Exam 4: Lecture 6 Flashcards
1
Q
Cone Cells in Drosophila Retina
A
- each unit eye contains four lens secreting cells called cones
- different than cone photoreceptors
- secrete overlying lens
2
Q
Lens of Drosophila
A
- made of roughly 30 protein layers
- main protein constituent of each layer is drosophila crystallin (dcy)
- homologous to crystallin proteins that make up human lens
- lens has different refractive index than surrounding air
- lens channels photons of light onto photoreceptors
3
Q
Development of Insect Retina
A
- photoreceptor cells instruct four undifferentiated cells to adopt a cone cell fate
- process of cone cell recruitment is thought to occur via EGF Receptor Pathway and Notch Pathway
- these pathways activate D-Pax2 gene in cells destined to become cone cells
4
Q
D-Pax2 gene
A
- key target of EGF Receptor Pathway and Notch Pathway
- required for formation of cone cells
5
Q
Photoreceptor cells
A
-produce ligands that bind to membrane bound receptors on presumptive cone cell surface
6
Q
Paired Box (Pax)
A
- founding member of Pax family of TF’s is Drosophila Paired protein
- contains special binding domain called PAIRED domain
- over years number of similar proteins have been identified in all organisms
- different Pax proteins grouped into distinct classes based on protein structure
7
Q
Pax1 and Pax9
A
-contain paired DNA binding domain and an eight amino acid octapeptide used in transcriptional repression
8
Q
Pax 2, Pax5, and Pax8
A
- contain paired DNA binding domain, octapeptide and first helix of homeo DNA binding domain
- lacks second and third helices so these proteins do not interact with DNA through this motif
- Drosophila D-Pax2 belongs in this class
9
Q
Pax4 and Pax6
A
- have intact paired and homeodomains
- can bind to DNA using both domains
10
Q
D-Pax2 in Cone Cells
A
- in wild-type retina is expressed in just the four cone cells
- loss of D-Pax2 expression leads to complete loss of all cone cells and roughening of external surface of compound eye
11
Q
Isolation of Rough Eye
A
-convenient way to screen for genes that affect eye development
12
Q
Lab Induced Mutation
A
- some use x-rays which will cause deletions within genome
- others use chemicals like EMS to induce single base changes
- some use transposable elements to jump around genome and inserting themselves into genes
- mutations that eliminate expression of D-Pax2 within cone cells are located in eye-specific enhancer
- D-Pax2 also expressed in other tissues-but these flies only eye enhancer has been disrupted
13
Q
D-Pax2 Eye Specific Enhancer
A
- approximately 500bp in length
- sufficient to drive expression of reporter construct just within cone cells
- contains binding sites for at least 4 proteins
- Pointed and Yan TF’s are downstream members of EGF REceptor signaling cascade
- Su(H) DNA BP is most downstream member of Notch Pathway
- Lozenge is TF that is expressed in all cells of developing retina (not developmentally regulated)
- binding sites make up only small fraction of DNA found within this enhancer
- some sequences conserved in other Drosophila species and may represent binding sites for additional proteins
14
Q
Sequences in D-Pax2
A
- some found solely in Drosophila melanogaster
- may simply be necessary for proper spacing between binding sites
- binding sites nearly always separated from each other since proteins that bind are large and bulky
- sites too close, DNA BP’s will not have enough room to bind onto enhancer
15
Q
Enhancers
A
- bound by DNA BP’s.
- some contain single recognition sites for multiple TF’s
- some contain multiple recognition sites for several different BP’s
- transcriptional output dependent upon number of TF binding sites on enhancer
16
Q
Pathways
A
- review EGF Receptor Pathway
- review Notch Pathway
17
Q
Activation of D-Pax2 Enhancer
A
- mutations that alter ability of Pointed (Pnt), Suppressor of Hairless (Su[H]) or Lozenge (Lz) to bind to D-Pax 2 eye enhancer results in complete loss of D-Pax2 expression within presumptive cone cells
- results in failure of cone cells to develop and structure of eye is disrupted (since lens is not secreted)
18
Q
Model
A
- combinatorial code of TF’s required to activate expression of target genes
- Lz DNA BP as well as EGF Receptor and Notch Pathways are required to activate expression of D-Pax2 gene and specify cone cell fates
- loss of any of the factors will inactivate D-Pax2 and presumptive cone cells will either remain undifferentiated or will be transformed into photoreceptor cell
19
Q
Complexity of D-Pax2 Enhancer
A
- within lies five sites for Su(H) binding, three sites for Lz binding and four sites for Pnt/Yan binding
- transcriptional reporter that preserves native spacing of these sites and nature of intervening sequences drives expression in all four cone cells
- construct in which the native spacing has been preserved, but sequence has been mutated fails to drive expression of reporter
- likewise, construct that eliminates intervening sequences also fails to drive expression of reporter
- results imply that DNA bases that lie between Lz, Pnt/Yan, and Su(H) sites are important for D-Pax2 expression in eye
20
Q
Order of Site
A
- also important
- scrambling the order while preserving the correct spacing is insufficient to maintain proper D-Pax2 enhancer expression
21
Q
Intervening Sequences
A
- important or just a subset?
- deleting or changing individual regions can be used to test requirements for each intervening segment
22
Q
Dissecting D-Pax2 Enhancer
A
- individual intervening segments either deleted or altered
- resulting modified enhancers are assayed for ability to drive expression of reporter construct in cone cells
- wild type construct used as control for comparison
- most intervening sequences are required for proper expression
- exceptions: deletions of 3rd intervening sequence (no effect)
- deletion of 5th intervening sequence leads to up-regulation of D-Pax2 expression
- suggests that during normal development this region is bound by a transcriptional repressor-in its absence the enhancer has increased activity
23
Q
Pax 3 and 7
A
- have all 3 motifs
- paired and homeodomains and octapeptide
