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In JoVE (1)

Other Publications (7)

Articles by Ouarda Taghli-Lamallem in JoVE

 JoVE Biology

Fluorescent Labeling of Drosophila Heart Structures

1Biology Department, San Diego State University, 2Development and Aging Program, NASCR Center, The Sanford Burnham Institute for Medical Research


JoVE 1423

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Other articles by Ouarda Taghli-Lamallem on PubMed

Direct Interaction Between Teashirt and Sex Combs Reduced Proteins, Via Tsh's Acidic Domain, is Essential for Specifying the Identity of the Prothorax in Drosophila

teashirt (tsh) encodes a zinc-finger protein that is thought to be part of a network that contributes to regionalization of the Drosophila embryo and establishes the domains of Hox protein function. tsh and the Hox gene Sex combs reduced (Scr) are essential to establish the identity of the first thoracic segment. We used the development of the first thoracic segment as a paradigm for Scr dependent regional morphological distinctions. In this specific context, we asked whether Tsh protein could have a direct influence on Scr activity. Here we present evidence that Tsh interacts directly with Scr and this interaction depends in part on the presence of a short domain located in the N-terminal half of Teashirt called "acidic domain". In vivo, expression of full length Tsh can rescue the tsh null phenotype throughout the trunk whereas Tsh lacking the Scr interacting domain rescues all the trunk defects except in the prothorax. We suggest this provides insights into the mechanism by which Tsh, in concert with Scr, specifies the prothoracic identity.

The Drosophila Muscle LIM Protein, Mlp84B, is Essential for Cardiac Function

Muscle LIM protein (MLP) is a cytoskeletal protein located at the Z-disc of sarcomeres. Mutations in the human MLP gene are associated with hypertrophic and dilated cardiomyopathy. MLP has been proposed to be a key player in the stretch-sensing response, but the molecular mechanisms underlying its function in normal and diseased cardiac muscle have not been established. A Drosophila homolog, Mlp84B, displays a similar subcellular localization at the Z-disc of sarcomeres throughout development and in the adult, suggesting Drosophila as a model to study MLP function. Here we employed genetic ablation and cardiac-specific RNA interference (RNAi) knockdown of mlp84B to investigate its role in heart function. We found that Mlp84B-deficient or heart-specific RNAi knockdown flies exhibit diastolic interval prolongation, heart rhythm abnormalities and a reduced lifespan, while showing no obvious structural phenotype. Our data demonstrate that Mlp84B is essential for normal cardiac function and establish the Drosophila model for the investigation of the mechanisms connecting defective cardiac Z-disc components to the development of cardiomyopathy.

Dystrophin Deficiency in Drosophila Reduces Lifespan and Causes a Dilated Cardiomyopathy Phenotype

A number of studies have been conducted recently on the model organism Drosophila to determine the function of genes involved in human disease, including those implicated in neurological disorders, cancer and metabolic and cardiovascular diseases. The simple structure and physiology of the Drosophila heart tube together with the available genetics provide a suitable in vivo assay system for studying cardiac gene functions. In our study, we focus on analysis of the role of dystrophin (Dys) in heart physiology. As in humans, the Drosophila dys gene encodes multiple isoforms, of which the large isoforms (DLPs) and a truncated form (Dp117) are expressed in the adult heart. Here, we show that the loss of dys function in the heart leads to an age-dependent disruption of the myofibrillar organization within the myocardium as well as to alterations in cardiac performance. dys RNAi-mediated knockdown in the mesoderm also shortens lifespan. Knockdown of all or deletion of the large isoforms increases the heart rate by shortening the diastolic intervals (relaxation phase) of the cardiac cycle. Morphologically, loss of the large DLPs isoforms causes a widening of the cardiac tube and a lower fractional shortening, a phenotype reminiscent of dilated cardiomyopathy. The dilated dys mutant phenotype was reversed by expressing a truncated mammalian form of dys (Dp116). Our results illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and other muscular-dystrophy-associated phenotypes.

