Reading for Monday’s lecture: ( genetic mosaics & chimeras ) p518 (“Aneuploid Mosaics…”) pp (“What cells…”) I will hold office hours during spring break, but at a different time than normal: during class time (11a-12N) on March 26 & March 30

Using them to follow chromosomes in mutant screens Balancer chromosomes: Aim: find genes that allow cells to know where they are so the cells can know what they should be expected lof mutant phenotype for “pattern formation” genes: (1) embryonic recessive lethal (2) alterred dentical belt pattern (exoskeleton) in dead embryos v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v wildtype v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v “bicaudal” Post. (dying fly embryos can still differentiate a lot) (genes generating positional information) Ant. Post. polarity>>> Consider the brute-force screen that led to the last fly Nobel Prize N-V & W:

Second chromosome (brute force) screen DTS / CyO females cn bw males mutagenize each son potentially carries a new mutant allele of interest …but a different new mutant in each Second take individual males and mate separately (10,000 crosses) DTS / CyO females single dominant temperature- sensitive lethal second- chromosome balancer second-chromosome “markers” (eye color = white) CyO / cn bw & let?? sons

DTS / CyO females single CyO / cn bw & let?? sons X CyO / cn bw let-a? sons daughters X each group of progeny separately (forced incest) : CyO / CyODTS / CyO DTS/ cn bw let? unwanted sibs all

CyO / cn bw let-a? cn bw let-a? / cn bw let-a? CyO / CyO to maintain any new let mutation do they all die? (no white eyes?) and if so, when? how? always die only after a 2nd generation of 10,000 crosses did they know which individual sons of mutagenized males carried a recessive lethal mutation of interest (value) CyO / cn bw let-a? sons daughters X each group of progeny separately (forced incest) :

Second chromosome screen DTS / CyO females cn bw males mutagenize each son potentially carries a new mutant allele of interest CyO / cn bw mut-a? sons daughters cn bw mut-a? / cn bw mut-a? DTS / CyO females single X CyO / cn bw & mut?? sons X X only after a 2nd generation of 10,000 crosses did they know which original F1 sons carried mutations of value F1 generation F2 generation Brute force keep populations separate! …and if looking for maternal-effect mutations, go blindly one generation more!

Second chromosome screen DTS / CyO females cn bw males mutagenize CyO / cn bw mut-a? sons daughters CyO / cn bw mut-a? cn bw mut-a? / cn bw mut-a? CyO / CyO DTS / CyO females single X sons X X CyO / cn bw & OR permissive DTS / cn bw & mut?? or Temperature for first cross doesn’t really matter: (1) have to hand- pick males anyway (2) males have no meiotic recombination (so DTS/mut OK)

Classic N-V&W screen illustrates two important points: (1) recessiveness (~lof) generally demands multiple generations of blind forced incest crosses (mating siblings) to recover mutant (2) recognizing an informative phenotype is a large part of the genetics game The N-V & W advantage: an informative phenotype that could be scored in dead embryos (didn’t demand survival -- or much else!). What if want to study something like eye development instead? ey 1 :recessive hypomorph, adults w/ no eyes ey -(null) : recessive embryonic lethal ey is pleiotropic (multiple “unrelated” phenotypes/functions) …can we overcome the limitations of recessiveness? Attractive features: interesting AND non-essential (and more), but consider: …can we overcome the limitations of pleiotropy? Got lucky with ey 1 how many other important eye genes missed? &Early

YES…we shall overcome (1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function) (2) use targetted genetic mosaics to screen for recessives in the F1 (homozygous clones in heterozygotes …in non-essential tissues only!) but first: already mentioned one way to deal with pleiotropy temperature-conditional mutant alleles ts muts. way too limited even in flies & worms FAR BETTER …can we overcome the limitations of pleiotropy?

(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function) sevenless/+ (wildtype) vs. sev/sev R7 photoreceptor missing (turned into cone cell) Illustrate with example from fly eye development studies: ? cone cell photo- recptr photo- recptr cone cell signal from R8 neighbor A developmental decision: R7 precursor cell Stemmed from original observation: goal: make genes “artificially” haploinsufficient bride-of-sevenless (null eye-specific) seven-in-absentia son-of-sevenless seven-up Other genes discovered to be involved in the R7 precursor decision: null sev allele: same thing (hence, eye-specific) null alleles not eye-specific: pleiotropic: How many other pleiotropic genes missed?

(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function) sev/sev R7 photoreceptor missing (turned into cone cell) ? cone cell photo- recptr photo- recptr cone cell signal from R8 neighbor R7 precursor cell make genes “artificially” haploinsufficient How many pleiotropic genes missed? sev - /sev - ; TM3,P{sev B4(ts) } / + designer ts allele sev encodes v-src homolog (human oncogene) screen for dominant mutations that make: 24.3 o C 22.7 o C R7 absent R7 present R7 present (Dominant suppressors) R7 absent (Dominant enhancers) growth temperature phenotype (3rd chromosome balancer)

(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function) screen for dominant mutations that make: 24.3 o C 22.7 o C R7 absent R7 present R7 present (Dominant suppressors) R7 absent (Dominant enhancers) growth temperature phenotype Found many pleiotropic lof alleles of both types (incl. recessive lethals). Poising sev + activity level on a phenotypic threshold made other genes haploinsufficient Wildtype fly must normally have an excess of sev + activity as insurance, so it can tolerate fluctuations in levels of other genes in pathway during development …if take away that cushion, now more sensitive to reductions in other gene levels but only with respect to sev function!

sevenless/+ (wildtype) vs. sev/sev R7 photoreceptor missing (turned into cone cell) made genes “artificially” haploinsufficient R7 precursor cell photo- recptr cone cell signal from R8 neighbor ….then look for newly induced dominant enhancer or suppressor alleles adjust level to poise system on phenotypic threshold sevenless/”+“ other genes in pathway NOT haploinsufficient now other genes in pathway ARE haploinsufficient

Point to keep in mind: …will not necessarily identify every relevant gene in pathway this way sevenless: receptor in R7 cell that responds to signal from R8 bride-of-sevenless: ligand (signal molecule) generated in R8 no new mutant alleles found in sev sensitized screen!

(2) use targetted genetic mosaics to screen for recessives in the F1 (homozygous clones in heterozygotes …in non-essential tissues only!) …recover new recessives in the F1??? Based on a phenominon discovered (‘30s) by former chair of U.C. Zoology Dept: mitotic recombination …only possible because of a very strange aspect of fly chromosome behavior: homologous chromosomes pair during mitotic interphase but improved upon enormously in modern times Another way around the limitations of pleiotropy in genetic screens: