Characterization of Corneal Lens of Drosophila melanogaster and its Genetic Mutants using Atomic Force Microscope and Scanning Electron Microscope

Abstract

Drosophila melanogaster (the fruit fly) is a widely used model organism for the study of human disease condition and genetic pathways due to the high degree of gene conservation between fly and human. The compound eyes of Drosophila comprise of a repetitive arrangement of ommatidia with a honey-comb like an appearance. Any mutation or genetic alteration disturbs the normal development of compound eye and results in 'rough eye' condition. Such rough eye conditions are used to study effects of the disease gene and in the identification of the modifiers that rectify the disease condition. Typically, phenotyping of the rough eye is carried out from the optical microscope and scanning electron microscopic observations of the corneal lens. However, this method is qualitative and very subjective. In order to arrive at a quantifiable parameter, we explore nanoscale morphology, elastic modulus and adhesion force of the corneal lens using atomic force microscope (AFM) and field-emission scanning electron microscope (FESEM). These methods have the advantage of the ease and less time consuming to obtain a large number of data for statistically meaningful evaluation.

Though AFM has emerged as an important tool in biology for its high-resolution imaging and mechanical property testing, the latter is not used widely compared to former due to high variability in the measurement. Therefore, parameters bringing variability in measurement of corneal lens properties like sample preparation, aging, usage of solvent, the location of indent are identified and a testing procedure has been drawn for reliable and repeatable measurements.

Rough eyes caused by different genetic mutants of Drosophila and human tau disease condition are tested to bring out their corneal lens characteristics. Effect of different levels of a tau protein, same levels of tau isoforms and effect of tau disease modifiers in corneal lens properties are studied. From this study, we identified corneal lens morphologies of tau disease condition is distinctly different from the normal fly condition. Adhesion force to be depended on the levels of the tau protein and isoform. Elastic modulus found to be lower in the tau disease condition and dependent on the type of mutant tau, levels of transcripts and isoform. Under disease suppressor condition, the normal formation of nanoscale morphology and elastic modulus close to that of wild type is observed. Study with genetic mutants of Drosophila enables to identify the molecular player involved in causing distinct nanoscale morphologies and molecules involved in bringing the changes in elastic modulus. Drosocrystallin, Wingless and Vein molecules found to play roles in corneal lens characteristics.

Based on these studies, we establish that AFM can be used as a quick tool in identifying quantifiable parameters that measure the physical variations in the corneal lens. In addition, the effect of disease protein, evaluation of disease status, determination of the modifiers, the study of molecular players in controlling the nanoscale morphology, corneal lens strength, identification of genetic players under a disease condition can be made quickly