MARCM

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Mosaic analysis with a repressible cell marker, or MARCM, is a genetics technique for creating individually labeled homozygous cells in an otherwise heterozygous Drosophila melanogaster . [1] It has been a crucial tool in studying the development of the Drosophila nervous system. This technique relies on recombination during mitosis mediated by FLP-FRT recombination. As one copy of a gene, provided by the balancer chromosome, is often enough to rescue a mutant phenotype, MARCM clones can be used to study a mutant phenotype in an otherwise wildtype animal.

Contents

Crosses

A visual depiction of MARCM Mosaic analysis with a repressible cell marker(MARCM).gif
A visual depiction of MARCM

In order to label small populations of cells from a common progenitor, MARCM uses the GAL4-UAS system. A marker gene such as GFP is placed under control of a UAS promoter. GAL4 is ubiquitously expressed in these flies, thus driving marker expression. In addition, GAL80 is driven by a strong promoter such as tubP. Gal80 is an inhibitor of GAL4, and will suppress GFP expression under normal conditions. This tubP-GAL80 element is placed distal to an FRT site. A second FRT site is placed in trans to the GAL80 site, usually with a gene or mutation of interest distal to it. Finally, FLP recombinase is driven by an inducible promoter such as heat shock.

When FLP transcription is induced, it will recombine the chromosomes at the two FRT sites in cells undergoing mitosis. These cells will divide into two homozygous daughter cells—one carrying both GAL80 elements, and one carrying none. The daughter cell lacking GAL80 will be labeled due to expression of the marker via the GAL4-UAS system. All subsequent daughter cells from this progenitor will also express the marker.

Labs will often have MARCM-ready lines which have the inducible FLP, GAL80 distal to a FRT site, GAL4, and UAS-Marker. These can be readily crossed with flies that have a mutation of interest distal to a FRT site. [2]

A Drosophila crossing scheme to produce progeny for MARCM studies Typical MARCM cross.jpg
A Drosophila crossing scheme to produce progeny for MARCM studies

Uses

By taking advantage of MARCM, one can easily trace all the cells that have been generated from a single progenitor. This is useful tool in tracking development and specific cell lineages in various environmental conditions. Applications for MARCM include studying neuronal circuits, [3] clonal analysis, [4] genetic screens, [5] spermatogenesis, [6] growth cone development, [7] neurogenesis, [8] and tumor metastasis. [9]

Many advances in the understanding of Drosophila development have been achieved through MARCM. The development, lineages, and characterizations of secondary axon tracts, [10] anatomical maps of cholinergic neurons in the visual systems, [11] lineages giving rise to a thoracic hemineuromere scaffold and the developmental framework for CNS architecture, [12] the role of Delta in developmental programming in the ventral nerve cord, [13] the wake-promoting octopaminergic cells in the medial protocerebrum, [14] genes involved in neuronal morphogenesis of the mushroom bodies, [15] and the regulation of commissural axon guidance [16] have all been identified through MARCM techniques.

Variations

There are many variations of MARCM. Twin-spot MARCM allows for labeling of sister clones with two separate markers, thus allowing for a higher resolution of lineage tracing. [17] In reverse MARCM, the mutation of interest is placed on the same chromosome as GAL80, so that the wild-type homozygous clones will be labeled. [18] Flip-Out MARCM highlights individual cells inside of mutant clones (ref "Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons," Neuron, 2002). The Q system allows for GAL4 independent MARCM by using the QF/QS system. [19] Lethal MARCM allows for the generation of large marked homozygous populations by including a lethal mutation near the GAL80 site. [20] Dual-expression control MARCM uses the LexA-VP16 transcriptional system in concordance with GAL4-UAS. [21] MARCM is also often used as a genetic screen. [15]

See also

Related Research Articles

The development of the nervous system, or neural development (neurodevelopment), refers to the processes that generate, shape, and reshape the nervous system of animals, from the earliest stages of embryonic development to adulthood. The field of neural development draws on both neuroscience and developmental biology to describe and provide insight into the cellular and molecular mechanisms by which complex nervous systems develop, from nematodes and fruit flies to mammals.

