A novel Drosophila, mef2-regulated muscle gene isolated in a subtractive hybridization-based molecular screen using small amounts of zygotic mutant RNA

It is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this differentiation process. I have therefore undertaken a novel molecular screen in Drosophila in a systematic search for such genes. The approach utilised subtractive hybridisation coupled to directional cDNA library construction. Libraries were made from as little as 20 microg total RNA isolated from hand-picked embryos of defined stage of development and genotype. In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA libraries, before subtracting twi from wild-type, increased the subtraction efficiency. It resulted in a cDNA population enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression pattern of each clone in a high throughput procedure and then DNA sequencing. The method, which is comprehensive and does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10 embryos. Analysis of one clone, Dmeso18E, that encodes a putative nuclear protein and fulfils the screen's aims is described. It is novel and its expression is mesoderm-specific, twi-dependent, and early during somatic, visceral, and heart muscle differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis of Dmeso18E expression revealed new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and genes like tin and Dmef2 or it lies in a parallel pathway of gene activation.     

WNT5 interacts with the Ryk receptors doughnut and derailed to mediate muscle attachment site selection in Drosophila melanogaster

In recent years a number of the genes that regulate muscle formation and maintenance in higher organisms have been identified. Studies employing invertebrate and vertebrate model organisms have revealed that many of the genes required for early mesoderm specification are highly conserved throughout evolution. Less is known about the molecules that mediate the steps subsequent to myogenesis, e. g. myotube guidance and attachment to tendon cells. We use the stereotypic pattern of the Drosophila embryonic body wall musculature in genetic approaches to identify novel factors required for muscle attachment site selection. Here, we show that Wnt5 is needed in this process. The lateral transverse muscles frequently overshoot their target attachment sites and stably attach at novel epidermal sites in Wnt5 mutant embryos. Restoration of WNT5 expression in either the muscle or the tendon cell rescues the mutant phenotype. Surprisingly, the novel attachment sites in Wnt5 mutants frequently do not express the Stripe (SR) protein which has been shown to be required for terminal tendon cell differentiation. A muscle bypass phenotype was previously reported for embryos lacking the WNT5 receptor Derailed (DRL). drl and Wnt5 mutant embryos also exhibit axon path finding errors. DRL belongs to the conserved Ryk receptor tyrosine kinase family which includes two other Drosophila orthologs, the Doughnut on 2 (DNT) and Derailed-2 (DRL-2) proteins. We generated a mutant allele of dnt and find that dnt, but not Drl-2, mutant embryos also show a muscle bypass phenotype. Genetic interaction experiments indicate that drl and dnt act together, likely as WNT5 receptors, to control muscle attachment site selection. These results extend previous findings that at least some of the molecular pathways that guide axons towards their targets are also employed for guidance of muscle fibers to their appropriate attachment sites.     

Isolation and characterization of the Xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata

Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated in many species representing a broad cross section of the major groups of living organisms, including the cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies.     

Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein.     

Expression and functional analysis of a novel Fusion Competent Myoblast specific GAL4 driver

In the Drosophila embryo, body wall muscles are formed by the fusion of two cell types, Founder Cells (FCs) and Fusion Competent Myoblasts (FCMs). Using an enhancer derived from the Dmef2 gene ([C/D]( *)), we report the first GAL4 driver specifically expressed in FCMs. We have determined that this GAL4 driver causes expression in a subset of FCMs and, upon fusion, in developing myotubes from stage 14 onwards. In addition, we have shown that using this Dmef2-5x[C/D]( *)-GAL4 driver to express dominant negative Rac in only FCMs causes a partial fusion block. This novel GAL4 driver will provide a useful reagent to study Drosophila myoblast fusion and muscle differentiation.     

