The InteractiveFly: Drosophila as a Model for Human Diseases


Rhabdomyosarcoma
Reviews | PubMed | Wikipedia | More in IF

Galindo, K.A., Endicott, T.R., Avirneni-Vadlamudi, U. and Galindo, R.L. (2014). A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets. G3 (Bethesda) 5: 205-217. PubMed ID: PubMed ID: 25491943

Abstract
Rhabdomyosarcoma (RMS) is an aggressive childhood malignancy of neoplastic muscle-lineage precursors that fail to terminally differentiate into syncytial muscle. The most aggressive form of RMS, alveolar-RMS, is driven by misexpression of the PAX-FOXO1 oncoprotein, which is generated by recurrent chromosomal translocations that fuse either the PAX3 or PAX7 gene to FOXO1. The molecular underpinnings of PAX-FOXO1−mediated RMS pathogenesis remain unclear, however, and clinical outcomes poor. This study reports a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation. With this system, a comprehensive deletion screen against the Drosophila autosomes was performed and it was demonstrated that mutation of Mef2, a myogenesis lynchpin in both flies and mammals, dominantly suppresses PAX7-FOXO1 pathogenicity and acts as a PAX7-FOXO1 gene target. Additionally, mutation of mastermind (mam), a gene encoding a MEF2 transcriptional coactivator, similarly suppresses PAX7-FOXO1, further pointing toward MEF2 transcriptional activity as a PAX-FOXO1 underpinning. These results show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease (Galindo, 2014).

Highlights

• PAX7-FOXO1 drives ectopic myogenesis in Drosophila embryos.
• A rapid, one-generation screen for PAX7-FOXO1 suppressors and enhancers.
• MEF2 as a PAX-FOXO gene target and a putative RMS effector.

Discussion
Given the critical role that the PAX-FOXO1 fusion oncoprotein plays in RMS, this study focuses on PAX-FOXO1 as an entry-point for designing a transgenic Drosophila RMS-related model that would be amenable to forward genetic screening and RMS gene discovery. To bypass the issue of cumbersome multigenerational screening schemes that would normally be required, a Gal80 X-linked chromosomal transgene was incorporated to generate a viable screening Gal4/UAS-PAX-FOXO1 master stock that allows for the rapid identification of PAX-FOXO1 genetic modifiers in a single genetic cross (Galindo, 2014).

With this platform, new PAX-FOXO1 pathogenesis underpinnings were probed. Though very similar in molecular structure, PAX3-FOXO1− and PAX7-FOXO1−positive RMS demonstrate differing clinical behaviors, as PAX3-FOXO1 tumors are more common and notoriously aggressive. Consequently, PAX3-FOXO1 is the PAX-FOXO1 fusion most commonly investigated in vertebrate models. This study focuses on PAX7-FOXO1 in the Drosophila system, which demonstrates phenotypes that are better penetrant and experimentally tractable due to the fact that human PAX7 demonstrates slightly greater sequence identity to fly PAX3/7 than does human PAX3. Additionally, as no other animal models of PAX7-FOXO1 presently exist, the fly PAX7-FOXO1 model also conveniently serves as a complement to vertebrate PAX3-FOXO1 models (Galindo, 2014).

The extent to which observations from the PAX7-FOXO1 fly model would impact the clinically more aggressive PAX3-FOXO1 RMS subtype, as well as PAX-FOXO1-negative (embryonal) RMS, is unknown. Notably, previous studies show that genetic modifiers identified from the Drosophila system impact PAX3-FOXO1 RMS oncogenesis and tumorigenesis. Furthermore, unpublished studies suggest that fly PAX7-FOXO1 genetic modifiers are similarly involved in Embryonal RMS. These findings provide marked validation for the applicability and value of this genetic fly system to human RMS (Galindo, 2014).

