes, brain and skeletal muscle in addition to a weak expression in the hatching gland. Incubation of transgenic zebrafish embryos with (dithiothreitol) DTT significantly induced XBP1Δ-GFP expression. Collectively, these studies unveil the presence of maternal xbp1 splicing in zebrafish oocytes, fertilized eggs and early stage embryos. The Tg(ef1α:xbp1δ-gfp) transgenic zebrafish provides a useful model for in vivo monitoring xbp1 splicing during development and under ER stress conditions.

The olive flounder (Paralichthys olivaceus) Pax3 homologues are highly conserved, encode multiple isoforms and show unique expression patterns.

Pax genes encode a highly conserved family of transcription factors that play crucial roles in the formation of tissues and organs during development. Pax3 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest and skeletal muscle. Here, we identified two spliced isoforms of Pax3a and three spliced isoforms of Pax3b and characterized their expression patterns. Both of flounder Pax3a-1 and Pax3b-1 contain the conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). But the PD domain in Pax3a-2 and Pax3b-3 is not intact and there is no HD in Pax3b-2 and Pax3b-3. Pax3a and Pax3b show distinct temporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that Pax3a and Pax3b are expressed in overlapping patterns in the dorsal central nervous system, with some subtle regional differences between the two genes. In addition, Pax3a is scattered in the somites while Pax3b is specifically expressed in the newly forming somites. RT-PCR results have shown that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated Pax3 genes. The duplicated Pax3 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis.

SMYD proteins: key regulators in skeletal and cardiac muscle development and function.

Muscle fibers are composed of myofibrils, one of the most highly ordered macromolecular assemblies in cells. Recent studies demonstrate that members of the Smyd family play critical roles in myofibril assembly of skeletal and cardiac muscle during development. The Smyd family consists of five members including Smyd1, Smyd2, Smyd3, Smyd4, and Smyd5. They share two highly conserved structural and functional domains, namely the SET and MYND domains involved in lysine methylation and protein-protein interaction, respectively. Smyd1 is specifically expressed in muscle cells under the regulation of myogenic transcriptional factors of the MyoD and Mef2 families and the serum responsive factor. Loss of function studies reveal that Smyd1 is required for cardiomyogenesis and sarcomere assembly in skeletal and cardiac muscles. Smyd2, on another hand, is dispensable for heart development in mice. However, Smyd2 appears to play a role in myofilament organization in both skeletal and cardiac muscles via Hsp90 methylation. A Drosophila Smyd4 homologue is a muscle-specific transcriptional modulator involved in the development or function of adult muscle. The molecular mechanisms by which Smyd family proteins function in muscle cells are not well understood. It has been suggested that members of the Smyd family may use multiple mechanisms to control muscle development and cell differentiation, including transcriptional regulation, epigenetic regulation via histone methylation, and methylation of proteins other than histones, such as molecular chaperone Hsp90.

Gne depletion during zebrafish development impairs skeletal muscle structure and function.

GNE Myopathy is a rare recessively inherited neuromuscular disorder caused by mutations in the GNE gene, which codes for the key enzyme in the metabolic pathway of sialic acid synthesis. The process by which GNE mutations lead to myopathy is not well understood. By in situ hybridization and gne promoter-driven fluorescent transgenic fish generation, we have characterized the spatiotemporal expression pattern of the zebrafish gne gene and have shown that it is highly conserved compared with the human ortholog. We also show the deposition of maternal gne mRNA and maternal GNE protein at the earliest embryonic stage, emphasizing the critical role of gne in embryonic development. Injection of morpholino (MO)-modified antisense oligonucleotides specifically designed to knockdown gne, into one-cell embryos lead to a variety of phenotypic severity. Characterization of the gne knockdown morphants showed a significantly reduced locomotor activity as well as distorted muscle integrity, including a reduction in the number of muscle myofibers, even in mild or intermediate phenotype morphants. These findings were further confirmed by electron microscopy studies, where large gaps between sarcolemmas were visualized, although normal sarcomeric structures were maintained. These results demonstrate a critical novel role for gne in embryonic development and particularly in myofiber development, muscle integrity and activity.

Expression and functional characterization of Smyd1a in myofibril organization of skeletal muscles.

BACKGROUND: Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos. CONCLUSION/SIGNIFICANCE: Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.

Characterisation and expression of myogenesis regulatory factors during in vitro myoblast development and in vivo fasting in the gilthead sea bream (Sparus aurata).

