Establishing Drosophila as a model to study the functional relevance of conserved heart genes

Background/Aims. Understanding the fundamental mechanisms underlying the development of congenital heart disease and cardiomyopathies is a goal of researchers worldwide, with the ultimate goal being the establishment of effective therapeutics for the amelioration of cardiac dysfunction. Unfortunately these disorders are often polygenic in aetiology, making it difficult for researchers to probe complex interactions that may contribute to the severity of the disease. Over the last decade, the adult fruit fly (Drosophila melanogaster) has emerged as an invaluable tool with which to study the genetic and molecular mechanisms underlying heart function. The aim of my thesis research was to establish the adult fruit fly as a model of human heart function, and to exploit this powerful genetic system to screen for conserved genes affecting the development and function of its cardiac syncytium. Methodology/Results Baseline measures of heart function and other factors contributing to variability in heart function (i.e. age, temperature, and the time of day) were assessed to establish the adult Drosophila heart model. I then performed an a priori RNAi screen, knocking down expression of individual conserved genes via cardiomyocyte-specific overexpression utilising the yeast GAL4/UAS system. Heart-specific ablation of Fermitin 1 and Fermitin 2 (Fit1, Fit2), the two Drosophila orthologs of Kindlin 2 (Kind2, a gene thought to be important for cardiomyocyte-cardiomyocyte junction integrity in human myocardium), caused severe cardiomyopathy characterised by the failure of cardiomyocytes to develop as a functional syncytium and loss of synchrony between cardiomyocytes. I generated a null allele of Fit1 via P-element mobilisation, but this had no impact on heart development or function. Similarly, the silencing of Fit2 failed to affect heart development or function. In contrast, the silencing of Fit2 in the cardiomyocytes of Fit1-null flies disrupted syncytium development, leading to severe cardiomyopathy. Temperature-sensitive cardiac-specific GAL4/GAL80ts lines were also generated, and knockdown of Fit (Fit1 and Fit2) function at different developmental stages was assessed. I observed the strongest effects of Fit knockdown on adult cardiac morphology during stages of heart development and remodelling, with significant cardiomyocyte decoupling. After 3-weeks of Fit knockdown during adulthood, cardiomyocytes were significantly decoupled, and these hearts were significantly arrhythmic compared to control animals. Conclusions/Discussion. My data provide clarity about the role of Kind2 by demonstrating a cell autonomous role for this family in the development of a functional cardiac syncytium in Drosophila. My findings also show that the Fermitins can functionally compensate for each other in order to control syncytium development. Therefore, my thesis demonstrates the power of the fruit fly as a model of human cardiac physiology, and supports the concept that abnormalities in cardiomyocyte KIND2 expression or function may contribute to cardiomyopathies in humans

Morphogenesis in Drosophila melanogaster : an in vitro analysis

The aim of this thesis was to investigate morphogenesis in the fruit fly Drosophila melanogaster using three in vitro tissue culture systems. Primary embryonic cultures derived from Drosophila melanogaster were used to study the effect of the moulting hormone ecdysone on cells in culture. The hypothesis was that the effect of ecdysone on these primary embryonic cells would parallel events which occur during metamorphosis in vivo and therefore the primary embryonic cultures could be used as an ‘in vitro’ model system. Transgenic fly lines expressing GFP were used to visualise and identify specific cell types and it was shown that cells in primary embryonic cultures respond to ecdysone morphologically. However due to the variability of cultures it was concluded that this culture system was not suitable for use as a model system. As defined cell types were observed the development of a protocol suitable for use with the primary embryonic culture system using dsRNA in order to demonstrate RNA interference was undertaken. Although this was unsuccessful, as cells in the primary embryonic cultures appeared to be resistant to dsRNA, some technical avenues remain to be explored. The Drosophila melanogaster cell line, Clone 8+, was used to investigate cell adhesion in tissue culture. Statistical analyses were carried out and it was established that derivatives of the parent cell line, Clone 8+, showed differential adhesion and proliferation characteristics. Analysis of microarray data was carried out in order to identify genes which may be responsible for the loss of cell adhesion in Clone 8+ cell lines and the potential roles of these genes in adhesion were discussed. A gene of interest, glutactin, was identified which may be responsible for loss of cell adhesion. Antibody staining was used to establish the expression of the protein glutactin in the Clone 8+ cell lines. The expression of glutactin suggested that the Clone 8+ cell line had maintained properties of the wing disc epithelial cell-type and disruption of cell polarity was considered as a possible mechanism. It was shown that f-actin colocalised with glutactin and the role of the cytoskeleton in glutactin secretion was discussed. It was concluded that glutactin was not responsible for loss of cell adhesion in the Clone 8+ cell lines. Further analysis of the microarray data revealed potential genes that could be responsible for the loss of cell polarity in the Clone 8+ cell lines and the possibility of cellular senescence was considered. It was hypothesised that the properties of adhesion and proliferation related to their ‘in vitro’ age. In the final investigation the movement of epithelial cells in Drosophila melanogaster third instar larval imaginal discs during morphogenesis was investigated. Firstly a lumen was identified in fixed imaginal disc tissue in association with cells expressing f-actin. This result was discussed in relation to the process of dorsal closure and wound healing. Further investigations involved live imaging of the dynamic process of evagination in the imaginal wing disc using transgenic flies expressing moesin-GFP. It was concluded that the lumen was not associated with the process of wound healing and it was concluded that the lumen appeared to be the mechanism directing peripodial epithelium contraction during morphogenesis of the imaginal wing disc. Dorsal closure and the process of invagination in relation to morphogenesis of the imaginal wing disc were discussed