Interestingly, though PAX7-FOXO1 induces expression of the late myogenic differentiation marker MHC, PAX-FOXO1 RMS myoblasts in culture and in vivo demonstrate only partial differentiation with little-to-no MHC expression. In considering this discrepancy, it should be first noted that PAX-FOXO1 is a relatively weak driver of RMS in culture and in vivo and requires additional/sequential genetic aberrations to induce oncogenic transformation. Thus, secondary mutations might be necessary to force the strength of RMS myoblast differentiation-arrest seen in human RMS tumors; by contrast, the PAX7-FOXO1 model of this study differs in that the system is free of any additional background mutations. Second, earlier studies show that expression of PAX3-FOXO1 in mouse embryonic cultured cells induces the formation of MHC-positive myocytes and myotube, similar to the Drosophila system in this study as the da-Gal4/UAS-PAX7-FOXO1 expression system targets undifferentiated embryonic primordia. Uncovering of the genetic/molecular sequence of RMS pathogenesis and the cell(s) origin will shed further insight into the underlying mechanisms that account for the myoblast differentiation arrest phenotypes seen in RMS in vivo (Galindo, 2014).

The differentiation and fusion of myoblasts into postmitotic, syncytial muscle requires that the bHLH myogenic regulatory factors (MRFs: Myf5, Mrf4, MyoD, and Myogenin) interact with E-proteins, which drive and regulate critical aspects of myogenic fate determination. The MRFs subsequently interact with the MEF2 transcription factors that, although lacking intrinsic myogenic activity, cooperate with the MRFs to synergistically activate muscle-specific genes and the downstream myogenic terminal differentiation program. Vertebrates possess four MEF2 family member genes (-A, -B, -C, -D), which demonstrate complex overlapping spatial and temporal expression patterns in embryonic and adult tissues, with greatest expression levels seen in striated muscle and brain. Because of genetic redundancy and overlapping expression patterns of the MEF2 genes, interrogating individual MEF2 gene activity in mammals is experimentally challenging, with loss-of-function mutation studies revealing only limited insights into MEF2 gene function in tissues in which the MEF2 genes do not overlap/compensate. Conveniently, flies possess only one Mef2 gene (D-Mef2) and serve as an excellent model system to delineate MEF2’s critical role in myogenesis. The study speculates that the lack of Mef2 redundancy in flies provides a marked experimental advantage in isolating D-Mef2 as a PAX7-FOXO1 effector. Similarly, the identification of mam was also likely facilitated by the fact that flies possess one mam gene, whereas mammals contain three mam orthologs. Thus, the study proposes that the comparative lack of genetic compensation/redundancy is an attractive advantage to Drosophila as a disease model system (Galindo, 2014).

The study suggests that further interrogation of MEF2 in RMS will open new avenues for RMS chemotherapy, which for high-risk disease has not improved for decades. For example, since MEF2 activity is tightly governed by class IIa histone deacetylases, histone deacetylase inhibitors are now ripe for preclinical testing as new anti-RMS agents. Additionally, it was found that the MEF2 cofactor Mastermind, which interacts with MEF2C and mediates crosstalk between Notch signals during myogenic differentiation, similarly influences PAX-FOXO1 pathogenicity in flies. Interestingly, Mastermind-specific, cell-permeable peptide inhibitors have been shown to block the progression of T-cell acute lymphoblastic leukemia in mice in vivo and thus are also new agents available for RMS preclinical testing. Further characterization of MEF2 in RMS cell and mouse models will continue to refine both our understanding and the potential targeting of MEF2 activity in RMS (Galindo, 2014).

In conclusion, the study postulates that: 1) The Drosophila PAX7-FOXO1 model is uniquely configured for the quick uncovering of new RMS genetic effectors with one simple genetic screening cross; 2) a putative PAX-FOXO1-to-MEF2/MASTERMIND axis underlies A-RMS; and 3) Drosophila conditional expression models are an efficient and powerful gene discovery platform for the rapid dissection of human disease (Galindo, 2014).

Go to top

Rodrigues, A., MacQuarrie, K. L., Freeman, E., Lin, A., Willis, A. B., Xu, Z., Alvarez, A. A., Ma, Y., White, B. E. P., Foltz, D. R. and Huang, S. (2023). Nucleoli and the nucleoli-centromere association are dynamic during normal development and in cancer. Mol Biol Cell 34(4): br5. PubMed ID: 36753381

Abstract

Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, this study examined nucleolar structure and centromeres at various differentiation stages using cell culture models, and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer (Rodrigues, 2023).

