| dc.description.abstract | Many genetic regulatory networks are required to define the overall characters in a living organism. Characters which are necessary for survival in the wild are often canalized, such that despite genetic and environmental perturbations, organisms can produce phenotypes resembling the corresponding wild type ones. Studying such gene regulatory phenomena would not only advance our understanding of how cells could respond to genetic and environmental perturbations but may also provide cues to effective therapeutic interventions, in cases where perturbations lead to disorder/disease conditions.
Striated muscles play an important role in mediating movement and locomotion in higher animals and hence are indispensable for survival and reproduction in the wild. Therefore, their development, which occurs in two phases, namely embryonic and adult phases, is very likely to be under robust genetic regulation. The development of both embryonic and adult muscles involves the expression and assembly of several myofibrillar proteins, leading to the formation of the highly ordered “sarcomere” structures, which are essential for normal muscle function. Even though the regulatory events involved in assembly of the sarcomeres have been widely addressed, the finer details of many of the molecular players involved, with respect to their functional roles, require a thorough investigation. Moreover, embryonic/foetal muscle development involves the expression and assembly of embryonic/foetal isoforms of several myofibrillar proteins, which are replaced by their adult counterparts in a tissue and spatio-temporal specific manner, during foetal to adult transition (postnatal isoform switching). The functional relevance and regulation of such developmental switching of isoforms also remains poorly understood.
One protein implicated in the development of striated muscle fibres is the Muscle LIM Protein (MLP). ‘LIM’ is an abbreviation derived from: Lin-11, Isl1 and Mec-3, the first three proteins of this family of proteins, to be identified. The LIM domain mediates protein-protein interactions by serving as an adaptor, thus facilitating the formation of macromolecular complexes. The most studied Muscle LIM protein is the vertebrate ortholog-CSRP3. This protein is a vital component of the Z-discs in mature fibres. CSRP3(-/-) mice show a complete disruption of the sarcomere structure in cardiomyocytes but only reduced sarcomere length in skeletal muscle myocytes. Mutations in this gene have been linked to the development of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). It has also been shown to be necessary for muscle differentiation. However, its role in the development of embryonic muscles, the regulatory aspects of its function and its precise role in the differentiation of striated muscle fibres have not yet been completely understood.
The Drosophila genome encodes for two Muscle LIM proteins- Mlp60A and Mlp84B. These have been named according to the genomic loci to which these were initially mapped, cytogenetically. Mlp84B function has been studied by using Mlp84B null mutants. These mutants do not show any phenotype till the larval developmental stages, thus showing that Mlp84B is dispensable for embryonic development. However, most of the Mlp84B null pupae are lethal. Those that do eclose are very weak and die within a few days post eclosion. These flies do not show any sarcomere structural defects, neither in the heart nor in the flight muscles. Mlp60A function on the other hand has not been addressed at all. We have undertaken the study of the Drosophila Muscle LIM Protein- Mlp60A, with regards to its involvement in the development of the larval body wall muscles (embryonic muscles) and the Indirect Flight Muscles (IFMs) of the adult. We found that this locus gives rise to two isoforms by alternative splicing, a shorter (279 bp) isoform and a longer (1461 bp) isoform. The short isoform is expressed throughout development, but the longer isoform is adult specific, being the dominant of the two isoforms in the IFMs. A muscle specific knock down of both the isoforms leads to late pupal lethality and severe flight defect in the population of surviving flies. The IFMs of these flies show no defects at the fascicular level, with normal IFM fascicle pattern and dimensions. However, at the sarcomere level, the Mlp60A knockdown flies show a reduced resting sarcomere length and fraying of few myofibrils, in longitudinal sections. An analysis of the IFM fascicle cross sections in these knockdown flies showed the presence of several actin blotches along the fascicle membrane and in the interior of the fascicles. These actin blotches could represent the frayed myofibrils seen in the longitudinal sections or actin aggregates which had been formed on the fascicular membrane, mainly. These results show that Mlp60A isoforms regulate the sarcomere length of myofibrils and may have an additional role as regulators of thin filament (mainly F-actin) dynamics and assembly.
We further performed a P-element based hop out mutagenesis screen to isolate isoform specific null or mutant alleles of Mlp60A. Our screen identified one Mlp60Anull allele which is homozygous recessive in nature for survival and function. The individuals homozygous for this allele are lethal, majorly in the larval developmental stages. These larvae show a significant defect in their locomotion, and in their body wall muscles. The specificity of these phenotypes was confirmed by genetic complementation test between the Mlp60Anull allele and a genomic deficiency of the Mlp60A region and more directly by rescuing the lethality of the Mlp60A null mutant through UAS/Gal4 based over-expression of the short isoform. These null larvae also showed a significantly reduced expression of thin filament proteins such as TnC, TnT, Act57B and Tm2, but not of the major thick filament protein MHC, pointing towards the involvement of Mlp60A-short isoform in the regulation of thin filament proteins.
