Exam 4: Lecture 6

Question 1

Cone Cells in Drosophila Retina

Answer 1

• each unit eye contains four lens secreting cells called cones
• different than cone photoreceptors
• secrete overlying lens

Question 2

Lens of Drosophila

Answer 2

• 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

Question 3

Development of Insect Retina

Answer 3

• 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

Question 4

D-Pax2 gene

Answer 4

• key target of EGF Receptor Pathway and Notch Pathway

- required for formation of cone cells

Question 5

Photoreceptor cells

Answer 5

-produce ligands that bind to membrane bound receptors on presumptive cone cell surface

Question 6

Paired Box (Pax)

Answer 6

• 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

Question 7

Pax1 and Pax9

Answer 7

-contain paired DNA binding domain and an eight amino acid octapeptide used in transcriptional repression

Question 8

Pax 2, Pax5, and Pax8

Answer 8

• 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

Question 9

Pax4 and Pax6

Answer 9

• have intact paired and homeodomains

- can bind to DNA using both domains

Question 10

D-Pax2 in Cone Cells

Answer 10

• 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

Question 11

Isolation of Rough Eye

Answer 11

-convenient way to screen for genes that affect eye development

Question 12

Lab Induced Mutation

Answer 12

• 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

Question 13

D-Pax2 Eye Specific Enhancer

Answer 13

• 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

Question 14

Sequences in D-Pax2

Answer 14

• 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

Question 15

Enhancers

Answer 15

• 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

Question 16

Pathways

Answer 16

• review EGF Receptor Pathway

- review Notch Pathway

Question 17

Activation of D-Pax2 Enhancer

Answer 17

• 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)

Question 18

Model

Answer 18

• 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

Question 19

Complexity of D-Pax2 Enhancer

Answer 19

• 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

Question 20

Order of Site

Answer 20

• also important
• scrambling the order while preserving the correct spacing is insufficient to maintain proper D-Pax2 enhancer expression

Question 21

Intervening Sequences

Answer 21

• important or just a subset?

- deleting or changing individual regions can be used to test requirements for each intervening segment

Question 22

Dissecting D-Pax2 Enhancer

Answer 22

• 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

Question 23

Pax 3 and 7

Answer 23

• have all 3 motifs

- paired and homeodomains and octapeptide