Go to top

Crose, L.E., Galindo, K.A., Kephart, J.G., Chen, C., Fitamant, J., Bardeesy, N., Bentley, R.C., Galindo, R.L., Chi, J.T. and Linardic, C.M. (2014). Alveolar rhabdomyosarcoma-associated PAX3-FOXO1 promotes tumorigenesis via Hippo pathway suppression. J Clin Invest 124: 285-296. PubMed ID: 24334454

Abstract
Alveolar rhabdomyosarcoma (aRMS) is an aggressive sarcoma of skeletal muscle characterized by expression of the paired box 3-forkhead box protein O1 (PAX3-FOXO1) fusion oncogene. Despite its discovery nearly two decades ago, the mechanisms by which PAX3-FOXO1 drives tumor development are not well characterized. Previously, it was reported that PAX3-FOXO1 supports aRMS initiation by enabling bypass of cellular senescence checkpoints. This study shows that this bypass occurs in part through PAX3-FOXO1–mediated upregulation of RASSF4, a Ras-association domain family (RASSF) member. RASSF4 expression is upregulated in PAX3-FOXO1–positive aRMS cell lines and tumors. Enhanced RASSF4 expression promotes cell cycle progression, senescence evasion, and tumorigenesis through inhibition of the Hippo pathway tumor suppressor MST1. It was also found that the downstream Hippo pathway target Yes-associated protein 1 (YAP), which is ordinarily restrained by Hippo signaling, is upregulated in RMS tumors. These data suggest that Hippo pathway dysfunction promotes RMS. This study provides evidence for Hippo pathway suppression in aRMS and demonstrates a pro-growth role for RASSF4. Additionally, the study also identifies a mechanism used by PAX3-FOXO1 to inhibit MST1 signaling and promote tumorigenesis in aRMS (Crose, 2014).

Highlights

• PAX3-FOXO1 promotes transcriptional changes in primary human myoblasts.
• RASSF4 is upregulated in PAX3-FOXO1–positive aRMS cells and tumors.
• dRASSF mutation dominantly suppresses PAX-FOXO1 pathogenicity in a Drosophila model of aRMS.
• PAX3-FOXO1 directly regulates a RASSF4 5' enhancer.
• RASSF4 inhibits cellular senescence in PAX3-FOXO1–expressing HSMMs.
• Hippo signaling is suppressed in aRMS.

Discussion
As an independent means of examining the relationship between PAX3-FOXO1 and RASSF4, this study took advantage of a Drosophila model of aRMS pathogenicity. In this model, misexpression of PAX3/7-FOXO1 in differentiating muscle causes myoblast fusion defects that result in larval lethality, manifested as only about 10% of F1 adults being composed of PAX7-FOXO1 adults. Although tumorigenesis is not detectable due to lethality, the misfused PAX3/7-FOXO1 myogenic cells act aggressively to infiltrate nonmuscle tissue. Forward genetic screening for PAX-FOXO1 enhancers/suppressors can then be used to identify novel PAX3/7-FOXO1 gene targets and effectors (Crose, 2014).

Using this Drosophila model, the expression data for transcriptional changes promoted by PAX7-FOXO1 was inspected. (Human PAX7 demonstrates a slightly higher sequence identity with Drosophila PAX3/7 than does human PAX3 and is used in this study.) It was found that dRASSF mRNA overexpresses 2.6-fold in PAX7-FOXO1 larval muscle, suggesting that similarly to the human myoblast model, this Drosophila model of aRMS shows upregulated RASSF. Next, it was examined whether dRASSF promotes PAX-FOXO1 phenotypes by testing the effect of Drosophila mutants that alter dRASSF levels or function. A Drosophila chromosomal deletion, Df(3R)Exel6193, that dominantly suppresses PAX7-FOXO1 semilethality was used. Interestingly, Df(3R)Exel6193 removed segments 94D3-94E4 on chromosome 3, which includes dRASSF. It was hypothesized that heterozygous deletion of dRASSF might account for Df(3R)Exel6193-mediated PAX7-FOXO1 suppression and that, consistent with PAX3-FOXO1 activation of RASSF4 in mammalian myoblasts, dRASSF might act as a PAX3/7-FOXO1 downstream target and effector. Next, two publicly available transposable-element insertion loss-of-function dRASSF alleles, DG30608 and A531, were also tested for suppression of PAX7-FOXO1. The DG30608 element inserts into the 5′UTR, which does not alter DRASSF protein structure but instead presumably alters dRASSF expression. The A531 insertion disrupts exon 5, resulting in a truncated dRASSF lacking the C-terminal SARAH domain, which mediates RASSF-MST1 physical association. Both dRASSF loss-of-function alleles were found to ameliorate PAX-FOXO1 pathogenicity, with A531 acting as the stronger suppressor. These data show that dRASSF and RASSF4 promote PAX-FOXO1 phenotypes in both Drosophila and mammalian models of aRMS (Crose, 2014).

