Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

South American Gymnotiform knifefish possess electric organs that generate electric fields for electro-location and electro-communication. Electric organs in fish can be derived from either myogenic cells (myogenic electric organ/mEO) or neurogenic cells (neurogenic electric organ/nEO). To date, the embryonic development of EOs has remained obscure. Here we characterize the development of the mEO in the Gymnotiform bluntnose knifefish, Brachyhypopomus gauderio. We find that EO primordial cells arise during embryonic stages in the ventral edge of the tail myotome, translocate into the ventral fin and develop into syncytial electrocytes at early larval stages. We also describe a pair of thick nerve cords that flank the dorsal aorta, the location and characteristic morphology of which are reminiscent of the nEO in Apteronotid species, suggesting a common evolutionary origin of these tissues. Taken together, our findings reveal the embryonic origins of the mEO and provide a basis for elucidating the mechanisms of evolutionary diversification of electric charge generation by myogenic and neurogenic EOs.

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord.

Somitic cells give rise to a variety of cell types in response to Hh, BMP, and FGF signaling. Cell position within the developing zebrafish somite is highly dynamic: how, when, and where these signals specify cell fate is largely unknown. Combining four-dimensional imaging with pathway perturbations, we characterize the spatiotemporal specification and localization of somitic cells. Muscle formation is guided by highly orchestrated waves of cell specification. We find that FGF directly and indirectly controls the differentiation of fast and slow-twitch muscle lineages, respectively. FGF signaling imposes tight temporal control on Shh induction of slow muscles by regulating the time at which fast-twitch progenitors displace slow-twitch progenitors from contacting the Shh-secreting notochord. Further, we find a reciprocal regulation of fast and slow muscle differentiation, morphogenesis, and migration. In conclusion, robust cell fate determination in the developing somite requires precise spatiotemporal coordination between distinct cell lineages and signaling pathways. Precise spatiotemporal control of signaling pathway activation is crucial for cell fate control and tissue patterning. Yin et al. map somitic cell fates in the developing zebrafish with four-dimensional imaging. FGF signaling imposes tight temporal control on Shh induction of muscle pioneers by non-autonomously regulating migration timing of slow-twitch muscles.

Myosin 18B is an unconventional myosin that has been implicated in tumor progression in humans. In addition, loss-of-function mutations of the MYO18B gene have recently been identified in several patients exhibiting symptoms of nemaline myopathy. In mouse, mutation of Myo18B results in early developmental arrest associated with cardiomyopathy, precluding analysis of its effects on skeletal muscle development. The zebrafish, frozen (fro) mutant was identified as one of a group of immotile mutants in the 1996 Tübingen genetic screen. Mutant embryos display a loss of birefringency in their skeletal muscle, indicative of disrupted sarcomeric organization. Using meiotic mapping, we localized the fro locus to the previously unannotated zebrafish myo18b gene, the product of which shares close to 50% identity with its human ortholog. Transcription of myo18b is restricted to fast-twitch myocytes in the zebrafish embryo; consistent with this, fro mutant embryos exhibit defects specifically in their fast-twitch skeletal muscles. We show that sarcomeric assembly is blocked at an early stage in fro mutants, leading to the disorganized accumulation of actin, myosin, and α-actinin and a complete loss of myofibrillar organization in fast-twitch muscles.

BACKGROUND: the transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. METHODS: the GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. RESULTS: Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. CONCLUSIONS: the Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation in the zebrafish, a role similar to that ascribed to its murine ortholog.

Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5'-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny.

The myosin heavy chain gene, MYH(M)₇₄₃₋₂, is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5'-flanking region of MYH(M)₇₄₃₋₂ into fertilized eggs of zebrafish and medaka. Although the -2.1kb 5'-flanking region of torafugu MYH(M)₇₄₃₋₂ showed no homology with the corresponding regions of zebrafish and medaka orthologous genes on the rVISTA analysis, the torafugu 5'-flanking region activated the GFP expression which was detected in the myotomal compartment for both zebrafish and medaka embryos. The GFP expression was localized to fast and slow muscle fibers in larvae as revealed by immunohistochemical analysis. In addition to the above tissues, GFP was also expressed in jaw, eye and pectoral fin muscles in embryos and larvae. These results clearly demonstrated that the 2.1 kb 5'-flanking region of MYH(M)₇₄₃₋₂ contains essential cis-regulatory sequences for myogenesis that are conserved among torafugu, zebrafish and medaka.

Sox6 has been proposed to play a conserved role in vertebrate skeletal muscle fibre type specification. In zebrafish, sox6 transcription is repressed in slow-twitch progenitors by the Prdm1a transcription factor. Here we identify sox6 cis-regulatory sequences that drive fast-twitch-specific expression in a Prdm1a-dependent manner. We show that sox6 transcription subsequently becomes derepressed in slow-twitch fibres, whereas Sox6 protein remains restricted to fast-twitch fibres. We find that translational repression of sox6 is mediated by miR-499, the slow-twitch-specific expression of which is in turn controlled by Prdm1a, forming a regulatory loop that initiates and maintains the slow-twitch muscle lineage.

