Analysis of Biological Development (K. Kalthoff)

Updates to Topic 22: Genetic and Molecular Analysis of Pattern Formation in the Drosophila Embryo


Answers to Questions in Text

Localization of Germ Cell and Posterior Determinants (p.562)

  1. Why was it important to replace the oskar trailer region with the bicoid trailer region in the transgene construct, rather than simply adding the bicoid trailer region to a complete oskar cDNA? Answer: In order to ensure that all of the hybrid mRNA is localized anteriorly and avoid the possibility that some of it is localized posteriorly.
  2. What phenotypes would you expect in embryos derived from females that carry the transgene but are lacking (a) bicoid+, (b) exuperantia+, or (c) oskar+? Answer: (a) as in transformed wild-type embryos, except that the anterior abdomen may be larger or more regular as there will be less anterior gap gene activity. (b) Since neither bicoid mRNA nor the bicoid/oskar hybrid mRNA will be localized, one would expect defective anterior segments, no ectopic pole cells but normal posterior segements and pole cells. (c) essentiall the oskar phenotype (defects in posterior segments and lack of posterior pole cells) combined with ectopic pole cells and abdominal segments anteriorly.

Control of the Pair-Rule Gene hairy+ (p. 569/570)

  1. In the first part of the experiment shown in Fig. 22.23, the researchers transformed wild-type flies. Wouldn't it have been better to transform a hairy null mutant in order to avoid a possible disturbance of the transgene expression by the resident hairy+ gene? Answer: No, if such “disturbance” existed, one would want it to operate in the experiment as well.
  2. How could the investigators have tested whether the Krüppel and knirps proteins that control hairy+ expression in stripes 6 and 7 bind directly to enhancer elements RK and RR? Answer: One option would be DNase footprinting as discussed on page 415. The investigators actually used another method known as immunoprecipitation. They incubated labeled RK and RR enhancer elements with knirps or Krüppel protein before adding antibodies against the protein. The RK and RR elements were precipitated along with knirps or Krüppel, indicating direct binding.
  3. As an analogy to the control of hairy+ gene expression, you could imagine that you are on a cross country trip in your car, and that you listen to the same National Public Radio program (hairy+ gene expression) broadcast on different frequencies by the local radio stations (the eight hairy expression domains) that you pass on your way (from the anterior to the posterior egg pole). Of course, you need to adjust the tuner of your radio as you move from one station to the next. In fact, you may imagine that instead of a tuner you have an array of fixed antennal circuits, with at least one circuit adjusted to the frequency of each station. In terms of this analogy, which part of the entire radio transmission system would correspond to (a) to the stripe-specific enhancers of the hairy+ gene and (b) to the gap gene proteins that drive hairy+ expression from these enhancers? Answers: (a) the fixed antennal circuits and (b) the radio waves emitted by the stations.

Ventralizing Effect of Toll Protein (pp. 586-588)

  1. Given that the recipients were dorsalized due to the lack of local Toll activity, how could the investigator tell whether he was injecting a recipient dosally, ventrally, or laterally? Answer: The dorsoventral polarity of the follicle cells, which is induced by the gurken/torpedo signal and unaffected by the lack of Toll, is sufficient to impose dorsoventral polarity on the vitelline membrane and chorion. This polarity is easily recognized by the dorsal appendages of the chorion and the stronger curvature of the chorion on the ventral side.
  2. Which control could be done to ascertain that the observed local ventralization is caused by the Toll activity of the injected cytoplasm and not some other stimulus associated with the injection procedure? Answer: Inject cytoplasm from donors derived from Toll- mothers. No local ventralization should occur.
  3. In the recipient embryos, the entire hierarchy of maternal effect genes down to the local generation of active spätzle ligand should work. Then why is the local ventralization occurring at the site of injection, rather than where spätzle is highest? Answer: A slight dorsoventral bias in the extent of the injection-induced ventralization was indeed observed. However, in contrast to wild-type embryos, where all spätzle ligand is immediately sopped up by an excess of Toll receptor, the mutant embryos used here did not have any Toll receptor except what was provided by the injected cytoplasm. Under these conditions, enough spätzle ligand can diffuse to the site of active Toll receptor near the site of injection.