Although the progrowth function of RASSF4 in aRMS found in this study may be distinct from the presumed canonical role of RASSFs in cancer, it does resolve a conflict between RASSF function in mammals versus Drosophila. While there are 10 RASSF genes in human cells, the Drosophila genome contains only two orthologs, known as dRASSF and dRASSF8. Of these genes, dRASSF is most similar to the C-terminal RASSF1-6, and most similar to RASSF4 within the Ras-association and SARAH domains. In contrast to the majority of mammalian RASSFs, dRASSF promotes cell proliferation and inhibits Hippo pathway activation through recruitment of phosphatases or competition with the SAV1 ortholog Salvador. While RASSF4 and DRASSF can inhibit Hippo signaling, rescue experiments for the RASSF4-knockdown phenotype with dRASSF expression were not successful in this study. Therefore, RASSF4 is likely not a true ortholog of dRASSF. However, the observations in this study on the effects of RASSF4 on aRMS may reflect one evolutionarily conserved role of RASSFs in cell proliferation (Crose, 2014).

Go to top

Avirneni-Vadlamudi, U., Galindo, K.A., Endicott, T.R., Paulson, V., Cameron, S. and Galindo, R.L. (2012). Drosophila and mammalian models uncover a role for the myoblast fusion gene TANC1 in rhabdomyosarcoma. J Clin Invest 122: 403-407. PubMed ID: 22182840

Abstract
Rhabdomyosarcoma (RMS) is a malignancy of muscle myoblasts, which fail to exit the cell cycle, resist terminal differentiation, and are blocked from fusing into syncytial skeletal muscle. In some patients, RMS is caused by a translocation that generates the fusion oncoprotein PAX-FOXO1, but the underlying RMS pathogenetic mechanisms that impede differentiation and promote neoplastic transformation remain unclear. Using a Drosophila model of PAX-FOXO1–mediated transformation, this study shows that mutation in the myoblast fusion gene rolling pebbles (rols) dominantly suppresses PAX-FOXO1 lethality. Further analysis indicates that PAX-FOXO1 expression causes upregulation of rols, which suggests that Rols acts downstream of PAX-FOXO1. In mammalian myoblasts, gene silencing of Tanc1, an ortholog of rols, reveals that it is essential for myoblast fusion, but is dispensable for terminal differentiation. Misexpression of PAX-FOXO1 in myoblasts upregulates Tanc1 and blocks differentiation, whereas subsequent reduction of Tanc1 expression to native levels by RNAi restorrs both fusion and differentiation. Furthermore, decreasing human TANC1 gene expression causes RMS cancer cells to lose their neoplastic state, undergo fusion, and form differentiated syncytial muscle. Taken together, these findings identify misregulated myoblast fusion caused by ectopic TANC1 expression as a RMS neoplasia mechanism and suggest fusion molecules as candidates for targeted RMS therapy (Avirneni-Vadlamudi, 2012).

Highlights

• PAX7-FOXO1 induces Rols misexpression in Drosophila.
• Tanc1 is essential for myoblast fusion, but not for myogenic differentiation.
• TANC1 silencing rescues differentiation arrest and failed fusion of PAX3-FOXO1 cells and marks RMS tumor cells.

