1. Announcements Exam this Wednesday: my “half” is 40%. Gerry Prody’s “half” is 60%. Exam regrade policy: if you have a question about how I graded an answer, send me an email outlining exactly how you felt I went wrong, and submit the exam for regrade. You MUST (1) make this request in writing, and you MUST (2) submit the exam, both within 5 business days of its return. PLEASE NOTE that I will regrade the entire exam – your grade may go up or it may go down (more likely).

2. Biol/Chem 473 Schulze lecture 4: Eukaryotic gene regulation: Early Drosophila development

3. Eukaryotic transcription is complex

4. Complexity of eukaryotic transcription

5. Insulators may function to organize the genome into transcriptionally autonomous domains

6. Linking complex gene regulation to nuclear organization

8. The best model organism in the whole world!!!! Drosophila melanogaster: “black bellied dew lover” – fruit fly

9. The early Drosophila embryo is a syncytium (a bag of nuclei)

11. (Maternal effect genes)

13. Patterning genes classified by mutant effect on segmentation The hierarchical nature of these interactions can be shown by genetics: mutations in earlier levels of the hierarchy severely disrupt later patterning.

14. Seminal publication and subsequent Nobel

15. Maternal genes Required before the embryo starts transcribing its own genes Mother expresses these genes during oogenesis (mum packs a lunch…) Localization of maternal gene products is the first step in the cascade… http://flymove.uni-muenster.de/Processes/Segmentation/

16. Earliest stages of development run on mRNA’s and proteins deposited by mum during oogenesis An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. How are mRNA’s localized? How are mRNA’s repressed? How are localized mRNA’s then, when required, de-repressed?

17. Maternal effect: Mum packs a lunch for the embryo Anterior endPosterior end

18. Maternal effect: Mum packs a lunch for the embryo The bicoid gene encodes a homeodomain transcription factor. The oskar gene encodes something we are not quite sure about. The gurken gene encodes TGFß homolog - EGF-R ligand, required for dorso / ventral patterning of the egg and embryo. The nanos gene encodes a translational repressor - zinc finger that targets Hunchback- and Bicoid-mRNAs to achieve posterior identity.

19. Maternal effect: Mum packs a lunch for the embryo Polarity of the oocyte cytoskeleton is essential for maternal factor localization (gurken plays a role here). Bicoid mRNA gets localized to anterior pole because another protein, called Staufen, recognizes a structure in the 3’UTR of the bicoid message (and Staufen protein is itself localized by the polarity of the cytoskeleton.

20. Maternal effect: Mum packs a lunch for the embryo Bicoid mRNA gets localized to anterior pole because another protein, called Staufen, recognizes a structure in the 3’UTR of the bicoid message (and Staufen protein is itself localized by the polarity of the cytoskeleton.

21. Maternal effect: Mum packs a lunch for the embryo Bicoid mRNA gets localized to anterior pole because another protein, called Staufen, recognizes a structure in the 3’UTR of the bicoid message (and Staufen protein is itself localized by the polarity of the cytoskeleton). These dsRNA UTR sequences are required for anterior localization of bicoid mRNA by Staufen protein

22. Gap genes The gap genes are expressed by the zygote (activated in the syncytial blastoderm) Their expression patterns are regulated by the maternal gene products, and they regulate each other They divide the embryo into broad, unique domains containing different combinations of transcription factors http://flymove.uni-muenster.de/Processes/Segmentation/

23. Pair-rule genes Pair-rule genes are expressed just before cellularization in patterns of 7 stripes (half the number of segments in a wild type larva) Some pair-rule genes respond to patterns already laid down by the maternal and gap genes, while others are regulated by other pair-rule genes Pair rule gene expression is very dynamic! (pretty pictures) http://flymove.uni-muenster.de/Processes/Segmentation/

24. Segment polarity genes Segment polarity genes are expressed at the onset of gastrulation They are expressed in 14 segment bands, refining the patterns of all the previous genes in the hierarchy. Maternal, gap and pair- rule gene expression begins to fade away http://flymove.uni-muenster.de/Processes/Segmentation/

25. Stripe boundaries are precisely defined

26. Control regions upstream of pair-rule genes are complex

27. ASSIGNED PAPER: Transcriptional regulation of a pair-rule stripe in Drosophila Small, S. et al., Genes & Development (1991) 5: 827-839

28. Main players Even-skipped (eve) is a homeo-box containing transcription factor. Hunchback (hb) has multiple Zn fingers. Bicoid (bic) is a homeo-box containing transcription factor. Kruppel (Kr) contains a Zn finger. Giant (gt) contains a Leucine zipper.

29. Wild type expression patterns of putative regulators of eve stripe 2 even-skipped protein is red hunchback protein is green overlap of expression is yellow

30. even-skipped protein is red Kruppel protein is green overlap of expression is yellow Wild type expression patterns of putative regulators of eve stripe 2

31. even-skipped protein is green giant protein is red Overlap of expression is yellow Wild type expression patterns of putative regulators of eve stripe 2

32. High levels of gt High levels of Kr

33. Wild type expression patterns of putative regulators of eve stripe 2 What predictions arise from this picture? High levels of gt High levels of Kr

34. eve-lacZ gene fusion This is a transgene: gets injected into the embryo (“P transformed embryos”), integrates into the genome and is expressed stably along with all the other genes. BUT: this transgene will express lacZ in an even-skipped-stripe 2 dependent manner!

35. Questions to ask about in vivo expression patterns How do we know the order in which patterning genes act in development? How were potential trans-acting regulators of stripe 2 identified? What would happen to the domain of expression of eve stripe 2 in giant or Kruppel mutant embryos? Do these vivo expression patterns indicate direct interaction between Kr and gt and the eve stripe 2 regulatory region?