We have also isolated another mutant allele from the same P-element screen, which consists of a 1.3 kb remnant of the full-length P-element insertion (named Mlp60AHFDE), at the original insertion site, which is within the 3rd exon of Mlp60A (a part of only the longer isoform). The imprecise excision leading to the generation of this allele did not excise any major part of the Mlp60A locus itself. We have characterized this allele along with the full length (7.987 kb) P-element insertion allele (named Mlp60AP-ele). The P-element insertion in both these alleles affects the long isoform transcript in the same manner. Due to the presence of the P-element insertion the usual 3’ splice site is avoided and a novel splice site from intron 3-4 is chosen, leading to the splicing out of the entire 3rd exon and inclusion of 17 bases from intron 3-4 i.e. 1726th base to 1742nd base in the Mlp60A gene sequence, along with the remaining exons:4-8, in the mature transcript. Hence, this alternative splicing does not disrupt the reading frame. Due to such de novo splicing, the translated product is devoid of 21 residues belonging to the 2nd LIM domain of the long isoform. But phenotypically, these two alleles are drastically different. The Mlp60AHFDE homozygous adult flies are severely flight defective (95% of the population tested) but those homozygous for Mlp60AP-ele show only a mild phenotype (around 40% of the population tested). Genetically, these complement each other indicating that the phenotype of either one or both could be contributed by a second site mutation. To investigate this further, complementation tests were performed between each of these and a genomic deficiency of Mlp60A region. Our data shows that the phenotype of the Mlp60AP-ele homozygous flies is due to a splicing regulatory mechanism which ensures that though the long isoform specific transcript is truncated, its reading frame is not completely disrupted, despite the presence of a 9.897 kb long P-element insertion in 3rd exon sequence of the primary transcript. When this P-element insertion is precisely excised (i.e. in the Precise excision allele, serving as the control in all our screens) the flight ability of the flies is completely restored to wild type, pointing towards the necessity of the longer isoform for flight.
We rescued the null mutants by transgenic expression of the shorter isoform of Mlp60A, through UAS-Gal4 system. We were able to rescue around a third of the Mlp60Anull individuals. These were tested for their flight ability. Around 40% of these rescued flies, flew normally and the remaining 60% had compromised flight, again pointing towards the requirement of the longer isoform for normal flight.
A very few of the Mlp60Anull individuals, when separated from the heterozygotes and grown on a protein rich medium (yeast paste) escape lethality and complete the development. These flies were majorly normal flighted. However, the fertilized eggs laid by these escapers were majorly (97%) embryonic lethal, with the remaining (3%) individuals also dying at the 1st instar larval stage. Hence our data uncovers a disparity between Mlp60A knockdown and knockout phenotype at the adult stage. A comparison between the isoform abundance levels, developmental lethality and flight ability of individuals from two different knockdown backgrounds, null mutants and null mutants rescued with short isoform transgenic expression unravels the existence of a Genetic Compensatory Response to Mlp60A depletion. This response ensures normal flight in the adult flies that escape the lethality caused by Mlp60A short isoform depletion, but it cannot compensate for the loss of the shorter isoform at the embryonic stage. Our data shows that this response could not be mediated by the Mlp60A paralogue-Mlp84B, as the latter was rather significantly down-regulated in the Mlp60Anull adult escapers.
The first chapter sheds light on the importance of the questions that this study deals with. We have described sarcomere structure complexity and how it is achieved during development. We have also given a detailed description of the existing scientific knowledge of the muscle LIM proteins and aspects of their function in striated muscle development that are yet to be addressed. We describe herein, the advantages which the IFMs of Drosophila offer us towards the addressing of such questions. Chapter three deals with the characterization of the different Mlp60A isoforms and their experimental verification. It deals with their developmental expression profile and isoform switching. Also, we provide evidence in this chapter of Mlp60A being an essential gene, which is necessary for normal flight in Drosophila, through knockdown studies. The fourth chapter deals with the generation and molecular characterization of mutants of this genetic locus. It also contains detailed description of the phenotypic characterization of the Mlp60Anull mutant allele, isolated by us. It also talks about the rescue of the null mutants by transgenic overexpression of the short isoform. The fifth chapter describes the studies conducted on long isoform mutants, demonstrating the functional relevance of Mlp60A isoform switching. In the fourth chapter we have described certain results obtained by us that point towards the existence of a genetic regulatory mechanism, which compensates for the loss of the longer isoform of Mlp60A. This chapter also describes results which give evidence of the importance of the maternally deposited short isoform, for development.
Overall, the present study provides a new insight into the genetic robustness of muscle development in the context of the Muscle LIM protein. Our results show that the Mlp60A-short isoform is indispensable for the development of larval body wall muscles and hence for survival, thus establishing Mlp60A as an essential gene in Drosophila melanogaster. We also provide functional insight into the biphasic nature of Mlp60A expression, first seen by Stronach et al., in the 1996 report on this gene. Our results also show the functional relevance of Mlp60A isoform switching, since we found that at-least one copy of the wild type long isoform encoding gene is necessary for normal flight. The muscle phenotypes shown by Mlp60A knockdown flies are reminiscent of the pathogenesis observed in several CSRP3 mutations that have been associated with the development of DCM or HCM in humans and provide a platform for dissecting the underlying mechanisms in future. | en_US |
