structure Ommatidial and eye-antenna disc
lens Pigment cells Cone cells photoreceptors Rhabdomeres (contain rhodopsin) Pigment cells
A
P
Cells often behave as pairs, only one member of pair shown temporal sequence of development assembly begins in mf R2, 3, 4, 5, 8 occupy apical regions Stays apical, becomes cone cell other cells are added progressively, rising apically as they are recruited into cluster those already apical move basally R1, 6 will be added slightly before R7
Downstream genes in the RTK pathway
sevenless (sev) Son of sevenless (sos)
drk
Rolled (rl)
Jun, Pointed Yan
Phyllopod (phyl) Seven in absentia: phyllopod degrades Tramtrack; the tramtrack 88k TF represses neuronal cell fate determination in Drosophila eye
Top Row: pattern of protein expression
Bottom Row: genetic analysis using various mutants
Cell protein requirements
Freeman M. 1996. Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell 87: 651-60. Like sevenless, DER is an RTK (EGF homolog) sev is needed for R7 recruitment, but ras pathway is required for all photoreceptors what is necessary for recruitment of other photoreceptors?
Determination not based on cell lineage
Cells not clonally related DER mutants suggest role in pathway
dominant alleles have defects in ommatidial spacing null alleles suggest role in photoreceptor formation the DER ligand, Spitz, needed for photoreceptor formation
DN form of receptor used to study role of DER in eye development
RTKs dimerize for signal transduction removal of intercellular domain creates dominant-negative, since mutant receptor can dimerize with + type, but cant signal DN receptor expressed only in eye using enhancers that allow for expression after proliferation
Conclusions
DER required for all cells in the ommatidium both DER and sev needed in R7 the two work interchangably in R7, suggesting that a common pathway is being used reiteratively in recruitment of different cell types
DER is required for determination of all photoreceptors
DN-DER expressed using the gal4/UAS system
DER is required for determination of all photoreceptors
DN-DER expressed using the gal4/UAS system 3 different Gal 4 drivers were used
GMR (opsin gene enhancer element) expressed in all cells behind MF sev-Gal4 expressed in subset of ommatidial cells (previous slide) - HS- Gal 4 controlled by heat shock pulse
DER is required for determination of all photoreceptors
GMR element expression leads to loss of all cells behind MF (1B) recruitment of photoreceptors blocked after first 3 cells (R8, 2 and 5), they die 24-30 hr later (1D) can be rescued by expression of + type DER (suggests other RTK not involved; 1E-F) expression with HS pulse leads to stripe with variable number of missing receptors (1H)
DER is required for determination of all photoreceptors
Sev-Gal 4 leads to loss of most photoreceptors it is expressed in(not shown)
DER is required for recruitment of cone and pigment cells
DN-DER also blocks cone and pigment cell differentiation
Is this a consequence of photoreceptor loss? No, monitored in eyes with normal number of other cellsdone by using different temporal pulses of HS-Gal4
early shocks; full complement of photoreceptors, fewer cone cells (2C-D) later shocks; loss of pigment cells (1o then Spitz 2o3o...)
Secreted causes overrecruitment of all cell types
Spitz is active ligand in eye only in cleaved form express both full length and extra-cellular fragmentonly fragment had effect expressed directly from sev promoter (not Gal4)
some ectopic photoreceptors (3A-B)
expressed from GMR-Gal4 (leaky;lethal)
extensive overrecruitment of photoreceptors (3C)
Secreted Spitz causes overrecruitment of all cell types
Expressed from HS-Gal4: more complex, posterior overrecruitment, then missing zone, then normal MF (3D) Expression from sev promoter directly leads go extra cone (4A) and pigment cells (4CD) shows posterior-anterior gradient of severity Halving the dose of DER can rescue (4E-F)
Why need for two RTKs in R7?
Does DER activate other pathways than ras? Are the two receptors interchangable? This would imply that they used the same pathway Activated forms of each receptor were used
cytoplasmic domain fused to constitutively active torso (RTK) domain (ligand independent) expressed under the sev or HS promoter
Are the two receptors interchangable?
torDDER causes extra recruitment of either R7 or other photoreceptors, depending on stage of cells (5A-B); done in sev mutants, so DER can replace sev outer PRs also recruited from mystery cells (5C-D) both torDDER and torDSev expressed under HS promoter can recruit cone and pigment cells (5E-H)
Are the two receptors interchangable?
torDSev can suppress hypomorphic DER mutations if expressed in the wing
Are the two receptors interchangable?
Taken together, these results suggest that the two receptors are interchangable HS experiments indicate the fate of a cell depends on its developmental stage when the activated receptor is expressed so it is the time the signal is received, not the signal, that is critical
Spitz overexpression can rescue sevenless
The chimeric constructs did not rely on normal receptor/ligand interactions Tested to see if the Spitz ligand could rescue sevenless mutants
Spitz expressed...