1. Genetics of Axis Specification in Drosophila: Anterior-Posterior Axis Determination
Gilbert - Chapter 9

2. Axis Specification Controlled by a variety of genes
Maternal effect genes
Gap genes
Pair-rule genes
Segment polarity genes
Homeotic selector genes

4. Genetic Screen for Genes involved in Drosophila Development
Nusslien-Volhard, Wieschaus
Fed mutagens to Drosophila
Then breed until mutation is homozygous recessive
Examined embryos for patterning defects
Used embryonic cuticles to do screens
Looked at pattern of denticles, shapes of segments

5. Goals of Genetic Screen
Create small mutations in fruit fly genome
Enough to mutate EVERY gene in the genome at least once
Identify EVERY mutation in the genome that affects embryonic development in the fruit fly
How many genes are there in fruit flies?
Note - At this time the D. melanogaster genome was not sequenced

6. How was the genetic screen performed?
Feed adult fruit flies with a mutagen
EMS - causes high mutation rate in offspring
Point mutations, short deletions, rearrangements
Can cause defective proteins, absence of proteins
THEN - breed the flies until the mutation is homozygous, and look for ANY embryo that has abnormalities
TENS OF THOUSANDS OF EMBRYOS ARE EXAMINED!!!

7. distinctive structures The genetic screen looked
The wild-type body
is segmented
and each segment
has a unique identity
and thus produces
distinctive structures
The genetic screen looked
for changes in this body plan

8. The molecular genetics of pattern formation in candy corn
Anterior
Borrowed from Mark Peifer, Ph.D.
One of my great prof’s. at UNC!
Posterior
The wild-type pattern

9. They collected a series of mutants affecting the body plan
Wild-type
Posterior
group
Anterior group
Morphogenesis
defects
Gap gene
Borrowed from Mark Peifer, Ph.D.
One of my great prof’s. at UNC!

10. Wieschaus and
Nüsslein-Volhard
removed single genes
and looked for effects
on the body plan

11. Genes involved in embryogenesis
Genes controlling embryonic development are either
maternal-effect genes
mRNA or protein already deposited in the egg
zygotic genes
Transcribed from nucleus of zygote

12. Maternal effect genes: Anterior - Posterior Polarity
Placed into developing oocyte by maternal cells
Nurse cells that surround the egg
Several genes were discovered
What phenotype would you be looking for in your embryos??

13. bicoid mutants have no head end!!
Wildtype larva
bicoid mutant
Gilbert Fig. 9.13

14. Bicoid Mutant
Lacks anterior structures, posterior structures are duplicated
Leads to lots of Molecular Biology questions!
How can we find this gene and its sequence?
What does the phenotype tell us about the function of bicoid?
What is the bicoid protein like in this mutant?

15. Bicoid and Nanos Bicoid mRNA Nanos mRNA
Concentrated in the future anterior end of the ovum by the nurse cells
Nurse cells are ovary cells of the mother
The mRNA is held in place by a network of microtubules
Nanos mRNA
Tethered to the cytoskeleton at the future posterior end of the egg

17. Bicoid and Specification of the Anterior pole
Bicoid appears to be essential for the formation of anterior structures
Further evidence
Bicoid mRNA is localized to the anterior end of the oocyte
As bicoid mRNA gets translated, a gradient of bicoid is created from A to P
More concentrated at most Anterior end

18. Localization of bicoid mRNA - What technique was used??

19. Bicoid Protein gradient: What technique is used?

20. Bicoid - More evidence
Bicoid mRNA (wild-type) can rescue the Bicoid mutant phenotype
Inject bicoid mRNA into anterior end of bicoid mutant embryo
Injection of WT bicoid mRNA anywhere into the early embryo turns that area into a head end!

23. What does bicoid do, and how?
Bicoid protein is arranged in a gradient
It represses the molecules that control posterior identity
Represses translation of a molecule that helps specify the posterior end - Caudal
Caudal mRNA is found in entire embryo
Caudal protein is found at posterior end

24. Caudal protein localization

25. What does bicoid do, and how?
Bicoid protein is a transcription factor
Enters nuclei, activates downstream gene expression
Hunchback - essential for anterior pole formation
Hunchback activates transcription of further head-specific gene products (swallow, exuperantia, buttonhead, orthodenticle)

28. A-P axis specification: a combination of protein gradients
Nanos - mRNA tethered to posterior pole
Forms a gradient from posterior to anterior
Inhibits translation of Hunchback mRNA in the posterior region
What would be the phenotype of a Nanos mutant?
Resulting gradients
A-P gradient of bicoid
A-P gradient of hunchback
P-A gradient of nanos
P-A gradient of caudal

33. Maternal effect genes and AP polarity - summary
Maternal mRNA’s are tethered to either A or P ends of oocyte
Proteins are translated to create a gradient
Activate or repress embryonic gene expression (hunchback, caudal)
Result is and anterior region and a posterior region

34. Hunchback protein localization