The aim of this study was to characterise a primary cell culture isolated from fast skeletal muscle of the gilthead sea bream. Gene expression profiles during culture maturation were compared with those obtained from a fasting-refeeding model which is widely used to modulate myogenesis in vivo. Myogenesis is controlled by numerous extracellular signals together with intracellular transcriptional factors whose coordinated expression is critical for the appropriate development of muscle fibres. Full-length cDNAs for the transcription factors Myf5, Mrf4, Pax7 and Sox8 were cloned and sequenced for gilthead sea bream. Pax7, sox8, myod2 and myf5 levels were up-regulated during the proliferating phase of the myogenic cultures coincident with the highest expression of proliferating cell nuclear antigen (PCNA). In contrast, myogenin and mrf4 transcript abundance was highest during the differentiation phase of the culture when myotubes were present, and was correlated with increased myosin heavy chain (mhc) and desmin expression. In vivo, 30days of fasting resulted in muscle fibre atrophy, a reduction in myod2, myf5 and igf1 expression, lower number of Myod-positive cells, and decreased PCNA protein expression, whereas myogenin expression was not significantly affected. Myostatin1 (mstn1) and pax7 expression were up-regulated in fasted relative to well-fed individuals, consistent with a role for Pax7 in the reduction of myogenic cell activity with fasting. The primary cell cultures and fasting-feeding experiments described provide a foundation for the future investigations on the regulation of muscle growth in gilthead sea bream.

Smyd1b is required for skeletal and cardiac muscle function in zebrafish.

Smyd1b is a member of the Smyd family that is specifically expressed in skeletal and cardiac muscles. Smyd1b plays a key role in thick filament assembly during myofibrillogenesis in skeletal muscles of zebrafish embryos. To better characterize Smyd1b function and its mechanism of action in myofibrillogenesis, we analyzed the effects of smyd1b knockdown on myofibrillogenesis in skeletal and cardiac muscles of zebrafish embryos. The results show that knockdown of smyd1b causes significant disruption of myofibril organization in both skeletal and cardiac muscles of zebrafish embryos. Microarray and quantitative reverse transcription-PCR analyses show that knockdown of smyd1b up-regulates heat shock protein 90 (hsp90) and unc45b gene expression. Biochemical analysis reveals that Smyd1b can be coimmunoprecipitated with heat shock protein 90 α-1 and Unc45b, two myosin chaperones expressed in muscle cells. Consistent with its potential function in myosin folding and assembly, knockdown of smyd1b significantly reduces myosin protein accumulation without affecting mRNA expression. This likely results from increased myosin degradation involving unc45b overexpression. Together these data support the idea that Smyd1b may work together with myosin chaperones to control myosin folding, degradation, and assembly into sarcomeres during myofibrillogenesis.

Comparative proteomic analysis reveals differentially expressed proteins correlated with fuzz fiber initiation in diploid cotton (Gossypium arboreum L.).

In this study, a comparative proteomic analysis was employed to identify fuzz fiber initiation-related proteins in wild-type diploid cotton (Gossypium arboreum L.) and its fuzzless mutant. Temporal changes in global proteomes were examined using 2-DE at five developmental time points for fuzz fiber initiation, and 71 differentially expressed protein species were identified by MS, 45 of which were preferentially accumulated in the wild-type. These proteins were assigned to several functional categories, mainly in cell response/signal transduction, redox homeostasis, protein metabolism and energy/carbohydrate metabolism. It was remarkable that more than ten key proteins with high-abundance were involved in gibberellic acid (GA) signaling and ROS scavenging, and increasing concentrations of active GAs and H2O2 were also detected approximately 5dpa in wild type ovules. Furthermore, in vivo GA and H2O2 treatments of ovules inside young bolls showed that these compounds can synergistically promote fuzz fiber initiation. Our findings not only described a dynamic protein network supporting fuzz initiation in diploid cotton fiber ovules, but also deepened our understanding of the molecular basis of cotton fiber initiation. BIOLOGICAL SIGNIFICANCE: Our study reported the identification of differentially expressed proteins in wild-type diploid cotton (G. arboreum L.) and its fuzzless mutant by comparative proteomic approach. In total, 71 protein species related to fuzz initiation were identified by MS. These proteins were assigned to several functional categories, mainly in energy/carbohydrate metabolism, protein metabolism, signal transduction, redox homeostasis etc. Importantly, a number of key proteins were found to be associated with GA signaling and ROS scavenging. In consistence with these findings, we detected the increase of GAs and H2O2 concentrations during fiber initiation, and our in vivo ovule experiments with GA and H2O2 injection and following microscopy observation of fuzz fiber initiation supported promoting effects of GA and H2O2 on cotton fiber initiation. These findings depicted a dynamic protein network supporting cotton fiber initiation in diploid cotton ovules. Our study is of major significance for understanding the molecular mechanisms controlling fuzz initiation and also provides a solid basis for further functional research of single nodes of this network in relation to cotton fiber initiation.

Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

Endogenous microbiota play essential roles in the host's immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host's development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -ß, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

Phylogenetic studies of three sinipercid fishes (Perciformes: Sinipercidae) based on complete mitochondrial DNA sequences.

The sinipercids are a group of 12 species of freshwater percoid fish endemic to East Asia and their phylogenetic placements have perplexed generations of taxonomists. We cloned and sequenced the complete mitochondrial DNA (mtDNA) of three sinipercid fishes (Siniperca chuatsi, S. kneri, and S. scherzeri) to characterize and compare their mitochondrial genomes. The mitochondrial genomes of S. chuatsi, S. kneri, and S. scherzeri were 16,496, 17,002, and 16,585 bp in length, respectively. The organization of the three mitochondrial genomes is similar to those reported from other fish mitochondrial genomes, which contains 37 genes (13 protein-coding genes, 2 ribosomal RNAs, and 22 transfer RNAs) and a major non-coding control region. Among the 13 protein-coding genes of all the three sinipercid fishes, three reading-frame overlaps were found on the same strand. There is an 81-bp tandem repeat cluster at the end of CSB-3 in the S. scherzeri control region. The complete mitochondrial genomes of the three sinipercids should be useful for the evolutionary studies of sinipercids and other vertebrate species.

Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles.

Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.

Smyd1b_tv1, a key regulator of sarcomere assembly, is localized on the M-line of skeletal muscle fibers.

BACKGROUND: Smyd1b is a member of the Smyd family that plays a key role in sarcomere assembly during myofibrillogenesis. Smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles and play a vital role in myofibrillogenesis in skeletal muscles of zebrafish embryos. METHODOLOGY/PRINCIPAL FINDINGS: To better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in transgenic zebrafish expressing a myc-tagged Smyd1b_tv1 or Smyd1b_tv2. The results showed a dynamic change of their subcellular localization during muscle cell differentiation. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, in mature myofibers, Smyd1b_tv1, and to a small degree of Smyd1b_tv2, exhibited a sarcomeric localization. Double staining with sarcomeric markers revealed that Smyd1b_tv1was localized on the M-lines. The sarcomeric localization was confirmed in zebrafish embryos expressing the Smyd1b_tv1-GFP or Smyd1b_tv2-GFP fusion proteins. Compared with Smyd1b_tv1, Smyd1b_tv2, however, showed a weak sarcomeric localization. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion encoded by exon 5, suggesting that some residues within the 13 aa insertion may be critical for the strong sarcomeric localization of Smyd1b_tv1. Sequence comparison with Smyd1b_tv1 orthologs from other vertebrates revealed several highly conserved residues (Phe223, His224 and Gln226) and two potential phosphorylation sites (Thr221 and Ser225) within the 13 aa insertion. To determine whether these residues are involved in the increased sarcomeric localization of Smyd1b_tv1, we mutated these residues into alanine. Substitution of Phe223 or Ser225 with alanine significantly reduced the sarcomeric localization of Smyd1b_tv1. In contrast, other substitutions had no effect. Moreover, replacing Ser225 with threonine (S225T) retained the strong sarcomeric localization of Smyd1b_tv1. CONCLUSION/SIGNIFICANCE: Together, these data indicate that Phe223 and Ser225 are required for the M-line localization of Smyd1b_tv1.

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Loss of Smyhc1 or Hsp90alpha1 function results in different effects on myofibril organization in skeletal muscles of zebrafish embryos.

BACKGROUND: Myofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90alpha1 (Hsp90alpha1) has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90alpha1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90alpha1 function or indirectly through the disorganization of myosin thick filaments. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide) in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1) resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90alpha1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines. CONCLUSION/SIGNIFICANCE: Together, these studies indicate that the hsp90alpha1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90alpha1 may play a role in the assembly and organization of other sarcomeric structures.