Lab Week 12
Drosophila Development #1
Genes controlling development in Drosophila
by Coral Warr and Derek Lessing
Background
Genes controlling body plan development
The combination of highly developed genetic methods and the more recent application of molecular genetics and cell biology techniques has made Drosophila melanogaster a particularly informative organism for the study of developmental genetics. The molecules that are important in the development and specification of the segmental body plan of the Drosophila embryo have been discovered and analyzed by looking for mutants that specifically disrupt that body plan. Most of these mutants were isolated in a large genetic screen performed by Ed Lewis, Christianne Nusslein-Volhard, Eric Weischaus and their colleagues and for this work they won the Nobel prize in Medicine in 1995. The understanding of these genes and their products was revolutionary in developmental biology and paved the way for much of our understanding of developmental genetics in vertebrates. The study of development in Drosophila continues to provide new insights and promote understanding of developmental genetics in all multicellular organisms. Probably the most amazing aspect of developmental biology has been the high degree of homology observed between genes involved in Drosophila and vertebrate development.
The analysis of these genes showed that the specification of the segments of the fly embryo is carried out through a cascade of transcription factors that delineate regions of the embryo and ultimately regulate the genes responsible for giving each segment its individual identity. These genes which encode transcription factors are expressed in overlapping but distinct patterns that delineate progressively smaller regions of the embryo's segments. A given gene can either activate or repress a gene downstream from it and it is the combination of activation and repression that results in the correct pattern of gene expression.
Maternal genes regulate the gap genes, which then divide the embryo into large sections. These genes in turn regulate genes known as pair rule genes because they are expressed in stripes that specify alternating segment boundaries. There are two classes of pair rule genes, primary and secondary, with the primary directly responsible for expression of the secondary. The pair rule genes regulate both the segment polarity genes, which give each segment its anterior posterior orientation, and the homeotic genes, which give each segment its specific identity.
Drosophila embryogenesis
Drosophila and other insects have an unusual early cleavage pattern in which nuclei divide without cytokinesis. The early divisions are rapid (<15 min), but well organized with pools of cytoplasm dividing and following around individual nuclei. During this phase of development, it is difficult to observe individual nuclei since the interior of the embryo is very yolky, but sometimes if you look carefully you can see the nuclei with their poles of cytoplasm as slightly lighter areas within the embryo. The first easily observable event occurs during the 13-14 division cycle when a small number of cells form at the posterior of the embryo. These cells are known as the pole cells and are the precursors of the germ line. The embryo at this point is known as a syncytial blastoderm.
As division proceeds, membrane furrows form around each nucleus, which have moved to peripheral positions around the embryo. The embryo is now a cellular blastoderm. One more round of proliferation expands this single cell layer of the embryo. Gastrulation then commences with a ventral furrow forming as the germ cells move to a more anterior position and then internalize. The presumptive endoderm and mesoderm involute through the ventral furrow (as opposed to other organisms that involute through dorsal surfaces). In Drosophila, the nervous system also forms on the ventral side, in a cell layer just underneath the ventral surface known as the mesectoderm. At the anterior and posterior end of the furrow, the ectoderm invaginates to form the anterior and posterior midgut and later the stomodeum (mouth) and hindgut. These invaginations eventually fuse to form the digestive tract of the embryo. A second predominant furrow that extends laterally at the anterior third of the embryo is the cephalic furrow, which delineates head segments.
The processes of gastrulation result in a structure referred to as a germ band that contains the precursors for all the body parts of the embryo. This germ band extends around the posterior until it almost meets the anterior, and then it retracts again to the posterior. During germ band extension and retraction, the segmental boundaries that characterize the larval stage form. A Drosophila larva has 3 head segments, 3 thoracic segments and 8 abdominal segments. The head segments of the developing embryo involute during late gastrulation and so they are internal to the thoracic segments. Later development during embryogenesis involves the differentiation of the various structures and organs of the larva. Most notably, movement begins in the gut at about 16 hr. and then in muscles about 17 hr. The trachea or respiratory apparatus of the embryo forms as darkly pigmented tubes that fill with air upon hatching. The embryo hatches as a larva at about 20-24 hr ( at 25 degrees C).
Using gene fusions as molecular probes
The expression of a specific gene can be followed and its regulation analyzed by a molecular technique known as reporter gene fusions. The flies you will be working with have a genetically engineered gene fusion inserted into one of their chromosomes. Gene X has its control and promoter regions fused to the lac Z gene, which encodes the beta-galactosidase enzyme (we will call this gene fusion construct gene X-lac Z). The enzyme is produced wherever the gene would normally be expressed in a wild type background. If the gene fusion were placed into a background where gene X's regulation is affected, then the expression of the enzyme would reflect that change in regulation. The enzyme is detected through a reaction in which a compound known as X-gal is broken down to produce a blue color.
Overall Aim
In the next two laboratory session, you will use the molecular technique of reporter gene fusions to study the role of genes involved in setting up the Drosophila body plan.
Objectives
2) To use molecular techniques, such as gene reporter fusions, to study the hierarchical relationships between genes that encode transcription factors responsible for the development of the Drosophila body plan.
Drosophila segmentation genes: I
BRIEF OVERVIEW:
This week we will perform two experiments:
2. We will examine reporter gene strains which are expressing the lacZ gene under the control of regulatory regions from three different segmentation genes. These genes represent examples of the gap genes and the pair rule genes.
MATERIALS:
Each pair of students will be given embryos from two of the following reporter gene strains:
The strains will be labeled A-C, and in the Analysis section below you will determine which strain is which based on the expression pattern you see for your strain, and the patterns your fellow students see in the other reporter strains.