Comprehensive in silico studies, based on the total fugu genome database, which was the first to appear in fish, revealed that torafugu Takifugu rubripes contains 20 sarcomeric myosin heavy chain (MYH) genes (MYH genes) (Ikeda et al. 2007). The present study was undertaken to identify MYH genes that would be expressed in adult muscles. In total, seven MYH genes were found by screening cDNA clone libraries constructed from fast, slow and cardiac muscles. Three MYH genes, fast-type MYH(M86-1), slow-type MYH(M8248) and slow/cardiac-type MYH(M880), were cloned exclusively from fast, slow and cardiac muscles, respectively. Northern blot hybridization substantiated their specific expression, with the exception of MYH(M880). In contrast, transcripts of fast-type MYH(M2528-1) and MYH(M1034) were found in both fast and slow muscles as revealed by cDNA clone library and northern blot techniques. This result was supported by in situ hybridization analysis using specific RNA probes, where transcripts of fast-type MYH(M2528-1) were expressed in fast fibres with small diameters as well as in fibres of superficial slow muscle with large diameters adjacent to fast muscle. Transcripts of fast-type MYH(M86-1) were expressed in all fast fibres with different diameters, whereas transcripts of slow-type MYH(M8248) were restricted to fibres with small diameters located in a superficial part of slow muscle. Interestingly, histochemical analyses showed that fast fibres with small diameters and slow fibres with large diameters both contained acid-stable myofibrillar ATPase, suggesting that these fibres have similar functions, possibly in the generation of muscle fibres irrespective of their fibre types.

Three embryonic myosin heavy chain (MYH) genes >> (MYHs) including MYH(emb1), MYH(emb2) and MYH(emb3) and encoding a C-terminal part of MYH were previously cloned and demonstrated to be expressed transiently in this order during development of common carp Cyprinus carpio embryos. The present study determined the full-length cDNA nucleotide sequences encoding the motor domain of the three MYHs, suggesting the implication of loop 1 and loop 2 sequences for the differences in the motor functions. Phylogenetic analysis based on the full-length amino acid sequences showed that MYH(emb1) and MYH(emb2) both belong to the fast types, though clearly differ from fast-type MYHs expressed in adult fast muscle previously reported. In contrast, MYH(emb3) was in a clade containing slow/cardiac type. Whole-mount immunostaining and in situ hybridization showed that the transcripts of the three embryonic MYHs are localized in the same or different cranial muscles of common carp larvae, suggesting that the three MYHs function cooperatively or individually in various cranial muscles.

Myosin heavy chain genes (MYHs) are the most important functional domains of myosins, which are highly conserved throughout evolution. The human genome contains 15 MYHs, whereas the corresponding number in teleost appears to be much higher. Although teleosts comprise more than one-half of all vertebrate species, our knowledge of MYHs in teleosts is rather limited. A comprehensive analysis of the torafugu (Takifugu rubripes) genome database enabled us to detect at least 28 MYHs, almost twice as many as in humans. RT-PCR revealed that at least 16 torafugu MYH representatives (5 fast skeletal, 3 cardiac, 2 slow skeletal, 1 superfast, 2 smooth, and 3 nonmuscle types) are actually transcribed. Among these, MYH(M743-2) and MYH(M5) of fast and slow skeletal types, respectively, are expressed during development of torafugu embryos. Syntenic analysis reveals that torafugu fast skeletal MYHs are distributed across five genomic regions, three of which form clusters. Interestingly, while human fast skeletal MYHs form one cluster, its syntenic region in torafugu is duplicated, although each locus contains just a single MYH in torafugu. The results of the syntenic analysis were further confirmed by corresponding analysis of MYHs based on databases from Tetraodon, zebrafish, and medaka genomes. Phylogenetic analysis suggests that fast skeletal MYHs evolved independently in teleosts and tetrapods after fast skeletal MYHs had diverged from four ancestral MYHs.

Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYHemb1, MYHemb2 and MYHemb3. MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10 degrees C (MYHS10) and 30 degrees C (MYHS30). Phylogenetic analysis demonstrated that MYHemb1 and MYHemb2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYHemb3 was similar to the adult slow muscle isoforms, MYHS10 and MYHS30. MYHemb1 and MYHemb2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYHemb1 continued to be expressed at low levels in 7-month-old juveniles when MYHemb2 was not detectable. MYHemb3 transcripts appeared at almost the same stage as MYHemb1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYHemb1 and MYHemb2 were expressed in the inner part of myotome, whereas MYHemb3 was expressed in the superficial compartment. MYHS10 and MYHS30 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYHS10 mRNA was expressed at an acclimation temperature of 10 and 20 degrees C, but not at 30 degrees C. In contrast, MYHS30 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10 degrees C-acclimated adult fish (MYHcard). MYHcard mRNA was first detected at 61 h.p.f. but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.