<span class="mw-page-title-main">Mosaic (genetics)</span> Condition in multi-cellular organisms

Mosaicism or genetic mosaicism is a condition in which a multicellular organism possesses more than one genetic line as the result of genetic mutation. This means that various genetic lines resulted from a single fertilized egg. Mosaicism is one of several possible causes of chimerism, wherein a single organism is composed of cells with more than one distinct genotype.

<span class="mw-page-title-main">DSCAM</span> Protein-coding gene in the species Homo sapiens

DSCAM and Dscam are both abbreviations for Down syndrome cell adhesion molecule. In humans, DSCAM refers to a gene that encodes one of several protein isoforms.

<span class="mw-page-title-main">FLP-FRT recombination</span>

In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 µ plasmid of baker's yeast Saccharomyces cerevisiae.

<span class="mw-page-title-main">GAL4/UAS system</span> Biochemical method

The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It is based on the finding by Hitoshi Kakidani and Mark Ptashne, and Nicholas Webster and Pierre Chambon in 1988 that Gal4 binding to UAS sequences activates gene expression. The method was introduced into flies by Andrea Brand and Norbert Perrimon in 1993 and is considered a powerful technique for studying the expression of genes. The system has two parts: the Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS, an enhancer to which GAL4 specifically binds to activate gene transcription.

<span class="mw-page-title-main">Brainbow</span> Neuroimaging technique to differentiate neurons

Brainbow is a process by which individual neurons in the brain can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein in individual neurons, it is possible to flag each neuron with a distinctive color. This process has been a major contribution to the field of neural connectomics.

<span class="mw-page-title-main">ROBO1</span> Protein-coding gene in the species Homo sapiens

Roundabout homolog 1 is a protein that in humans is encoded by the ROBO1 gene.

<span class="mw-page-title-main">SLIT1</span> Mammalian protein found in Homo sapiens

Slit homolog 1 protein is a protein that in humans is encoded by the SLIT1 gene.

<span class="mw-page-title-main">Roundabout family</span>

The Roundabout (Robo) family of proteins are single-pass transmembrane receptors that are highly conserved across many branches of the animal kingdom, from C. elegans to humans. They were first discovered in Drosophila, through a mutant screen for genes involved in axon guidance. The Drosophila roundabout mutant was named after its phenotype, which resembled the circular traffic junctions. The Robo receptors are most well known for their role in the development of the nervous system, where they have been shown to respond to secreted Slit ligands. One well-studied example is the requirement for Slit-Robo signaling in regulation of axonal midline crossing. Slit-Robo signaling is also critical for many neurodevelopmental processes including formation of the olfactory tract, the optic nerve, and motor axon fasciculation. In addition, Slit-Robo signaling contributes to cell migration and the development of other tissues such as the lung, kidney, liver, muscle and breast. Mutations in Robo genes have been linked to multiple neurodevelopmental disorders in humans.

Andrea Hilary Brand is the Herchel Smith Professor of Molecular Biology and a Fellow of Jesus College, Cambridge. She heads a lab investigating nervous system development at the Gurdon Institute and the Department of Physiology, Development and Neuroscience. She developed the GAL4/UAS system with Norbert Perrimon which has been described as “a fly geneticist's Swiss army knife”.

Genetic ablation occurs when a gene is deemed “null” through the homologous genetic recombination of a gene. It is utilized in the selective suppression of a specific cell line or cell type. This genetic engineering technique does not limit growth suppression to just the activity of an individual gene. Specific cell ablation enables the examination of the in vivo activity of cells. An example of this method in action can be seen through the production of a knockout mouse. This is accomplished through the administration of one or more transgenes into a fertilized mouse oocyte’s pronucleus. Afterwards, it is reimplanted into a host mother, who then births a transgenic mouse. The transgenic mouse carries one copy of the transgene3 out of several hundred. From these mice, a homozygous colony can be created through breeding.

