Fluorescent tagging to separate cell types representative of germ layers

Model Organism: Worms Methods & Reagents
1

I’m a new worm researcher, so thanks in advance for the help, and apologies if my question is naive. :slight_smile: I’ve been told that the worm community is very collegial and open, and it certainly seems that way from the forums, so here goes…

I’m interested in testing whether expression levels of my gene of interest are similar across germ layers in adults following a chemical exposure.

To do this, I’d like to choose three cell types that are representative of the three germ layers (one belonging to ectoderm, one to endoderm, one to mesoderm)
and separate the cells using FACS to test the different cell types individually.

I think I should be able to use fluorescent tags (three different colors?) that are linked to cell type-specific promoters/genes, so that my cell types of interest show up as different colors and are easy to sort.

Ideally, I’d be able to purchase strains that contain cell type-specific genes tagged with different fluorescent proteins for this purpose.

Any ideas would be greatly appreciated! Many thanks in advance!

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Hi, welcome to the forum. While we are waiting for the ‘gastrulation stars’ to come on stage, I can fill in as the warm up act.

If you are interested in the expression of your GOI in response to something like bisphenol A (isn’t google wonderful?) in ecto-, meso- and endoderm, then my first suggestion would be to look at the embryo as it has all three in a nicely laid out pattern. I assume that you are interested in early development, so I guess also that your GOI would be expressed in the embryo? You could either use a transgenic strain carrying your gene construct or you could isolate individual germ cells from embryo cultures. Then you don’t need to go down the complicated road of FACS for three different cell types.

Of course, FACS is also a commonly used approach…

In any case, I assume you have seen/read the following?:

http://www.wormbook.org/chapters/www_gastrulation/gastrulation.html

and more generally:

http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm

(see the section on the embryo)

For embryonic cell culture:

http://www.wormbook.org/chapters/www_culture/culture.html

Now, if you are really committed to FACS, then you just have to sit tight and wait for the flood of suggestions for cell type-specific fluorescent markers and contemplate the amount of work necessary to get three distinguishable labels into your worms!

Steve