Farewell to Professor David Yaffe - A pillar of the myogenesis field.

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel's War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a "David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe's earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".

Identification of an RNA-dependent RNA polymerase in Drosophila establishes a common theme in RNA silencing.

In lower eukaryotes, such as A. thaliana, C. elegans, S. pombe and N. crassa, RNA-dependent RNA polymerase (RdRP) is a required component of the RNA silencing pathway. Remarkably, even though robust RNA silencing occurs in Drosophila in response to exogenous dsRNA and siRNAs, no RdRP homolog has been identified in the Drosophila genome or in any other higher eukaryote characteristic of the known cellular RdRPs. We showed recently that the largest subunit of the Drosophila RNA polymerase II core elongator complex, called D-elp1, has RdRP activity capable of using unprimed or primed synthesis mechanisms to convert single stranded RNA templates into double stranded RNA (dsRNA) that can be cleaved by Dcr-2. Loss of D-elp1 inhibits both siRNA and dsRNA directed RNAi in S2 cells but does not affect micro RNA targeting. Transposon RNA levels also increase with the loss of D-elp1 while the corresponding endo siRNAs, critical for transposon suppression, are dramatically reduced and this is correlated with a reduction in transposon antisense RNA levels. D-elp1 associates tightly with Dicer-2, similar to the Dicer-RdRP interaction observed in lower eukaryotes. With the exception of S. cerevisiae, which lacks the RNAi machinery altogether, RdRP activity is conserved in the elp1 homologs from S. pombe to human. This commentary focuses on the importance and universality of RdRP in RNA silencing.

Identification of an RNA-dependent RNA polymerase in Drosophila involved in RNAi and transposon suppression.

Here, we show that recombinant Drosophila elp1 (D-elp1) produced in Sf9 cells or Escherichia coli, corresponding to the largest of the three subunits in the RNA polymerase II core elongator complex, has RNA-dependent RNA polymerase (RdRP) activity. D-elp1 is a noncanonical RdRP that can synthesize dsRNA from different ssRNA templates using either a primer-dependent or primer-independent initiation mechanism. Of the three core subunits, only D-elp1 depletion inhibits RNAi in S2 cells but does not affect micro RNA function. Furthermore, D-elp1 depletion results in increased steady state levels of representative transposon RNAs and a decrease in the corresponding transposon antisense transcripts and endo siRNAs. In contrast, although Dcr-2 depletion results in increased transposon RNA levels and a reduction in the corresponding endo siRNAs, there is no change in the transposon antisense RNA levels. In D-elp1 null third instar larvae transposon RNA levels are also increased and the corresponding transposon antisense RNAs are reduced. D-elp1 associates tightly with Dcr-2, similar to the Dicer-RdRP interaction observed in lower eukaryotes. These results identify an aspect of the RNAi pathway in Drosophila that suggest transposon derived endo siRNAs, critical for transposon suppression, are produced, in part, in a D-elp1 dependent step that converts transposon RNA into dsRNA that is subsequently processed by Dcr-2. The generality of this mechanism in genome defense and RNA silencing in higher eukaryotes is suggested.

A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis.

Satellite cells are skeletal muscle stem cells that provide myogenic progeny for myofiber growth and repair. Temporal expression of muscle regulatory factors (MRFs) and the paired box transcription factor Pax7 defines characteristic phases of proliferation (Pax7(+)/MyoD(+)/myogenin(-)) and differentiation (Pax7(-)/MyoD(+)/myogenin(+)) during myogenesis of satellite cells. Here, using bromodeoxyuridine (BrdU) labeling and triple immunodetection, we analyzed expression patterns of Pax7 and the MRFs MyoD, Myf5, or myogenin within S phase myoblasts prepared from posthatch chicken muscle. Essentially, all BrdU incorporation was restricted to Pax7(+) cells, of which the majority also expressed MyoD. The presence of a minor BrdU(+)/Pax7(+)/myogenin(+) population in proliferation stage cultures suggests that myogenin up-regulation is alone insufficient for terminal differentiation. Myf5 was detected strictly within Pax7(+) cells and decreased during S phase while MyoD presence persisted in cycling cells. This study provides novel data in support of a unique role for Myf5 during posthatch myogenesis.

Myostatin promotes the terminal differentiation of embryonic muscle progenitors.

Myostatin, a TGF-beta family member, is an important regulator of adult muscle size. While extensively studied in vitro, the mechanisms by which this molecule mediates its effect in vivo are poorly understood. We addressed this question using chick and mouse embryos. We show that while myostatin overexpression in chick leads to an exhaustion of the muscle progenitor population that ultimately results in muscle hypotrophy, myostatin loss of function in chick and mouse provokes an expansion of this population. Our data demonstrate that myostatin acts in vivo to regulate the balance between proliferation and differentiation of embryonic muscle progenitors by promoting their terminal differentiation through the activation of p21 and MyoD. Previous studies have suggested that myostatin imposes quiescence on muscle progenitors. Our data suggest that myostatin's effect on muscle progenitors is more complex than previously realized and is likely to be context-dependent. We propose a novel model for myostatin mode of action in vivo, in which myostatin affects the balance between proliferation and differentiation of embryonic muscle progenitors by enhancing their differentiation.

