Phenotype (Function) Genetics Gene A Gene B Gene C Proteins A B C P

Four-winged fruit fly Mutations in ultrabithorax regulatory region transform the 3rd thoracic segment into 2nd one. Edward B. Lewis

Question Lewis’s homeotic mutations shows that there is an correlation between mutant phenotype and gene functions. Each gene is responsive for a specific function, thus, generating mutations in individual genes is sufficient to uncover gene functions in development. A: Agree B: not Agree

- Homeotic phenotype, pleiotropic phenotypes and no phenotypes

The genomes: - the number of genes - yeast 6,400 - rice32,000 - worm 20,000 - fly13,000 - Human 30,000 What is your reaction to this number: A: There are too few genes B: There are too many genes C: Both Gene number Functional diversity

Mutations Specific phenotypes most genes act in multiple developmental processes, making it difficult to isolate mutations for a specific role. Malor problem #1 - Isolate conditional mutations Approaches to deal with it: - Using sensitized genetic screens to isolate partial loss-of-function or hyperactive mutations - Genetic mosaic screens/tissue specific knockouts

Pleiotropy often prevents the observation of phenotypes of null alleles in specific tissue or at specific stages. Heterozygous mother Early phenotypes of a null allele masks the late phenotypes null/+ null/null Dead embryo rf/+ rf/rf uncoordinated homozygous progeny m/+ m/m Larval lethal or sterile Late phenotypes of a null allele may masks the early phenotypes Heterozygous mother homozygous progeny m/+ m/m Dead embryo Elimination of maternal activity by creating germline mosaics or by RNAi Maternal product provides early essential functions

An F1 screen for lethal or sterile mutations. Dead embryo or larvae, or sterile adults m+m+ mmmm m + Po F1 clone F2 Pick WT siblings to individual plates No longer see lethal progeny Discard 1/4 1/2 1/4 Continue to see lethal progeny. Repeat the step to keep the strain EMS Po 20 plates F1 20 plates Screen for lethal or sterile phenotypes 400 plates; 800 mutagenized genomes F2 3 days Isolate 20 F1s from each Po plate …

lethal screen in fly X-ray X TM * * X F1 * * X * * F3 homozygotes, lethal

Maintaining lethal or sterile mutations let uncdpy Select wild type progeny let uncdpy let Balancer let Keep the animals that continue to segregate Dpy and Unc progeny Recombination within the balanced region is suppressed Balancer

Maternal effect lethal gene null/+ null/null Dead embryos null/+ Dead embryos null/null+/+ X A: Dead embryos B: wild type C: not sure

Mutations Specific phenotypes A large % of genes have no obvious knockout phenotypes Malor problem #2 Yeast: 40% genes Worm: KOs of a large % have no obvious phenotypes Mouse: >30% knockouts have no drastic phenotypes Even for gene with mutant phenotypes, they have other functions not manifested by the phenotypes

Vote A: Genes with no robust knockout phenotypes have no important biological functions. B: Genes with no robust knockout phenotypes have less important functions than those with robust phenotype. C: Genes with no robust knockout phenotypes have just as important functions.

Why are there genetic redundancies associated with our genome? The genomes use the strategy to increase the resilience to mutational effects. A: yes B: no We may discuss more at the end.

Redundancy provided by duplicated genes Homologs: genes with common ancestry. - paralogs: some kind of common ancestry (seen in structure or sequence), but different functions, the consequences of "parallel evolution" - ortholog = common ancestry and function

Redundancy provided by duplicated genes A B Same biochemical functions Function GenotypePhenotype H2B 1(-); H2B2 (+)wild type H2B 1(+); H2B2 (-)wild type H2B 1(-); H2B2 (-)dead Yeast Histone H2B has two genes encoding essentially the same protein H2B is an essential component of nucleosomes Syne1 Syne12 Nuclear membrane functions GenotypePhenotype syne1(-); syne2 (+)wild type syne1(+); syne2 (-) wild type syne1(-); syne2 (-) die at birth, Mouse Syne 1/2 genes

Redundancy provided by duplicated genes Example or C. elegans Notch receptors lin-12(-) epidermal tissue defect glp-1(-) germline defects lin-12(-) & glp-1(-) embryonic lethal LIN-12 GLP-1 LAG-1 Function C LIN-12 GLP-1LAG-1Function B LAG-1Function A GenotypePhenoytpe lin-12(-); glp-1 (+)phenotype A lin-12(+);glp-1 (-) phenotype B lin-12(-); glp-1 (-) phenotype C Question: The differences between lin-12 and glp-1 functions reflect A: the differences between the LIN-12 and GLP-1 protein structures. B: the differences in their expression pattern.