Mef2s are required for thick filament formation in nascent muscle fibres

During skeletal muscle differentiation, the actomyosin motor is assembled into myofibrils, multiprotein machines that generate and transmit force to cell ends. How expression of muscle proteins is coordinated to build the myofibril is unknown. Here we show that zebrafish Mef2d and Mef2c proteins are required redundantly for assembly of myosin-containing thick filaments in nascent muscle fibres, but not for the earlier steps of skeletal muscle fibre differentiation, elongation, fusion or thin filament gene expression. mef2d mRNA and protein is present in myoblasts, whereas mef2c expression commences in muscle fibres. Knockdown of both Mef2s with antisense morpholino oligonucleotides or in mutant fish blocks muscle function and prevents sarcomere assembly. Cell transplantation and heat-shock-driven rescue reveal a cell-autonomous requirement for Mef2 within fibres. In nascent fibres, Mef2 drives expression of genes encoding thick, but not thin, filament proteins. Among genes analysed, myosin heavy and light chains and myosin-binding protein C require Mef2 for normal expression, whereas actin, tropomyosin and troponin do not. Our findings show that Mef2 controls skeletal muscle formation after terminal differentiation and define a new maturation step in vertebrate skeletal muscle development at which thick filament gene expression is controlled.     

The nesprins are giant actin-binding proteins,orthologous to Drosophila melanogaster muscle protein MSP-300

Nesprin-1 and nesprin-2 (also known as Syne-1 and Syne-2,) are large ( approximately 3300-residue) vertebrate proteins associated with emerin and lamin A at the nuclear envelope of muscle cells and other cell types. We show that the previously described nesprins are short isoforms of giant proteins comprising an actin-binding amino-terminus connected to a carboxy-terminal klarsicht-related transmembrane domain by a massive ( approximately 6000-8000 amino acid) spectrin-like rod domain, making full-length nesprin-1, at one megadalton, the largest non-titin protein hitherto described in humans. We find that MSP-300, a 7000-residue Drosophila melanogaster protein whose disruption results in defects of muscle development, corresponds to the N-terminal two-thirds of the Drosophila nesprin ortholog. A nesprin-like protein is also encoded by the nematode genome. Moreover, we demonstrate that the larger isoforms of nesprin-1, like MSP-300, are localized to the sarcomeric Z-line of both skeletal and cardiac muscle. The recognition that a characteristic muscle-specific mutant phenotype in the fly results from a disruption of its nesprin ortholog reinforces the candidacy of the human proteins for involvement in genetic diseases of skeletal and cardiac muscle.     

Even-skipped, acting as a repressor, regulates axonal projections in Drosophila

Nervous system-specific eve mutants were created by removing regulatory elements from a 16 kb transgene capable of complete rescue of normal eve function. When transgenes lacking the regulatory element for either RP2+a/pCC, EL or U/CQ neurons were placed in an eve-null background, eve expression was completely eliminated in the corresponding neurons, without affecting other aspects of eve expression. Many of these transgenic flies were able to survive to fertile adulthood. In the RP2+a/pCC mutant flies: (1) both RP2 and aCC showed abnormal axonal projection patterns, failing to innervate their normal target muscles; (2) the cell bodies of these neurons were positioned abnormally; and (3) in contrast to the wild type, pCC axons often crossed the midline. The Eve HD alone was able to provide a weak, partial rescue of the mutant phenotype, while both the Groucho-dependent and -independent repressor domains contributed equally to full rescue of each aspect of the mutant phenotype. Complete rescue was also obtained with a chimeric protein containing the Eve HD and the Engrailed repressor domain. Consistent with the apparent sufficiency of repressor function, a fusion protein between the Gal4 DNA-binding domain and Eve repressor domains was capable of actively repressing UAS target genes in these neurons. A key target of the repressor function of Eve was Drosophila Hb9, the derepression of which correlated with the mutant phenotype in individual eve-mutant neurons. Finally, homologues of Eve from diverse species were able to rescue the eve mutant phenotype, indicating conservation of both targeting and repression functions in the nervous system.     

Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton

Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca^b1086 loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal–ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.     

