Lab Week 13

Drosophila Development #2

Genes controlling development in Drosophila Part 2
by Coral Warr and Derek Lessing

Brief overview
The purpose of this lab is to examine the expression of the fushi tarazu (ftz) gene in the presence of mutations in genes that normally regulate ftz expression. In the embryos you will examine, the ftz upstream regulatory region has been fused to the coding sequence for b-galactosidase (the lacZ gene from E. coli) and reintroduced into flies by P element-mediated germ line transformation. The presence of lacZ enzyme will be detected by staining with X-gal.

You will be given live embryos from at least two of the fly strains (hopefully more if we get enough embryos!):

Kruppel-; ftz-lacZ
hairy-; ftz-lacZ
fushi tarazu-; ftz-lacZ
hedgehog-; ftz-lacZ

The parents of the embryos were homozygous for the ftz-lacZ construct, but heterozygous for the mutation. (Remember, most of the genes that affect segmentation are recessive and embryonic lethals, so the mutant stocks are heterozygous.) If the gene that has been mutated affects ftz exprssion, one quarter of the embryos should be homozygous mutants that show this misexpression.

You will be given a group of embryos that will be from 0 to 5 hours old. These will be used for ftz-lacZ staining following the same protocol you used last week.

Protocol for X-gal staining of ftz-lacZ embryos

1. Egg collections
Eggs are collected on apple juice-agar plates that have been smeared with live yeast. The younger collections were started this morning. You should take a look at the eggs that have been collected for you under a dissecting scope. Notice the "rabbit ear" respiratory appendages.

2. Dechorionation
Remember that bleach ruins clothes, so be careful!

Add approximately 2 ml 50% bleach into egg collection basket. Use enough bleach to cover embryos. Dechorionation should be complete in 3 minutes. You can watch the respiratory appendages dissolve under the dissecting scope. When they are gone, dechorionation is complete.

Transfer basket to PBT and place plate on shaker to wash eggs for 5 minutes. Transfer embryos to another 2mLs of PBT and repeat wash.

3. Fixation
(Caution: Wear gloves for this step)

Remove PBT using a yellow tip on the end of a disposable pipet and discard PBT (the smaller bore of the yellow tip prevents you from sucking up too many embryos when removing the PBT). Pipette 2 mLs of glutaraldehyde-saturated heptane (fix) into a fresh well (the heptane is the upper phase). Mark the well that contains the fix solution as it will need to be discarded into a special discard jar later. Also pipette 2 mLs PBT into a fresh well ready for immediate washing. Transfer basket to the fix solution and place plate on shaker for 4 minutes (this time is critical!). Heptane permeabilizes the vitelline membrane and vigorous agitation helps dislodge it. Glutaraldehyde covalently cross-links proteins to preserve morphology. It is important not to fix embryos too long! You're trying to fix long enough to preserve morphology, but not so long that you kill the b-galactosidase enzyme.

Transfer basket into PBT and wash for 5 mins on shaker. Repeat wash 2 times (total 3 washes). Before proceeding to staining, pipette the used heptane into the marked waste beaker using your 1mL Pipetteman.

4. X-gal staining
Add 1.75 ml X-gal staining solution into an empty well and transfer basket. Put tray into the 37 degree C incubator/shaker. The blue staining reaction should occur quickly (~30-60 mins minutes). You can monitor the staining reaction under the dissecting scope.

5. Mounting embryos for examination
When staining reaction is complete, move basket to a well of PBT to rinse. Use a blue pipette tip to transfer embryos into an Eppendorf tube. Try to get as many as possible. Let embryos settle to bottom of tube. Remove PBT and add 100 microliters of 70% glycerol mounting medium. Gently flick tube to mix glycerol and residual PBT. Use a Pasteur pipet to transfer embryos from the tube to a microscope slide. Gently place a coverslip over embryos and view by bright field microscopy. (You can seal the coverslip with fingernail polish to make semipermanent preparation.)

ANALYSIS AND INTERPRETATIONS:

1. Describe the reporter gene expression pattern(s) present in each batch of embryos. Make sketches of a few representative embryos that show good staining. Use the embryo staging series to stage your stained embryos. For staging, use handouts or the Interactive Fly website. The url for this is: flybase.bio.indiana.edu:82/allied-data/lk/interactive-fly/aimain/1aahome.htm.

2. How can you tell which are wild-type embryos, and which one are the mutants?

3. What would you predict about the effect of a loss of function mutation of each of the four genes: Krupple, hedgehog, hairy and fushi tarazu on the expression of ftz in a mutant embryo? (See References to help you make these predictions.)

4. Which mutations did you receive?

References:

**Wolpert, L. et al (2002) Principles of Development, 2nd ed. Oxford University Press

**S. F. Gilbert (1997) Developmental Biology, 5th Edition. Sinauer Associates.

**S.B. Carroll and M.P. Scott (1986) Zygotically active genes that affect the spatial expression of the fushi tarazu segmentation gene during early Drosophila embryogenesis. Cell 45: 113-126--initial report of effects of gap and pair-rule gene mutations on ftz expression. Includes pictures of anti-ftz antibody staining of Kruppel, hunchback, knirps, hairy and eve mutant embryos among others. Click here for a PDF file of Carroll and Scott (1986)

This page was posted 18 April 2003

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