Selector Gene Properties of engrailed+ (p. 589/590)

  1. In Fig. 22.44, why is the wing outline in part (c) drawn differently from the outlines in parts (a) and (b)? Also, why has the labeled clone hitting the posterior wing margin a border of strong bristles in part (e) but not in part (b)? Answers: Parts (a) and (b) show wings of flies that are heterozygous engrailed1/+ and therefore show normal wing morphology. Part (c) shows the wing of a homozygous engrailed1/1 fly. The posterior wing compartment therefore resembles a mirror image duplication of the anterior compartment. The clone hitting the posterior wing margin is heterozygous engrailed1/+ in part (b) but homozygous engrailed1/1 in part (e). In the latter case, posterior clones acquire anterior morphology, including the strong bristles at the wing margin.
  2. In subsequent work with an engrailed allele (engrailedC2) causing a more severe loss of function, the investigators generated (engrailed1/ engrailedC2) heterozygous clones. If such clones originated in the posterior compartment but near the compartment border, they crossed into the anterior compartment, as seen in Figure 22.44e. However, when such a clone originated away from the compartment border, the margin of the clone constricted, and the narrowing ring cleanly pinched out the clone so that it separated from the wing blade as a vesicle (Fig. 22.44f). How do you explain these results? Answer: Cells with engrailed+ activity sort out from cells without engrailed+ activity, due to different cell surface properties.

Compartment Interactions Across Boundaries in the Wing (pp. 594-596)

  1. In the experiment diagrammed in Fig. 22.49, why would the removal of the yellow+ segment from the transgene by flp recombinase cause a phenotypic effect? Should not the resident gene copies of yellow+ mask the loss of this gene from the transgenic construct? Answer: The transgenic flies must lack endogenous yellow+ activity to make this experiment work.
  2. The flp-out technique described above can be used similarly to generate marked cell clones that constitutively express engrailed+. Based on the model diagrammed in Fig. 22.48, what phenotypic effects would you expect if such clones form (a) in the posterior compartment and (b) deep in the anterior compartment? Answer: Clones in the posterior compartment have no morphological effect because engrailed+ is expressed there normally. Clones in the anterior compartment cause formation of additional wing structures resembling those resulting from ectopic hh or dpp synthesis (see Fig. 22.50; see also Fig. 1 of Zecca et al. (1995) Development 121: 2265-2278).
  3. By mitotic recombination, induced by X-rays (see Method 6.1) or flp, one can generate marked cell clones that are lacking endogenous engrailed+ activity. Suppose you generate such clones in flies that contain a lacZ reporter gene driven (a) by a hh promoter or (b) by a dpp promoter. How should the expression of the lacZ reporter transgene change if clones without engrailed+ activity form deep in the posterior wing compartment? Answer: Such clones should fail to express the hh-lacZ transgene, in contrast to surrounding cells, which do. All cells in such clones should express the dpp-lacZ transgene, in contrast to surrounding cells, which do not.

Comments

Some reviewers of the 1st as well as the 2nd edition found this chapter excessively long. Of course I feel vindicated by the award of the 1995 Nobel Prize to Lewis, Nüsslein-Volhard, and Wieschaus. Students in my course groan about the many gene names to learn, but they also appreciate the power of genetic and molecular analysis on display here. How did this chapter work in your course? (KK)

Clarifications and Corrections

New Review Articles and Research Articles

on Maternal Genes Affecting the Anteroposterior Body Pattern

on Segmentation Genes

on Homeotic Genes

on The Dorsoventral Body Pattern

on The Compartment Hypothesis and Appendage Formation

on Patterning Genes in Other Insects


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Last modified: 07 October 2002