Drosophila RNA Interference (RNAi) Using a Gal-4 Inducible Transgene Vector.

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a method for RNAi in vivo using tissue-specific Gal-4 transgenes to induce dsRNA synthesis from an upstream activator sequence (UAS) vector. This vector contains the desired exonic inverted sequences representing the target gene (preferably more than 400 bp) separated by a unique spacer, the first intron of the actin 5C gene. The inverted repeats are stable during cloning in E. coli with this intronic spacer and the intron is spliced out to produce an almost perfect dsRNA target for Dicer cleavage and the production of siRNAs.

Preparation of Double-Stranded RNA for Drosophila RNA Interference (RNAi).

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. It has been used in our laboratory to phenocopy a series of known mutations in Drosophila, including twist, engrailed, daughterless, Dmef2, and, to a lesser extent, white in the adult eye. This protocol describes the preparation of dsRNA by in vitro transcription of complementary strands of a cloned DNA fragment that codes for all or a portion of the gene of interest, followed by annealing of the transcribed RNA.

Collection of Drosophila Embryos for RNA Interference (RNAi).

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a method for collection of Drosophila embryos for RNA interference (RNAi) experiments. The embryos are collected in a simple, homemade apparatus, arrayed on prepared glass slides, and readied for injection. It is important to keep the embryos moist and oxygenated. Work expeditiously, because embryos should be injected within 30-60 min after collection.

Injection of dsRNA into Drosophila Embryos for RNA Interference (RNAi).

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a technique by which Drosophila embryos can be injected with dsRNA in order to disrupt targeted gene function. The approach is straightforward, utilizing improved methods for injecting the dsRNA directly through the chorion of the embryo. This strategy minimizes problems normally associated with desiccation of the dechorionated embryo and facilitates post-injection analysis of gene expression.

Stereotypic founder cell patterning and embryonic muscle formation in Drosophila require nautilus (MyoD) gene function.

nautilus is the only MyoD-related gene in Drosophila. Nautilus expression begins around stage 9 at full germ-band extension in a subset of mesodermal cells organized in a stereotypic pattern in each hemisegment. The muscle founder cell marker Duf-LacZ, produced by the enhancer trap line rP298LacZ, is coexpressed in numerous Nautilus-positive cells when founders first appear. Founders entrain muscle identity through the restricted expression of transcription factors such as S59, eve, and Kr, all of which are observed in subsets of the nautilus expressing founders. We inactivated the nautilus gene using homology-directed gene targeting and Gal4/UAS regulated RNAi to determine whether loss of nautilus gene activity affected founder cell function. Both methods produced a range of defects that included embryonic muscle disruption, reduced viability and female sterility, which could be rescued by hsp70-nautilus cDNA transgenes. Our results demonstrate Nautilus expression marks early founders that give rise to diverse muscle groups in the embryo, and that nautilus gene activity is required to seed the correct founder myoblast pattern that prefigures the muscle fiber arrangement during embryonic development.

Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells.

The realization that short double-stranded RNA (dsRNAs) 21-25 bp in length represent the basis for posttranscriptional gene silencing (PTGS) in plants, quelling in N. crassa, and RNA interference (RNAi) in C. elegans and Drosophila has given insight into one of the most evolutionarily conserved pathways in eukaryotes. dsRNA that arises due to viral infection, transposon mobilization, random insertion of transgenes near active promoters, transcripts from repetitive elements in the genome, or introduction of exogenous dsRNA directly is processed by one of the RNase III-related enzymes, known as the Dicers, to produce 21- to 25-bp short dsRNAs or short interfering RNAs (siRNAs) that target the degradation of the cognate RNA sequence (Denli and Hannon, 2003; Hannon, 2002; Plasterk, 2002). Proteins in the RNAi pathway and siRNA-like RNAs have also been recently demonstrated to play a role in the formation and maintenance of heterochromatin in S. pombe as well as in transgene-induced PTGS in Drosophila (Hall et al., 2002; Pal-Bhadra et al., 2004; Volpe et al., 2002). An understanding of siRNA function in these crucial regulatory pathways requires biochemical approaches to study siRNAs and their role in gene silencing as well as the formation and maintenance of heterochromatin. This chapter describes simple methods for using Drosophila embryo extracts and cultured insect cells to study siRNA function in the RNAi pathway in vivo and in vitro. We describe the most recent protocols for the preparation and use of Drosophila embryo extracts used in gene targeting studies. We present methods we have used to assay siRNA function in Drosophila embryo extracts and in cultured SL2 cells that demonstrate a combined role for siRNAs and RNA-dependent RNA polymerase (RdRp) activity in Drosophila RNAi.

The Drosophila selenoprotein BthD is required for survival and has a role in salivary gland development.

Selenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability.

Analysis of the 3(')-hydroxyl group in Drosophila siRNA function.

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3'-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed "degradative PCR" and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3'-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.