Pigment dispersing factor (pdf) is a gene that encodes the protein PDF, which is part of a large family of neuropeptides. Its hormonal product, pigment dispersing hormone (PDH), was named for the diurnal pigment movement effect it has in crustacean retinal cells upon its initial discovery in the central nervous system of arthropods. The movement and aggregation of pigments in retina cells and extra-retinal cells is hypothesized to be under a split hormonal control mechanism. One hormonal set is responsible for concentrating chromatophoral pigment by responding to changes in the organism's exposure time to darkness. Another hormonal set is responsible for dispersion and responds to the light cycle. However, insect pdf genes do not function in such pigment migration since they lack the chromatophore.

Slit-Robo is the name of a cell signaling protein complex with many diverse functions including axon guidance and angiogenesis.

Norbert Perrimon is a French geneticist and developmental biologist. He is the James Stillman Professor of Developmental Biology in the Department of Genetics at Harvard Medical School, an Investigator at the Howard Hughes Medical Institute, and an Associate of the Broad Institute. He is known for developing a number of techniques for used in genetic research with Drosophila melanogaster, as well as specific substantive contributions to signal transduction, developmental biology and physiology.

<span class="mw-page-title-main">Cell lineage</span> Developmental history of a tissue or organ

Cell lineage denotes the developmental history of a tissue or organ from the fertilized embryo. This is based on the tracking of an organism's cellular ancestry due to the cell divisions and relocation as time progresses, this starts with the originator cells and finishing with a mature cell that can no longer divide.

Q-system is a genetic tool that allows to express transgenes in a living organism. Originally the Q-system was developed for use in the vinegar fly Drosophila melanogaster, and was rapidly adapted for use in cultured mammalian cells, zebrafish, worms and mosquitoes. The Q-system utilizes genes from the qa cluster of the bread fungus Neurospora crassa, and consists of four components: the transcriptional activator (QF/QF2/QF2w), the enhancer QUAS, the repressor QS, and the chemical de-repressor quinic acid. Similarly to GAL4/UAS and LexA/LexAop, the Q-system is a binary expression system that allows to express reporters or effectors in a defined subpopulation of cells with the purpose of visualising these cells or altering their function. In addition, GAL4/UAS, LexA/LexAop and the Q-system function independently of each other and can be used simultaneously to achieve a desired pattern of reporter expression, or to express several reporters in different subsets of cells.

The Gal4 transcription factor is a positive regulator of gene expression of galactose-induced genes. This protein represents a large fungal family of transcription factors, Gal4 family, which includes over 50 members in the yeast Saccharomyces cerevisiae e.g. Oaf1, Pip2, Pdr1, Pdr3, Leu3.

James "Jim" William Truman is an American chronobiologist known for his seminal research on circadian rhythms in silkmoth (Saturniidae) eclosion, particularly the restoration of rhythm and phase following brain transplantation. He is a professor emeritus at the University of Washington and a former senior fellow at Howard Hughes Medical Institution Janelia Research Campus.

Susan M. Dymecki is an American geneticist and neuroscientist and director of the Biological and Biomedical Sciences PhD Program at Harvard University. Dymecki is also a professor in the Department of Genetics and the principal investigator of the Dymecki Lab at Harvard. Her lab characterizes the development and function of unique populations of serotonergic neurons in the mouse brain. To enable this functional dissection, Dymecki has pioneered several transgenic tools for probing neural circuit development and function. Dymecki also competed internationally as an ice dancer, placing 7th in the 1980 U.S. Figure Skating Championships.

<span class="mw-page-title-main">Reinhard F. Stocker</span> Swiss biologist

Reinhard F. Stocker is a Swiss biologist. He pioneered the analysis of the sense of smell and taste in higher animals, using the fly Drosophila melanogaster as a study case. He provided a detailed account of the anatomy and development of the olfactory system, in particular across metamorphosis, for which he received the Théodore-Ott-Prize of the Swiss Academy of Medical Sciences in 2007, and pioneered the use of larval Drosophila for the brain and behavioural sciences.

References

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