Context-dependent Regulation of Hox Protein Functions by CK2 Phosphorylation Sites

Variations in Hox protein sequences and functions have been proposed to contribute to evolutionary changes in appendage shape and number in crustaceans and insects. One model is that insect Hox proteins of the Ultrabithorax (UBX) ortholog class evolved increased abilities to repress Distal-less (Dll) transcription and appendage development in part through the loss of serine and threonine residues in casein kinase 2 (CK2) phosphorylation sites. To explore this possibility, we constructed and tested the appendage repression function of chimeric proteins with insertions of different CK2 consensus sites or phosphomimetics of CK2 sites in C-terminal regions of Drosophila melanogaster UBX. Our results indicate that CK2 sites C-terminal to the homeodomain can inhibit the appendage repression functions of UBX proteins, but only in the context of specific amino acid sequences. Our results, combined with previous findings on evolutionary changes in Hox protein, suggest how intra-protein regulatory changes can diversify Hox protein function, and thus animal morphology.

Tinman/Nkx2-5 Acts Via MiR-1 and Upstream of Cdc42 to Regulate Heart Function Across Species

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.

Electrophysiological Mapping of Embryonic Mouse Hearts: Mechanisms for Developmental Pacemaker Switch and Internodal Conduction Pathway

Electrical Mapping of Embryonic Mouse Hearts. Introduction: Understanding sinoatrial node (SAN) development could help in developing therapies for SAN dysfunction. However, electrophysiological investigation of SAN development remains difficult because mutant mice with SAN dysfunctions are frequently embryonically lethal. Most research on SAN development is therefore limited to immunocytochemical observations without comparable functional studies. Methods and Results: We applied a multielectrode array (MEA) recording system to study SAN development in mouse hearts acutely isolated at embryonic ages (E) 8.5-12.5 days. Physiological heart rates were routinely restored, enabling accurate functional assessment of SAN development. We found that dominant pacemaking activity originated from the left inflow tract (LIFT) region at E8.5, but switched to the right SAN by E12.5. Combining MEA recordings and pharmacological agents, we show that intracellular calcium (Ca(2+) )-mediated automaticity develops early and is the major mechanism of pulse generation in the LIFT of E8.5 hearts. Later in development at E12.5, sarcolemmal ion channels develop in the SAN at a time when pacemaker channels are down-regulated in the LIFT, leading to a switch in the dominant pacemaker location. Additionally, low micromolar concentrations of tetrodotoxin (TTX), a sodium channel blocker, minimally affect pacemaker rhythm at E8.5-E12.5, but suppress atrial activation and reveal a TTX-resistant SAN-atrioventricular node (internodal) pathway that mediates internodal conduction in E12.5 hearts. Conclusions : Using a physiological mapping method, we demonstrate that differential mechanistic development of automaticity between the left and right inflow tract regions confers the pacemaker location switch. Moreover, a TTX-resistant pathway mediates preferential internodal conduction in E12.5 mouse hearts. (J Cardiovasc Electrophysiol, Vol. pp. 1-10).

Over-expression of DSCAM and COL6A2 Cooperatively Generates Congenital Heart Defects

A significant current challenge in human genetics is the identification of interacting genetic loci mediating complex polygenic disorders. One of the best characterized polygenic diseases is Down syndrome (DS), which results from an extra copy of part or all of chromosome 21. A short interval near the distal tip of chromosome 21 contributes to congenital heart defects (CHD), and a variety of indirect genetic evidence suggests that multiple candidate genes in this region may contribute to this phenotype. We devised a tiered genetic approach to identify interacting CHD candidate genes. We first used the well vetted Drosophila heart as an assay to identify interacting CHD candidate genes by expressing them alone and in all possible pairwise combinations and testing for effects on rhythmicity or heart failure following stress. This comprehensive analysis identified DSCAM and COL6A2 as the most strongly interacting pair of genes. We then over-expressed these two genes alone or in combination in the mouse heart. While over-expression of either gene alone did not affect viability and had little or no effect on heart physiology or morphology, co-expression of the two genes resulted in ≈50% mortality and severe physiological and morphological defects, including atrial septal defects and cardiac hypertrophy. Cooperative interactions between DSCAM and COL6A2 were also observed in the H9C2 cardiac cell line and transcriptional analysis of this interaction points to genes involved in adhesion and cardiac hypertrophy. Our success in defining a cooperative interaction between DSCAM and COL6A2 suggests that the multi-tiered genetic approach we have taken involving human mapping data, comprehensive combinatorial screening in Drosophila, and validation in vivo in mice and in mammalian cells lines should be applicable to identifying specific loci mediating a broad variety of other polygenic disorders.

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