Pelota controls self-renewal of germline stem cells by repressing a Bam-independent differentiation pathway

In the Drosophila ovary, germline stem cell (GSC) self-renewal is controlled by both extrinsic and intrinsic factors. The Bmp signal from niche cells controls GSC self-renewal by directly repressing a Bam-dependent differentiation pathway in GSCs. pelota (pelo), which has been previously shown to be required for Drosophila male meiosis, was identified in our genetic screen as a dominant suppressor of the dpp overexpression-induced GSC tumor phenotype. In this study, we reveal the unexpected new role of Pelo in controlling GSC self-renewal by repressing a Bam-independent differentiation pathway. In pelo mutant ovaries, GSCs are lost rapidly owing to differentiation. Results from genetic mosaic analysis and germ cell-specific rescue show that it functions as an intrinsic factor to control GSC self-renewal. In pelo mutant GSCs, Bmp signaling activity detected by Dad-lacZ expression is downregulated, but bam expression is still repressed. Furthermore, bam mutant germ cells are still able to differentiate into cystocytes without pelo function, indicating that Pelo is involved in repressing a Bam-independent differentiation pathway. Consistent with its homology to the eukaryotic translation release factor 1alpha, we show that Pelo is localized to the cytoplasm of the GSC. Therefore, Pelo controls GSC self-renewal by repressing a Bam-independent differentiation pathway possibly through regulating translation. As Pelo is highly conserved from Drosophila to mammals, it may also be involved in the regulation of adult stem cell self-renewal in mammals, including humans.     

An interspecific functional complementation test in Drosophila for introductory genetics laboratory courses

Introductory genetics courses often include evolutionary genetics concepts such as sequence homology and functional conservation. It is usually assumed that two sequences showing homology (i.e., sharing a common ancestral sequence) perform the same molecular function. The correlation, however, does not always hold true, and evidence for functional conservation must come from functional studies. In this study we describe a genetics laboratory class that demonstrates functional conservation between the Drosophila protein Muscleblind (Mbl) and its human ortholog MBNL1. We use the Gal4/UAS system to express MBNL1 in a Drosophila mutant background and measure the in vivo activity of the human protein by rescue of mbl mutant phenotype in embryos. As a control, ubiquitous expression of Drosophila MblC, one of the four protein isoforms encoded by the gene, increased by 71% the viability of mbl mutant embryos and greatly reduced the hypercontracted abdomen of mutant larvae. In a parallel experiment, human MBNL1 provided a robust rescue of the embryonic lethality (78%) and improved abdomen hypercontraction as well. Under both conditions, rescued larvae die as first instars, probably due to overexpression effects, lack of alternative protein isoforms, or incomplete expression in critical tissues such as the nervous system. The use of two constructs in the rescue experiment (UAS-mblC and UAS-MBNL1) and the incomplete rescue prompt several questions for students. The fact that a human protein works in a Drosophila cellular context illustrates the use of an in vivo test to prove functional conservation.     

Myogenin is required for late but not early aspects of myogenesis during mouse development

Mice with a targeted mutation in the myogenic basic helix-loop-helix regulatory protein myogenin have severe muscle defects resulting in perinatal death. In this report, the effect of myogenin's absence on embryonic and fetal development is investigated. The initial events of somite differentiation occurred normally in the myogenin-mutant embryos. During primary myogenesis, muscle masses in mutant embryos developed simultaneously with control siblings, although muscle differentiation within the mutant muscle masses was delayed. More dramatic effects were observed when secondary myofibers form. During this time, very little muscle formation took place in the mutants, suggesting that the absence of myogenin affected secondary myogenesis more severely than primary myogenesis. Monitoring mutant neonates with fiber type-specific myosin isoforms indicated that different fiber types were present in the residual muscle. No evidence was found to indicate that myogenin was required for the formation of muscle in one region of the embryo and not another. The expression patterns of a MyoD- lacZ transgene in myogenin-mutant embryos demonstrated that myogenin was not essential for the activation of the MyoD gene. Together, these results indicate that late stages of embryogenesis are more dependent on myogenin than early stages, and that myogenin is not required for the initial aspects of myogenesis, including myotome formation and the appearance of myoblasts.     

aph-1 and pen-2 are required for Notch pathway signaling,gamma-secretase cleavage of betaAPP,and presenilin protein accumulation

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.