Discussion
This study uses a Drosophila chromosomal deletion, Df(3L)vin5, that dominantly suppresses PAX7-FOXO1–induced lethality. Human PAX7 demonstrates slightly higher sequence identity to Drosophila PAX3/7 than does human PAX3 and is therefore used in flies in this study. Df(3L)vin5 deletes segments 68A2–69A1 on chromosome 3, which includes the muscle-patterning gene rols, located at 68F1. rols encodes an essential adaptor molecule that links the Kirre transmembrane receptor with the machinery that drives myoblast cell-cell fusion and syncytial muscle formation; therefore, rols expression in the somatic mesoderm temporally coincides precisely with embryonic myoblast fusion. However, it was found by mRNA expression profiling that rols is misexpressed in PAX7-FOXO1 larval muscle. Thus, the study hypothesizes that heterozygous deletion of the rols locus might account for Df(3L)vin5-mediated PAX7-FOXO1 suppression and that rols might act as a PAX7-FOXO1 target gene (Avirneni-Vadlamudi, 2012).

Of the 2 alternative transcripts expressed from the rols locus, only one of which is expressed in myoblasts; expression of the second is restricted to endodermal/ectodermal precursors. In this study, 2 rols homozygous-lethal, P-element insertion loss-of-function alleles, P1027 and P1729, were tested for suppression of PAX7-FOXO1. Of these 2 alleles, only the P1729 insertion disrupts expression of the myoblast rols transcript (myoblast expression of rols is unperturbed in P1027); accordingly, only the rolsP1729 allele suppresses PAX7-FOXO1–induced lethality and muscle pathogenicity (Avirneni-Vadlamudi, 2012).

To investigate whether rols acts as a downstream PAX-FOXO1 target, the daughterless-Gal4 transgene was used to drive ubiquitous embryonic expression of UAS-PAX7-FOXO1 and probed for Rols misexpression. Since native Rols expression initiates at embryonic stage 11, the study focused only on embryos stage 10 or earlier. Diffuse expression of PAX7-FOXO1 and Rols is observed in blastoderm (stage 4–5) embryos, which consist of uncommitted precursor cells, and expression persists in all examined cells — including nonmyogenic ectodermal and endodermal cells — of gastrulated (stage 9–10) embryos. Taken together, these Drosophila studies reveal that rols acts as a PAX7-FOXO1 downstream target gene, direct or indirect, and as a bona fide genetic effector (Avirneni-Vadlamudi, 2012). 

RMS model systems conveniently promote insights into not only neoplasia, but also muscle development. Although ultrastructural studies suggest that myoblast fusion biology is conserved, few of the Drosophila fusigenic genes have been identified as essential in mammals, and none of these are from the founder subfamily. As the name implies, founder myoblasts are seminal to Drosophila myogenesis, uniquely dictating the location and physiology of each individual muscle. With rols and Tanc1, it was shown that founder gene function is conserved in mammals and, furthermore, participates in human disease. How founder gene activity influences other forms of neuromuscular disease now becomes an intriguing issue (Avirneni-Vadlamudi, 2012). 

Genetic screening in a Drosophila model and loss-of-function/gain-of-function studies in mammalian platforms have collaboratively uncovered a PAX-FOXO1-to-TANC1 neoplasia axis, a finding that the study suggests to be novel. Results from this study also argue that the relationship between myogenesis transcription factor (e.g., MyoD) signaling and myoblast fusion genes is intricate. In the presence of altered fusion potential, both Drosophila and mammalian myoblasts transition to differentiated myocytes, which suggests that later aspects of myogenesis signaling must uncouple from the TANC1 fusigenic pathway. Yet correcting PAX-FOXO1–mediated overexpression of rols/TANC1 rescues PAX-FOXO1–induced differentiation and arrest. These results intimate that correction of the TANC1 fusigenic axis feeds back to and rescues PAX-FOXO1–mediated misregulation of myogenic signaling, raising fascinating questions regarding the mechanisms by which this occurs. The observation in this study that PAX3-FOXO1 protein levels remain unchanged in TANC1-silenced cells argues that rescue does not originate from decreased expression of PAX3-FOXO1 from the PAX3 promoter. Thus, the study speculates that rescue occurs epistatically downstream of PAX3-FOXO1 (Avirneni-Vadlamudi, 2012).

