Roles of the troponin isoforms during indirect flight muscle development in Drosophila

Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, amajority of themyofibrillar proteins in the Indirect Flight Muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform

Beadex function in the motor neurons is essential for female reproduction in Drosophila melanogaster.

Drosophila melanogaster has served as an excellent model system for understanding the neuronal circuits and molecular mechanisms regulating complex behaviors. The Drosophila female reproductive circuits, in particular, are well studied and can be used as a tool to understand the role of novel genes in neuronal function in general and female reproduction in particular. In the present study, the role of Beadex, a transcription co-activator, in Drosophila female reproduction was assessed by generation of mutant and knock down studies. Null allele of Beadex was generated by transposase induced excision of P-element present within an intron of Beadex gene. The mutant showed highly compromised reproductive abilities as evaluated by reduced fecundity and fertility, abnormal oviposition and more importantly, the failure of sperm release from storage organs. However, no defect was found in the overall ovariole development. Tissue specific, targeted knock down of Beadex indicated that its function in neurons is important for efficient female reproduction, since its neuronal knock down led to compromised female reproductive abilities, similar to Beadex null females. Further, different neuronal class specific knock down studies revealed that Beadex function is required in motor neurons for normal fecundity and fertility of females. Thus, the present study attributes a novel and essential role for Beadex in female reproduction through neurons

Beadex, a Drosophila LIM domain only protein, function in follicle cells is essential for egg development and fertility

LIM domain, constituted by two tandem C2H2 zinc finger motif, proteins regulate several biological processes. They are usually found associated with various functional domains like Homeodomaini kinase domain and other protein binding domains. LIM proteins that are devoid of other domains are called LIM only proteins (LMO). LMO proteins were first identified in humans and are implicated in development and oncogenesis. They regulate various cell specifications by regulating the activity of respective transcriptional complexes. The Drosophila LMO protein (dLMO), Beadex (Bx), regulates various developmental processes like wing margin development and bristle development. It also regulates Drosophila behavior in response to cocaine and ethanol. We have previously generated Bx null flies and shown its essential function in neurons for multiple aspects of female reproduction. However, it was not known whether Bx affects reproduction through its independent function in ovaries. In this paper we show that female flies null for Bx lay eggs with multiple defects. Further, through knock down studies we demonstrate that function of Bx in follicle cells is required for normal egg development. We also show that function of Bx is particularly required in border cells for Drosophila fertility

Transcription factor erect wing (EWG) is involved in indirect flight muscle patterning, development and maintenance in Drosophila

Muscle development is a multistep process which includes myoblast diversification, proliferation, migration, fusion, differentiation and growth. A hierarchical exhibition of myogenic factors is important for dexterous execution of progressive events in muscle formation. EWG (erect wing) is a transcription factor known to have a role in indirect flight muscle development (IFM) in Drosophila. We marked out the precise spatio-temporal expression profile of EWG in the myoblasts, and in the developing muscles. Mutant adult flies null for EWG in myoblasts show variable number of IFM, suggesting that EWG is required for patterning of the IFM. The remnant muscle found in the EWG null flies show proper assembly of the structural proteins, which implies that some myoblasts manage to fuse, develop and differentiate normally indicating that EWG is not required for differentiation program per se. However, when EWG expression is extended beyond its expression window in a wild type background, muscle thinning is observed implying EWG function in protein synthesis inhibition. Mis-expression studies in wing disc myoblasts hinted at its role in myoblast proliferation. We thus conclude that EWG is important for regulating fusion events which in turn decides the IFM pattern. Also IFM in EWG null mutants show clumps containing broken fibres and an altered mitochondrial morphology. The vertebrate homolog of EWG is nuclear respiratory factor1 (NRF1) which is known to have a function in mitochondrial biogenesis and protection against oxidative stress. Gene expression for inner mitochondrial membrane protein, Opa1-like was found to be absent in these mutants. Also, these flies were more sensitive to oxidative stress, indicating a compromised mitochondrial functioning. Our results therefore demonstrate that EWG functions in maintaining muscles’ structural integrity by ensuing proper mitochondrial activity

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs. (C) 2013 Elsevier Inc. All rights reserved

<i>Bx</i> null females show compromised reproduction.

<p><i>Bx</i> null allele was generated for assessing their reproductive ability. (A) Genomic location of G14-1 P[GawB] element which was mobilized using Δ2–3 transposase. (C, C') <i>Bx</i> null flies (<i>Bx⁷</i>) generated showed held-up wings. (D) RT-PCR analysis showed complete absence of <i>Bx</i>-RA and <i>Bx</i>-RB transcript products. (E) PCR amplification of genomic DNA of <i>Bx⁷</i> flies showed deletion of ∼2 kb from <i>Bx</i> gene sequence. Position of primers used for the genomic PCR is depicted in A and genomic region deleted in <i>Bx⁷</i> is represented in B. (E) <i>Bx⁷</i> females have highly reduced fecundity (F, ***, p<0.0001; **, p = 0.0011) and fertility (G, ***, p<0.0001) (no of eggs tested>800 (<i>w¹¹¹⁸</i> and controls), ∼200 (<i>Bx⁷</i>)) (Students unpaired t-test with Welch's correction) compared to wild type and other controls. (H) almost 100% of the eggs are laid on the surface of the media by <i>Bx⁷</i> mutant females compared to control females which deposit upto 90% of the eggs into the media (schematic of eggs laid by control and <i>Bx⁷</i> mutant females is shown below the X-axis of the graph) (***, p<0.001, Two way anova with Bonferroni post tests).</p