I. upon the relevance of such scientific explorations.

I.                             ABSTRACT

This paper aims to validate and further explore the morphogenesis of the heart tube, dorsal vessel, in embryonic Drosophila melanogaster due to its shared developmental homology to that of the mailman cardiac system. This endeavor is executed via imaging immunoflurescently stained structures in Drosophila embryos with the use of laser confocal microscopy.

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One of the specific proteins that were targeted for imaging included Fasciclin III, which is found in the cell membrane of visceral mesodermal cells, which finally give rise to the dorsal vessel. The second principal protein of interest in this experiment is Pericardin, which is found exclusively in pericardial cells – cells that surround the forming dorsal vessel.


Ultimately, the experiment was partially successful in imaging the desired structures. Beyond the acquisition of the images, the paper reflects on ways to improve replications of this experiment, suggests future directions of research, and also touches upon the relevance of such scientific explorations.  
















II.                         INTRODUCTION


Pursuing research on specimens other than Homo sapiens always has the ultimate purpose of understanding our biological, psychological, or sociological manifestation to a superior extent than before. When working with specimens that are very evolutionarily removed from us, such as the fruit fly, Drosophila melanogaster (henceforth referred to as “Drosophila”), a visceral sense of skepticism organically arises at the thought that we can enrich our understanding of ourselves from such experimental subjects. However, according to modern evolutionary biology, drosophila and Homo sapiens share a noteworthy amount of genetic similarities.


Specifically, seventy-five percent of of genes that cause disease in in humans are shared with the fruit fly 1, and according to the National Human Genome Institute, the fruit fly is more similar to humans than any other animal sequenced so far, its genetic information can be directly related to humans in many cases.” 2 This model organism has been used in pioneering studies in classical genetics and for the past one hundred years and played an integral role in genetic research.


Due to its wide use in research, drosophila is more relevant to current research than ever. One remarkable reason why this species is so efficient in studies is the public availability of its complete genomic sequence 3. This availability stands as testament to the importance placed on this information being available for future research endeavors. Another benefit of fruit fly use in the lab is that mutants are readily available on demand, allowing for the experimental exploration of specific genotypic populations. Then, its short life cycle of ten to fifteen days, its mall size, and its aggressive reproductive rate make it time efficient to culture and statistically analyze over generations 4.  


            Further yet, this species and H. sapiens demonstrate parallelism beyond genetics and into early embryological development as well. Such is the case with the development of the cardiac structures. Despite the simple structure and function of the Drosophila dorsal vessel, the organ shares several similarities with the early-stage hearts of vertebrates. The steps that lead to the formation of these structures appear to be highly conserved in vertebrate and invertebrate embryos. Homologues of Drosophila genes function during vertebrate cardiogenesis, indicating a conservation of molecular mechanisms in the formation of these essential circulatory organs 5.


            Early in vertebrate development, uncommitted part of the mesoderm residing on each lateral half of the developing embryo becomes specified to a cardiogenic fate by diffusible factors released from the underlying endoderm. Once specified, the cardiogenic precursors migrate to fuse at the midline of the embryo forming the linear heart tube. The tube, subsequently, undergoes looping morphogenesis, which does not occur in Drosophila 5.


Next, the embryonic mesoderm and ectoderm in Drosophila contain cardiogenic regions that engage coordinated migration that culminates in a fusion event at the midline of the embryo. The extracellular matrix has been found to be responsible, at least in part, for for this orchestration. This is not surprising as morphogenic signals commonly arise from extracellular matrix and these environmental cues to cells affect all aspects of cell behavior. 5


            Specifically, the extracellular protein pericardin, a Drosophila extracellular matrix protein, has been of high interest. Pricardin, a type IV collagen a-chain, has been shown to be involved in the heart tube formation due to its expression in the pericardial cells at the onset of the dorsal closure. Another indicator that perocardin plays a role in orchestrating this migration is its high concentration at the basal surface of the cardioblasts and pericardial cells, close to the dorsal ectoderm, but absent from the lumen 6.


Other evidence comes from genetic silencing of the protein. With pericardin silenced, the formation of the heart epithelium exhibits significant defects. These type of embryos had disorganized heart epithelium during their migration to the dorsal midline, indicating pericardin’s involvement in regulating these events 6. 