Go to top

Galindo, R.L., Allport, J.A. and Olson, E.N. (2006). A Drosophila model of the rhabdomyosarcoma initiator PAX7-FKHR. Proc Natl Acad Sci U S A 103: 13439-13444. PubMed ID: 16938866 

Abstract
Alveolar rhabdomyosarcoma (ARMS) is an aggressive myogenic-type tumor and a gain-of-function disease, caused by misexpression of the PAX3-FKHR or PAX7-FKHR fusion oncoprotein from structurally rearranged chromosomes. PAX3-FKHR misexpression in terminally differentiating mouse myofibers can cause rhabdomyosarcoma at a low frequency, suggesting that skeletal muscle is an ARMS tissue of origin. Because patterned muscle is widely viewed as irreversibly syncytial, questions persist, however, regarding this potential pathogenetic mechanism for ARMS tumor initiation. To further explore this issue, this study generated transgenic Drosophila lines that conditionally express human PAX-FKHR. It was shown that PAX7-FKHR causes nucleated cells to form and separate from syncytial myofibers, which then spread to nonmuscular tissue compartments, including the central nervous system, and that wild-type PAX3 demonstrates similar potential. Further, Ras, which is known to interfere with the differentiation of myogenic cells, genetically interacts with PAX7-FKHR: constitutively activated Ras enhances PAX7-FKHR phenotypes, whereas loss-of-function ras alleles dominantly suppress PAX7-FKHR activity, including rescue of lethality. These results show that PAX-FKHR can drive the generation of discrete nucleated cells from differentiated myofibers in vivo, argue for syncytial muscle as an ARMS tissue of origin, and demonstrate that Drosophila provides a powerful system to screen for genetic modifiers of PAX-FKHR (Galindo, 2006).

Highlights

• Targeted expression of PAX7-FKHR in Drosophila causes muscular phenotypes.
• PAX7-FKHR generates nucleated cells from syncytial myofibers.
• Liberated PAX7-FKHR myogenic cells demonstrate invasive behavior.
• Ras is a PAX7-FKHR genetic modifier.

Discussion
This study used the fruit fly Drosophila melanogaster as a model organism to explore the pathogenicity of the ARMS initiator PAX7-FKHR. It was shown that (i) PAX7-FKHR, when expressed in differentiated muscle, causes discrete nucleated cells to form from synyctial myofibers, (ii) these cells, freed from myofiber attachment, spread to the CNS, (iii) these properties are not unique to the PAX7-FKHR chimera, as human wild-type PAX3 demonstrates similar activity in fly muscle, and (iv) activated Ras enhances and diminished Ras activity suppresses the PAX7-FKHR muscular phenotype and associated lethality (Galindo, 2006).

Despite intensive study, the tissue of origin for ARMS has been puzzling. Because skeletal muscle is irreversibly postmitotic and syncytial, the origin has long been hypothesized to be a muscle precursor cell or stem-like cell. Yet, transgenic expression of PAX3-FKHR in muscle precursor cells or muscle-specific satellite stem cells demonstrates no evidence of tumorigenesis, whereas expression of PAX3-FKHR in terminal differentiating myofibers causes rhabdomyosarcomagenesis. Because no evidence exists, either in cell culture or in vivo, that PAX-FKHR can induce individual cells to form de novo from synytial tissue, questions persist regarding whether the mouse model has undetectably generated tumors from an unknown cell. Because most PAX3-FKHR mice in the ARMS model do not grow tumors, this study predicted that exhaustively surveying mouse muscle for focal or subtle cellular changes would be difficult. In contrast, it was postulated that Drosophila would provide a practical approach for this type of study, given the amenability of the organism to rapid, thorough, serial examination of living muscle. Additionally, because fly muscle contains no known mechanism for repair (including satellite cells), it was predicted that muscle dysmorphology would not be obfuscated by physiologic regeneration. With this approach, this study was able to document evidence of cells generating de novo from syncytial tissue. These results show that PAX-FKHR can specify this process and that cells can form from differentiated muscle in vivo. Thus, in a complementary fashion, the ARMS mouse and PAX7-FKHR fly strongly argue that muscle can serve as a RMS tissue of origin (Galindo, 2006).