Note: The parents of two of the types of embryos were homozygous for the lacZ construct, thus all the embryos will contain the construct. These two are the hairy-lacZ and the Kruppel-lacZ constructs. The parents of the embryos containing the other construct (even-skipped-lacZ) were heterozygous for the construct, thus only three-quarters of the embryos will contain the construct. Therefore, one-quarter of the embryos from this two strain would not be expected to exhibit any staining.
In addition, younger embryos (0-2 hours) will not stain and can be ignored.
You will be given one basket of embryos from your reporter strain, which will have 0 to 5 hour old embryos that will be used for lacZ staining. You will also have another basket of wild type embryos of mixed ages (0-24 hours), which we will use to examine Drosophila development in live embryos. Embryogenesis in Drosophila can easily be observed in living embryos using the compound microscope. In order to view the embryos, the outer layer of the egg, the chorion, must be removed.
Embryos were collected on apple juice-agar plates that have been smeared with live yeast. The younger collections from the reporter gene strains were started this morning, while the older collections of wild type embryos were made over the previous 24 hours. The embryos have been collected off the plates for you, and stored in PBT in a staining basket in a 12 well plate.
Today you will be making two kinds of preps. In the first prep, you will use your reporter gene strain to look at expression of the lacZ construct at an embryonic stage (cellular blastoderm) prior to morphologically apparent segmentation. The second prep will involve making slides of wild type embryos to allow you to observe different developmental stages.
METHOD:
Step 1 is the same for both preps, therefore can be done with both sets of embryos simultaneously. When you finish step 1, leave your wild type embryos sitting in PBT in an eppendorf tube while you continue with the X-gal staining protocol with your reporter gene strain. Once you have these staining, you can return to the wild type embryos for mounting on slides and examination using the microscope.
Observe the embryos using your dissecting microscope at highest magnification. At the anterior, the eggs have two thread-like structures known as dorsal appendages ("rabbit ears") which are used for respiration. The outside layer of the embryo is called the chorion. In the older embryos, you may be able to see the black denticle belts on the late larvae showing through the chorion.
***Sketch the appearance of the wild type embryos before proceeding to dechorionate them.
Perform step 1 with both sets of embryos.
1. Dechorionation (removal of eggshell)
Pipette 2mLs of 50% bleach into a well of your plate (Remember that bleach ruins clothes, so be careful!). Also pipette 2mLs of PBT into the next well ready for immediate washing. Transfer the basket containing the embryos to the well containing bleach. 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.
For the wild type embryos, use your 1mL Pipetteman and a blue tip to suck them up and transfer them to an eppendorf tube. Leave them sitting in PBT while you complete the X-gal staining with your reporter gene embryos.
Prep 1: X-gal staining of geneX-lacZ embryos
2. Fixation.
(Wear gloves for this step)
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 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 beta-galactosidase enzyme.
Transfer basket into PBT and wash for 5 mins on shaker. Repeat wash 2 times. Before proceeding to staining, pipette the used heptane into the marked waste beaker using your 1mL Pipetteman.
3. X-gal staining
(wear gloves for this step)
Add 1.75 mLs X-gal staining solution to a new well. Place basket into the staining solution and put plate at 37 degrees C (in the 37 degree C incubator in the lab). The blue staining reaction should occur in between 30 and 60 minutes. After 60 minutes check your staining under your dissecting scope. Consult with an instructor whether staining is complete and you should proceed to washing, or whether you should stain for another 30 minutes.
While you are waiting for the staining reaction to occur, proceed to mount and observe your wild type embryos as outlined in Prep 2 below.
4. Observation
When staining reaction is complete, remove the basket from the staining solution and place into 2mLs PBT. Wash on the shaker for 5 minutes. Repeat the wash twice. Using your 1 mL Pipetteman, suck up the embryos into a blue tip and transfer them to an empty well (no basket). View under your dissecting scope.
Note: Sometimes some non-specific staining occurs, resulting in embryos that appear blue all over. Ignore these.
*** Sketch the reporter gene expression pattern(s) that you observe.
*** Observe (and sketch if necessary) the reporter gene expression patterns obtained by your fellow students or by the instructor.
Prep 2: Examination of wild type embryos
2. Slide preparation
Remove as much PBT as possible (don't worry if you lose some embryos, it is unavoidable). Add 100 microliters of 70% glycerol to tube and flick to mix with residual PBT. Using a blue tip, transfer embryos to a microscope slide. Cover the embryos with a coverslip and examine using your compound microscope.
Observe the embryos under 10x magnification with your compound microscope. You can orient yourself by observing that the anterior of the egg has structure known as the micropyle while the posterior is slightly blunted. The ventral side is more convex than the dorsal side.
*** Using the staging series showing the stages of embryo development, identify four embryos at different developmental stages, and draw them. Label any apparent structures, furrows or cells. It is interesting to continue to observe your embryos as they proceed through development. In later stages you may see the embryos moving.
ANALYSIS AND INTERPRETATIONS:
2. For your two reporter gene embryos, what proportion of the embryos showed the staining pattern? What are the reasons only some of the embryos stain?
3. From your sketches of the different reporter gene staining patterns determine which of strains A-C represents each reporter gene. You may find the eve-lacZ and hairy-lacZ strains difficult to distinguish based on their expression patterns. What piece of information that you were given about the strains enables you to distinguish them?
4. Describe the reporter gene expression pattern of each of your two genes, and relate it to which class of segmentation gene each gene belongs to.
***Next week you will be examining the effect of mutations in several genes (Krupple, hedgehog, hairy and fushi-tarazu) on the expression of the fushi-tarazu gene. An excellent reference for this is the Interactive Fly website. The (very long!) url for this is: flybase.bio.indiana.edu:82/allied-data/lk/interactive-fly/aimain/1aahome.htm.
This page was posted 13 April 2003