Furthermore, the dorsal vessel is surrounded by several types of pericardial cells. These cells are loosely associated with cardiac cells and do not express muscle proteins. The precise roles pericardial cells play during heart development have been poorly understood. What is known, however, is that during late stage 11 and stage 12 of embryonic development, precursor cells to the dorsal vessel acquire a polarity and reorganize their shape to form a continuous epithelial layer on each side of the dorsal opening. Later in the process of dorsal closure, the two rows of cardiac cells, together with the pericardial cells, which are attached to the basal membrane of cardiac cells, migrate dorsally and fuse at the dorsal midline to form the dorsal vessel tube enclosing a lumen. 7


            The aim of this experiment is to successfully image the morphological development of the dorsal vessel and to visualize that in fact the stated tissues behave as described and depicted in previous experiments. With proper procedural execution, the images collected in this experiment should add to the scientific body of evidence in support of the homological morphogenesis between the mammalian cardiac structures and the development of the open cardio-lymphatic system found in Drosophila, as well as the proteins involved.


III.                     METHODS


Embryos Used


The embryos used for the execution of this experiment were obtained from wild type Dorsophila melanogaster. These embryos ranged in age from 0 hours to 22 hours old at at the time of fixation.


Fixation Procedure


In preparation for the fixation procedure, the embryos were first transferred into a mesh containing vial by washing the plate with embryo wash. Next, the process of dechorionating the embryos was executed via washing them with fifty percent bleach solution for two minutes using a plastic pipette. Then, after the two minutes, we began washing the dechrionated embryos with distilled water until the bleach odor was no longer present. To ensure that the embryo batch was successfully dechrionated, they were quickly placed under a microscope and visually confirmed to be lacking the chorion. Following this, the embryos were transferred to a scintillation vial containing 3 mL of heptane. To achieve this transfer, the mesh containing the batch of embryos was first dried of excess water with paper towels, and then with the aid of a paint brush, the embryos were brushed into the heptane solution.


For the execution of the fixation, 3mL of formaldehyde fix (3.7% formalehyde in 1xPBS – 3.7% PFA) were added to the scintillation vial and placed on a laboratory rocker for ten minutes. After the aforementioned time elapsed, the fixation solution (the bottom layer) was removed with a glass pipette and then discarded as waste.


Next, the process of removing the vitelline began by the addition of 3 mL of methanol to the scintillation vial congaing the heptane and the embryos. After being shook the embryos lost their vitelline membrane, they began to sink to the bottom, from the heptane-methanol interface. This procedure was repeated while removing and adding fresh methanol and heptane, and keeping them at a volume ratio of approximately one-to-one.


After this, the heptane was removed with the embryos remaining at the heptane-methanol interface, and the majority of the methanol. Next, 1 mL of fresh methanol was added to the remaining embryos and was afterwards transferred in a Eddpendorf tube to be stored at -20°C. 8


Fluorophores Used


In order to be able to visualize the structures involved in the morphogenesis of the dorsal vessel, fluorescent stains were carefully chosen from a given class catalogue in such a way to not overlap or interact. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain DNA, given its high affinity to A-T rich religions of DNA and would’ve fluoresced with 405nm laser excitation 9. This dye would’ve worked for the samples partitioned for the spinning disk confocal.  Next, TO-PRO-3 fluorescent dye was used as well to target dsDNA, and had 642?661 nm excitation/emission values which was compatible with the ranges available on the laser scanning confocal microscope 10. Concanavalin A-Tetramethylrhodamine (ConA-TMR) selectively binds to ?-mannopyranosyl and ?-glucopyranosyl residues, has an absorption and emission maxima 555 and 580 nm, respectively, and was included in the experiment for the purpose of acting as a background dye 11.


            For the primary antibodies used for the experiment included anti-Pericardin harvested in mice, anti-Actin harvested in rabbit, anti- Fasciclin III harvested in mice. Anti-Pericardin was chosen because it was the antibody most directly and exclusively targeting the cells involved in the morphogenesis of the dorsal vessel of the drosophila embryos. Next, anti-Actin antibody was also selected for the execution of the experiment because it would serve as a background to its paired primary. It is noteworthy to mention that no slide contained anti-Pericardin with anti-Fasciclin III due to both being harvested in mice and causing indiscriminant targeting of both by any secondary antibody. Finally, anti-Fasciclin III was used because Fasciclin III marks the visceral mesoderm 12.


            As for the secondary antibodies, the goal was to target the primary antibodies via the animals they were harvested in, while also differentiating the primaries from one another by having considerably distinct excitation wavelength values. Spesifically, different permutation of 647 nm, 555nm, 488nm, and 405nm were used.