Cloning of two neurally expressed genes found adjacent to P[GAL4]307, a marker of the giant fibre circuit, in Drosophila melanogaster

The enhancer trap line P[GAL4]307 was isolated on the basis of expression in the giant fibres. It has proved to be an elegant marker of giant fibres in adult Drosophila. The giant fibres (GFs) are part of a simple circuit in the fly's central nervous system (eNS) that mediate a light-off escape response involving a jump and wing beat. Detailed analysis of the adult expression pattern, using antibodies to the products of the genetic elements used in enhancer trapping, has revealed that all the major components of the escape circuit are clearly detectable. Subsequently, the development of this circuit has been traced using the same antibody staining techniques, revealing the timing of circuit establishment and the developmental profiles of each component neuron. To complete this profile the birthdating of the GFs has been attempted. The overall aim of this section of work was to map the circuit from the first cell division producing the major neurons in embryos, through establishment of the circuit in pupae to full development of the active circuit in adults. The second major section of work has been the cloning of genes from DNA surrounding the P-element insertion. An important facet of P-element design is the ability to use it to remove the genomic DNA from around the site of insertion. A probe created from a fragment of this genomic DNA has been used to screen a library. This yielded a eDNA that, in combination with subsequently isolated cDNAs, forms a contiguous stretch of DNA approximately 4Kb in length. A translation of the eDNA has revealed homology with proteins from a range of eukaryotes from yeast to humans. These may form a family of elongation initiation co-factors. On the basis of homology with an Arabidopsis thaliana gene argonaute, this new Drosophila gene has been named diomedes. The genomic region has also been sequenced, revealing another gene close to diomedes. This new gene, LD07701, appears to be full length, and is unusual because it may be a non-coding RNA. Both genes isolated have neural expression in embryos, larvae and pupae, although it appears neither is the gene whose expression is reflected by the enhancer trap pattern

A P Element Excision-Derived Mutation in Drosophila melanogaster

The purpose of the work described in this Thesis was to isolate and mutate novel genes essential in the development of the Drosophila melanogaster nervous system. Two marked P element enhancer trap strains were found to express the reporter gene lacZ in spatially and temporally regulated patterns in the nervous system. Excisions of the P element construct in one of these strains, A22, were detected in approximately 68% of the A22-derived progeny which were recognised by the loss of the rosy gene. 19 strains were subsequently found to have a recessive mutant phenotype believed to be a result of imprecise excision of the P element. This represents a mutation rate of approximately 24%. Each of these 19 strains had a recessive mutation that resulted in abnormal wing development. Other mutant phenotypes were not detected in any of the excision-derived strains. No similar mutations are known to be located at the site of P element insertion of the ancestral A22 strain and therefore these excision-derived mutants are considered novel. Four excision-derived mutants were selected for further characterisation and these were found to be cold-sensitive. The severity and frequency of the mutant phenotype was increased when these four strains were raised at 2

Analysis of molecular forces transmitted by Talin during muscle development in vivo

