High rates of plasmid cotransformation in E. coli overturn the clonality myth and reveal colony development

DNA transformation methods, pioneered by Griffith in 1928 and made commonplace by Hanahan in the 1980s 1, ushered in the dawn of molecular cloning of DNA. It is accepted that a typical transformation produces clonal bacterial colonies. To the contrary, using low concentrations of several fluorescent plasmids, under the same selective antibiotic, we find that E. coli bacteria readily accept multiple plasmids, resulting in widespread aclonality and surprisingly uncover a complex pattern of colony development. Cotransformation of plasmids occurs by either CaCl2 or by electroporation methods of bacterial transformation. A bacterium rod transformed with three plasmids - each expressing a high level of a unique fluorescent protein - and replated on agar, appears to reassign a random number of the three fluorescent plasmids to its daughter cell during cell division until an equilibrium is reached whereby ensuing progeny carry a specific distribution of the three plasmids. Thus, the potential to follow multiple lineage tracings in a bacteria colony simultaneously lends itself to mosaic analysis of gene function. We observe that clonally related bacterium rods self-organize in a fractal growth pattern can remain linked during colony development revealing a potential target against microbiota growth.

Invivo and systematic analysis of random multigenic deletions associated with human diseases during epithelial morphogenesis in Drosophila

Random loss of multigenic loci on chromosomes, a crucial drive for evolution, occurs frequently in all living organisms. Analysis of such chromosomal disruption and understanding the consequences of their impact on the growth and development of multicellular organisms is challenging. In this report, we have addressed this issue using invivo mosaic analysis of deficiency lines in Drosophila. Genes on fly deficiency lines were compared with human orthologs for their implications in disease development during cytoskeletal processes and epithelial morphogenesis. The cytoskeletal phenotypes from the fly has been utilized to predict the function of human orthologs. In addition, as these Drosophila deficiency lines are equivalent to human microdeletions, based on the clonal behaviour and phenotypes generated, a systematic analysis has been carried out to establish the critical loci that correspond to Microdeletion Syndromes and Mendelian Disorders in humans. Further we have drawn the synteny that exists between these chromosomes and have identified critical region corresponding to defects. A few potential candidates that might have an implication in epithelial morphogenesis are also identified.

Versatile CRISPR/Cas9-mediated mosaic analysis by gRNA-induced crossing-over for unmodified genomes

Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. Here, we report mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for 1 chromosome arm, we demonstrate the method’s application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites into the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can in principle be applied in any organism that is compatible with CRISPR/Cas9.

Clonal Analysis of Gliogenesis in the Cerebral Cortex Reveals Stochastic Expansion of Glia and Cell Autonomous Responses to Egfr Dosage

Development of the nervous system undergoes important transitions, including one from neurogenesis to gliogenesis which occurs late during embryonic gestation. Here we report on clonal analysis of gliogenesis in mice using Mosaic Analysis with Double Markers (MADM) with quantitative and computational methods. Results reveal that developmental gliogenesis in the cerebral cortex occurs in a fraction of earlier neurogenic clones, accelerating around E16.5, and giving rise to both astrocytes and oligodendrocytes. Moreover, MADM-based genetic deletion of the epidermal growth factor receptor (Egfr) in gliogenic clones revealed that Egfr is cell autonomously required for gliogenesis in the mouse dorsolateral cortices. A broad range in the proliferation capacity, symmetry of clones, and competitive advantage of MADM cells was evident in clones that contained one cellular lineage with double dosage of Egfr relative to their environment, while their sibling Egfr-null cells failed to generate glia. Remarkably, the total numbers of glia in MADM clones balance out regardless of significant alterations in clonal symmetries. The variability in glial clones shows stochastic patterns that we define mathematically, which are different from the deterministic patterns in neuronal clones. This study sets a foundation for studying the biological significance of stochastic and deterministic clonal principles underlying tissue development, and identifying mechanisms that differentiate between neurogenesis and gliogenesis.