How might PAX-FKHR cause muscle “dedifferentiation?” Because both wild-type PAX and PAX-FKHR demonstrate activity in fly muscle and, therefore, presumably share gene targets and cofactors, wild-type PAX can be used for providing mechanistic insights. Specifically, wild-type PAX performs at least two distinct developmental functions: (i) PAX proteins regulate early organogenesis and lineage determination; and (ii) PAX functions late in lineage development to repress the terminal differentiation of tissue-specific precursors. For example, in melanocyte development, PAX3 inhibits the terminal differentiation of maturing melanoblasts, thereby maintaining a population of partially differentiated precursors available for damage response. PAX7 likewise inhibits late steps in the terminal differentiation of muscle satellite cells, where PAX7 and myogenin expression are mutually exclusive, and overexpression of PAX7 interferes with MyoD-dependent transcriptional activation and down-regulates MyoD expression. PAX5 has also been characterized as a negative modulator of plasma cell terminal differentiation. This study proposes that PAX-FKHR, upon chromosomal rearrangement and misexpression, reacquires its inhibitory activity on terminal differentiation and interferes with the integrity of differentiated myofibers (Galindo, 2006).

For ARMS tumorigenesis to originate from postmitotic muscle, newly generated PAX-FKHR cells would need to re-enter the cell cycle to proliferate. The fact that PAX3-FKHR in the ARMS mouse model requires loss of cell-cycle regulation for appreciable levels of tumorigenesis argues that PAX3-FKHR alone is not sufficient to cause cell-cycle reentry. Consistent with this observation, this study did not find evidence of cell-cycle reentry in myofiber nuclei or individual GFP-positive myogenic cells in either PAX7-FKHR or PAX7-FKHR, RasV12 animals after staining with phosphohistone H3 antibody. These results are similar to earlier findings that activated Ras does not promote proliferation of either mammalian or fly myogenic precursor cells. Genetic gain-of-function and loss-of-function ras experiments in this study suggest, nonetheless, that Ras signaling is involved in PAX7-FKHR-mediated dedifferentiation of muscle. In this regard, it is noteworthy that constitutively activated Ras strongly interferes with the terminal differentiation of mammalian myoblasts, whereas ras gain-of-function or loss-of-function alters the differentiation of Drosophila founder cell myoblasts, the patterning pioneers of fly muscle. Therefore, it is possible that the interaction observed between PAX7-FKHR and Ras results not from proliferation but from the fact that both molecules functionally perturb muscle differentiation (Galindo, 2006).

Outcomes for advanced ARMS remain dismal despite intensive therapy, underscoring the need to characterize the pathogenetic mechanisms underlying tumorigenesis. As presented in this study, the PAX7-FKHR Drosophila phenotypes are sensitive to both genetic suppression and enhancement. Therefore, this PAX-FKHR model is well situated for unbiased genetic screens. This approach, unexplored with regards to PAX-FKHR, should allow for the isolation of previously unknown PAX-FKHR gene targets and cofactors. Uncovering these molecular entities should represent a starting point for the conceptual development of therapeutics to target these interactions and poison ARMS (Galindo, 2006).

Go to top

Rodrigues, A., MacQuarrie, K. L., Freeman, E., Lin, A., Willis, A. B., Xu, Z., Alvarez, A. A., Ma, Y., White, B. E. P., Foltz, D. R. and Huang, S. (2023). Nucleoli and the nucleoli-centromere association are dynamic during normal development and in cancer. Mol Biol Cell 34(4): br5. PubMed ID: 36753381

Abstract

Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, this study examined nucleolar structure and centromeres at various differentiation stages using cell culture models, and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.

Go to top

Reviews

Go to top

More in IF

daughterless homologs, myogenesis and rhabdomyosarcoma 

Pax3 in rhabdomyosarcoma

Back to Drosophila as a Model for Human Diseases

Date revised: 20 June 2015

Home page: The Interactive Fly © 2015 Thomas Brody, Ph.D.

The Interactive Fly resides on the web server of the Society for Developmental Biology