The muscle-tendon system built during the development of an animal is essential to allow the body to move, breath or keep the heart beating for a lifetime. The muscle is the most important force producing tissue in an animal and, at the same time, it is also dependent on forces built up in the muscle-tendon tissue, especially during its development. Using the Drosophila musculature as a model system, it had been shown that tension is built up in the muscle-tendon tissue during development and that this tension is required for myofibrillogenesis, the process of building myofibrils, which are long chains of the contractile units of muscles called sarcomeres. The main focus of this thesis was to analyze how tension in tissues is transmitted across proteins at the molecular level to understand how proteins sense and respond to mechanical forces in vivo. As a model system, the developing Drosophila flight muscles were used that form in the pupal stage of the Drosophila life cycle. During development, these muscles attach to tendon cells and the connections between these two cells, called muscle attachment sites, need to bear the forces built up in the tissue. Muscle attachments are cell-extracellular matrix (ECM)-cell contacts that require receptor molecules in the cell membrane called integrins to connect the ECM between the cells with the contractile actin cytoskeleton inside the cells. Since integrins cannot directly connect to actin themselves, they require an adaptor protein called Talin that can bind to both integrin and actin filaments. Thus, Talin is in the ideal position to transmit and sense forces at muscle attachments. Previous studies on Talin force transduction demonstrated that Talin indeed bears forces in the piconewton (pN) range using Förster resonance energy transfer (FRET)-based molecular tension sensors. However, these studies were based on analyzing Talin in focal adhesions in cells cultured in vitro in an artificial environment. Therefore, we aimed to analyze Talin force transmission for the first time in vivo in the natural mechanical environment in the intact organism. In a first step, different FRET-based tension sensor modules and various control constructs were inserted in Drosophila into the endogenous talin (rhea) gene, taking advantage of the newly established clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system to achieve precise modification of the genome. After demonstrating that the Talin protein is still fully functional after insertion of the tension sensor modules, forces across Talin were first quantified—as a proof of concept—in primary muscle fibers in vitro using fluorescence lifetime imaging microscopy (FLIM) to measure FRET. In a second step, forces transmitted by Talin at muscle attachments during flight muscle development were analyzed in detail in living pupae. We discovered that a surprisingly small proportion of Talin molecules at developing muscle attachments transmit forces at the same time (Paper I). Nevertheless, a large pool of Talin molecules need to be recruited to muscle attachment sites during development, as quantified by fluorescence correlation spectroscopy (FCS), to prepare for the forces generated by active muscle contractions in the adult fly. If the accumulation of Talin at flight muscle attachments is reduced during development by RNA interference (RNAi), the muscle attachments rupture in young adults, likely during the first flight attempts. In conclusion, recruitment of a high number of Talin molecules during development is physiologically relevant to enable the muscle to adapt to sudden changes in tissue forces, likely by dynamically sharing the load among the Talin molecules. This mechanical adaptation concept is important to ensure that the muscle-tendon connections are stable and last for a lifetime. During the course of the thesis, I also discovered that flight muscles contract spontaneously during development. Characterization of these contractions in wild-type animals compared to a knockdown condition provided a functional readout for myofibrillogenesis during development (Paper IV). Furthermore, a review article on the role of mechanical forces during muscle development (Paper II) and a video article explaining how to perform in vivo imaging in Drosophila pupae (Paper III) were published

Isolation and characterisation of thyroid hormone-responsive genes of amphibian tail

Amphibian metamorphosis is a post-embryonic process that systematically transforms different tissues in a tadpole. This transformation requires extensive remodelling of almost every tissue in the animal. Thyroid hormone plays a causative role in this complex process by inducing a cascade of gene regulation. One of the more dramatic effects of thyroid hormone (triiodothyronine T 3) is to induce a complete regression of tadpole tail in culture in a simple chemically defined medium. The technique of differential display proposed by Liang and Pardee in 1992, has been applied in an attempt to isolate and then characterise responsive genes induced by thyroid hormone triiodothyronine (T3). Library screens using the PCR fragment xL52 as a probe allowed the isolation of -2.5kb clone termed xth-2. Sequence analysis and database searches at the amino acid level revealed that this clone (xth-2) showed approximately 91% identity to some of the members of a recently discovered family of tissue-specific transmembrane proteins called Hem proteins. Temporal expression of xth-2 using RT-PCR technique revealed that this gene is developmentally regulated. Whole mount in situ hybridisation used for detecting the location of this mRNA in Xenopus laevis embryos at different developmental stages indicated that xth-2 protein was highly expressed in the brain and the pattern of expression has been extended along the central nervous system (CNS) and the caudal region (tail bud). Expression of xth-2 protein in Xenopus embryos, did not show any significant effect on the phenotypic features of the embryos examined. The Nap1 protein, a member of Hem family proteins has recently been found to associate with the SH3 domain of Nck protein, and is thought to play an important role in signalling transduction. We could therefore, speculate that protein xth-2 will have the same function as does the Nap1 on the basis of their sequence similarity, tissue distribution and also the expression pattern

The Dachsous/Fat/Four-jointed Signalling Coordinates the Uniform Orientation of Planar Cell Alignement in the Drosophila Abdominal Epithelium

