Mosaic Analysis

Usha Nagarasan, Ph.D. is an Assistant Professor in the School of Chemical and Biotechnology(SCBT) at SASTRA University, Thanjavur, India THANJAVUR TAMILNADU India where she researches epithelial morphogenesis and tumorigenesis using Drosophila melanogaster as a model.  MORE ABOUT THE AUTHOR

The Recent review by Germani et al., on “Mosaic Analysis in Drosophila”, which appeared in January edition of Flybook of Genetics (http://www.genetics.org/content/208/2/473) discusses mosaicism, one of the finest and sophisticated techniques, which is not only well-utilized and established in Drosophila model organism but also extended to other organisms and revolutionized the field of Developmental biology.

The authors have described well mosaics as individuals with at least two different genotypes, which arise because of mutations within a cell to either remain “silent” without obvious effect to the individual or impose a catastrophic consequence leading to developmental disorders as mosaicism progresses.

In this review, authors have discussed how mosaics can be generated spontaneously by abnormal mitotic events, by chromosomal fusion, exposure to DNA damaging agents and associated activities of repair mechanisms and so on. They have also detailed how mosaics can either naturally occur or experimentally induced. Unlike the experimentally induced mosaics, naturally occurring ones may go unnoticed due to lack of associated markers to follow them.

The authors have  captured well the scenario of Thomas Hunt Morgan’s famous Fly Room to show how (i) gynandromorphs, mosaic of male and females were generated due to changes in X chromosome inheritance, (ii) ring-X chromosomes were formed due to instability in X chromosome. These findings have consequently led to HJ Muller’s Nobel prize winning discovery of producing mutations/mosaics by irradiation and Curt Stern’s crucial discovery of somatic crossover between sister chromatids of different genotypes.

Image result for twin spots

Somatic recombination can lead to mosaics with recombinant clones being visible with the right assemblage of genetic markers.

These discoveries paved way for analyzing mosaic flies carrying visible markers, two adjacent clones of bristles for two different mutations, called the twin-spots. Authors have rightly mentioned that genetic mosaics are invaluable and indispensable tool to characterize several essential genes, understand interaction between two distinct cell types, undertake lineage analysis etc..

The power of  clonal analysis lies in the fact that generation of mutant clones and analysis can be utilized for functional characterization of genes which otherwise would cause lethality in whole animals. Therefore, clonal analysis is utilized broadly to characterize essential genes which cause embryonic lethality, distinguish cell-autonomous and non-autonomous role of gene. Thus, this technique has served as a powerful tool in the hands of geneticists.

A Drosophila melanogaster gynandromorph.

The authors have also depicted the ideal features of cell markers, essential components of the mosaic analysis which is used to detect and analyze mutant clones. Further, the review describes the FLP/FRT recombination system and explains how identification of this system has made clonal analysis technique inducible, site-specific and therefore effective and highly efficient. Here, how recombination or excision and rejoining occurs based on the position and orientation of FRT sites is well described and illustrated. The authors have explained how spatio-temporal regulation is modulated by changing the FLP expression. In addition, the authors have detailed about further improvements of FLP/FRT systems and various clonal analysis kits available to meet the experimental requirements.

Image result for drosophila mosaics

An example of a mosaic wing imaginal disc from Drosophila melanogaster. This is an image from Genetic mosaic techniques for studying Drosophila development by Seth S. Blair published in Development 2003 130: 5065-5072; doi: 10.1242/dev.00774

They have also discussed various forms of mosaics, methods of generating mosaics (from twin-spot to multi-coloring), means of analyzing mosaicism, advantages and disadvantages of available variations to generate and analyze mosaics.

While mosaic analysis has been used almost exclusively in Drosophila the technologies for establishing mosaic inducing systems in other insects are now available.  Furthermore, the recent advent of CRISPR/Cas9 has enabled mosaics to be readily generated and in some cases this has precluded the need to create transgenic lines to analyze gene function by creating knockout mutations.

 

Trans Recombination Recombination between two chromosomes; FRT sites placed at the same location on each sister chromatid

Advantage/Salient feature

Twin spot clones Positively marked Control and negatively marked mutant clones are generated adjacent to each other
Twin Spot Generator(TSG) Hybrid Chromosomes with fluorescent proteins to detect clones immediately
Mitotic Clones in germline X-linked Dominant Female Sterile (DFS) ovoD1 mutation to study essential maternal genes
Minute Technique Utilize Minutes (M) mutant to provide growth advantage to weak clones
MARCM Permits positive cell labeling allows expression of transgene only within the marked clone while suppresses its expression in all other cells
Cis Recombination FRT sites are placed in the same orientation on the same chromosome

Advantage/Salient feature

FLP-out Techniques Generate clones in any phase of cell cycle with spatio-temporal regulation
“Memory” experiments and G-Trace Labeling method that clubs real-time analysis and spatio-temporal information to differentiate ongoing and transient expression pattern
CoinFLP Differentiate the ratio of mutant and non-mutant cells
Colorful Mosaics Labeling the whole tissue with various markers to differentiate various population of cells

Advantage/Salient feature

Flybow and dBrainbow (like Brainbow in mammals) Differentially label multiple cell lineages simultaneously in a variety of tissues
TIE-DYE Whole-tissue-labeling tool in fixed samples to mark most of the cells in a target tissue or organ
Raeppli Whole-tissue-labeling tool for live imaging
Multicolor FlpOut (MCFO) Stochastic multicolor labeling with modified fluorescent proteins
Additional tools for Conditional Mutant Clonal Analysis Analyze gene placed between FRT and centromere; Genes on 4th chromosomes with no FRT sites

Advantage/Salient feature

MARGE Rescues the homozygous mutation and permits to study the effect of mutation on differentiated cells
FlpStop Presence of “FLEx-switch” cassette permits to generate both disrupting(D) and nondisrupting(ND) alleles
Flip-Flop Permits mosaic analysis in post-mitotic cells

 

Germani F,  Bergantinos C and Johnston LA (2018) Mosaic Analysis in Drosophila

GENETICS February 1, 2018 vol. 208 no. 2 473-490; https://doi.org/10.1534/genetics.117.300256

 

 

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