Movies


Optogenetics


This movie demonstrates the effect of activating the ion channel channelrhodopsin-2 in Drosophila larvae. Channelrhodopsin-2, encoded by a foreign gene (a transgene), was expressed in neurons containing the excitatory neurotransmitter substance glutamate. These neurons include the motor neurons that control the body wall muscles. 

Channel opening induced by blue light triggers action potentials (nerve impulses) in the motor neurons, causing a visible "mass contraction" of the body wall that shortens and broadens the larva. Characteristically, the contraction is immediately followed by a relaxation when the light is turned off, so that the larva again appears long and slender. 

Dietary supplementation of the cofactor all-trans-retinal is necessary for channelrhodopsin to function. Therefore, the mass contraction cannot be evoked by light in control animals that have been not been fed retinal.

Two examples of responses to blue light stimulation in a control larva is shown first, followed by three responses of an experimental animal raised on food containing retinal. Note that the control animal may sometimes also respond to light. Drosophila larvae have vision and thus display a "normal" behavioral response when elucidated. However, the mass contraction seen in the experimental animal, which is temporally closely linked to the period where the light is on, is missing in the control animal.




For more information about how to perform this and similar experiments, see the following articles:

  • Honjo et al. 2012. Optogenetic manipulation of neural circuits and behavior in Drosophila larvae. Nature Protocols  7, 1470-78.
  • Pulver et al. 2011.Optogenetics in the teaching laboratory: using channelrhodopsin-2 to study the neural basis of behavior and synaptic physiology in Drosophila. Advances in Physiology Education 35, 82-91.


Generation of clones by mitotic recombination





This movie illustrates one way of producing homozygous mutant cell clones (x/x) on a heterozygous (x/+) genetic background, where x and + indicates a mutant allele and a wild-type allele, respectively. First, a pair of homologous chromosomes is shown. The blue chromosome carries a reccesive mutation (red cross). The red chromosome carries a transgenic insertion encoding both the red marker mini-white (labeled " w+ ") and a recessive lethal allele of a “killer” gene (indicated with a cross on blue background). The following events are shown in sequence:

1. DNA replication in preparation for mitosis.

2. Recombination between two chromatides, one from each of the two homologous chromosomes. Recombination does not normally happen in mitosis but can be induced by expressing FLP recombinase. This enzyme exchanges the chromosome arms distal to FRT recognition target (FRT) sites, shown as white triangles. The centromeres are shown as black dots.

3. Chromosome alignment. Only the most interesting of two possible orientations is shown.

4. Separation of the chromosomes and cleavage  (cytokinesis) of the mother cell into two daughter cells. One of these (topmost cell on the video) is homozygous for the recesive mutant allele (x/x) and does not carry the red mini-white marker. If expression of the FLP recombinase (and thereby mitotic recombination) has been targeted to the eye, cells with this genotype will display the mutant phenotype and will also be white (assuming that the experiment is performed on a white mutant background) .

The second daughter cell (lowermost) has the mini-white in double dose and will in principle be red. However…

5. …because the second daughter cell is also homozygous for the recessive lethal “killer” gene, it dies, leaving only the mutant daughter cell.

In other experimental designs, the allele of the “killer” gene is dominant rather than recessive as shown here. This ensures that heterozygous cells (x/+) that may produced if the FLP recombinase fails or induces recombination an even number of times, are killed as well as the wild-type homozygotes (+/+).

Tissue-specific induction of mitotic recombination is very helpful when working with recessive alleles that are lethal if all cells in the organism have it on both chromosomes. If only the eye (or another non-vital organ) is made homozygous (x/x) while the rest of the animal remains heteroxygous (x/+), the impact of the allele can be revealed by studying changes in the structure or function of the eye.




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