[eng] Within multicellular organism, mature tissues and organs reach high degrees of order in the arrangement of their constituent cells. During morphogenesis the emergence of long-range order is subjected to multiple and multilevel developmental constrains. Complex series of temporal and spatial instructions must be integrated to account for reproducible and stereotyped mature tissue arrangements. A remarkable example is given by mature epithelial monolayers were cells are ringed together in specific morphologies via cell-cell adhesion and show highly organized planar patterns. Cells in epithelial tissues acquire a precise planar geometry that is often, if not always, evenly aligned with the tissue axes. Little is still known on the cellular mechanisms governing the axial orientation of cell arrangement and planar polarity. The research presented in this Thesis addressed these issues through the analysis of the developing abdominal epithelium of Drosophila melanogaster. We found that the abdominal epithelial cells reach their final arrangement within about 2 days of pupation. During this time, the abdominal epithelial tissue undergoes extensive morphogenesis by tissue expansion and cellular remodelling. During expansion, epithelial cells divide randomly relative to the tissue axis while migrating dorsally, while during remodelling cell adjust their final position within the plane of the epithelium while migrating anteriorly. When cell movements arrest, a stable arrangement of the cells within the plane of the segmental field is attained. At this time, epithelial cells are oriented and aligned among each other and throughout the tissues invariably in parallel to the A/P axis. We indicated as uniform orientation of planar cell alignment (PCA). We found that the axis orientation of PCA is evolving in a spatiotemporal precise manner along the A/P axis, and that this dynamic oriented behaviour was progressively modulated through expansion and remodelling. We found that the Dachsous/Fat/Four-jointed planar polarity pathway was specifically involved in the orientation of PCA. The steps followed to reach the axial orientation of PCA over developmental times correlates with the pattern of expression of the Dachsous/Fat/Four- jointed pathway. Such correlation was sustained by genetic interferences with the pathway components. We found that both the dynamics of the axis orientation of PCA and the attainment of its uniformity along the A/P axis were disrupted rearrangements in dachsous, fat or four-jointed mutants. We further found that loss of the axial uniform orientation of PCA in these mutants is accompanied by an overall reduction in mutual cell alignment and in cell shape elongation. These effects were also sustained through local interference in the activity of the pathway through clonal analyses. Local changes in pathway components induce the mutant clones to minimize cell-cell contacts with surrounding wild-type cells, suggesting differential adhesive properties between dachsous, fat, four-jointed mutant cells and the rest of the tissue. Surprisingly, we found that this effect was also directional and that Dachsous has an instructive role in driving the axis orientation of PCA, possibly by regulating the Fat localization across the cells/tissue. Therefore, from these findings, we propose that the Dachsous/Fat/Four-jointed pathway guide the orientation of PCA by favouring oriented cell-cell contact adhesiveness between abdominal epithelial cells. In particular, different adhesive properties imposed throughout the cell perimeter by Ds-Ft heterodimeric interaction at opposite edges of the cell might align epithelial cells changing and orienting their shape. Over time this biased interaction responding to the Dachsous gradient would be reinforced in response to the activity of Dachsous and Four-jointed onto Fat.[spa] En los organismos multicelulares, tejidos y órganos tienen una disposición altamente organizada de las células que los constituyen. En particular, los tejidos epiteliales están constituidos por células que se disponen de manera ordenada con respecto a los ejes del cuerpo aunque los mecanismos que gobiernan esta disposición ordenada son poco conocidos. En esta tesis nos hemos focalizado en el estudio de los mecanismos que guían la orientación del eje celular en el plano de un epitelio en crecimiento. El epitelio abdominal de Drosophila constituye un sistema modelo para estudiar in vivo las dinámicas celulares que ocurren a lo largo de la morfogénesis tisular. Nuestros resultados demuestran que las células del epitelio abdominal empiezan a orientarse progresivamente llegando a una disposición espacial altamente ordenada. En esta disposición las células demuestran una preferencia en alinearse con el eje anteroposterior del tejido dando lugar a una orientación uniforme del alineamiento celular. Por otro lado, hemos descubierto que la vía de señalización de Dachsous/Fat/Four-jointed está involucrada en guiar el eje de orientación celular con respecto a el eje anteroposterior. Nuestros datos indican que los patrones de expresión de los genes dachsous, fat y four-jointed juegan un papel clave para el correcto alineamiento celular con el eje anteroposterior. De hecho, mutaciones en estos genes alteran el alineamiento espacial y afectan la elongación celular. Además, alteraciones locales en la actividad de cada uno de estos genes indican que están involucrados en la modulación orientada de los contactos adhesivos entre células vecinas. En resumen, estos resultados sugieren que la vía de señalización de Dachsous/Fat/Four- jointed tiene un papel clave en el alcance de una orientación celular uniforme a lo largo del tejido abdominal y, posiblemente, en otros epitelios