Redundancy provided by duplicated genes Experiment (Greenwald and Strul): LIN-12 GLP-1 LAG-1 Function C LIN-12 GLP-1LAG-1Function B LAG-1Function A GenotypePhenoytpe lin-12(-); glp-1 (+)phenotype A lin-12(+);glp-1 (-) phenotype B lin-12(-); glp-1 (-) phenotype C glp-1 coding region lin-12 promoter acts as lin-12 acts as glp-1 lin-12 coding region glp-1 promoter

"Redundancy" by structurally unrelated genes? Question : Majority of the genetic redundancy we observed (for example the “no phenotype” situation with 40% of the yeast genes) are due to functional redundancy provided by duplicated genes A: Yes B: No C: do not have a clue

Genetic redundancy due to protein activities on different targets in the same pathway C D Function AB GenotypePhenoytpe ark-1(lf); gap-1(+)wild type ark-1(+); gap-1(lf) wild type ark-1(lf); gap-1(lf)Multivulva (90%) EFGR ARK-1 RAS GAP-1 Vulval induction EGF (signal) P. Sternberg lab

RTKRAS GRB2SOSRAF UNC-101ARK-1SLI-1 (CBL) GAP-1 Ark-1Sli-1Gap-1 Unc-101 Ark-1 Sli-1 Ark-1 Gap-1 Ark-1 Unc % Multivulva Sternberg lab

Discovery of synMuv genes Wild type Multivulva mutagen Genotype Phenotype lin-8(-)wild type lin-9(-)wild type lin-8(-) & lin-9(-)Multivulva Horvitz and Sulston 1980

ClassB synMuv genes (20) ClassA synMuv genes (4) Vulval differentiation Synthetic Muv phenotype define redundant genetic pathways Ferguson and Horvitz, 1989 Later papers No structural similarity between genes

Mutations Specific phenotypes A large % of genes have no obvious knockout phenotypes Malor problem #2 Methods to deal with it: - Multiple knockouts - Genetic screens in sensitized background - Synthetic screens

The concept and usage of mosaic analysis - What is the problem? Why do we need mosaic analyses? - Germline mutations vs. somatic mutations - Mosaic analysis in Drosophila - Mosaic analysis in C. elegans - Genetic mosaic screens in fly - Mosaic analysis in mouse

About mosaic analysis A genetic mosaic is an organism carrying cells of different genotypes Question: A somatic mutation in our body leads to a mosaic genotype regarding the gene containing the mutation. A: agree. B. disagree. C. not sure.

About mosaic analysis Most of the classical geneticists have been doing germ-line mutagenesis. Therefore, all cells in a given animal have the same genotype (non-mosaic). A: agree. B. disagree. C. not sure.

About mosaic analysis The vast majority of cancers are caused by somatic mutations. A: agree. B. disagree. C. not sure. What about other human diseases?

Multiple steps (multiple mutations) in cancer formation

A concept Phenotypes from mutations in somatic cells in a specific tissue are often different from phenotypes of animals that contain the mutation in every cell. A: Yes B: No C: not sure

Why do we need mosaic analyses? 1. Determine the site of gene action. Q: Does expression pattern tells us a gene’s action site? A: Yes, always. B. Only sometimes. C. Tells us essentially nothing about the action site. The cell or cells in which a gene is expressed is not necessarily where the gene expression is needed for a specific function

A concept Where the abnormality caused by a mutation in a gene is seen is not necessarily where the expression of the gene is needed for the function. Genetic mosaics permit a correlation between cellular genotype and cellular phenotype

Mosaic analysis can be used to determine the site of gene action Wild type (cell death) Mutant (cell survives) Mosaics (Genotype of the ced-3 gene) Phenotype of the middle cell conclusion The gene being tested acts (A) cell autonomously (B) cell non-autonomously in the middle cell for its function in programmed cell death.

Cell-nonautonomous Hunter and Wood, Cell 1990 Drawing from ergitol.com

2. Determine gene functions in specific tissues zygote (-) (-) Germ Cells ( - ) Somatic Cells (+) (+) Germ-line mosaic All progeny are mutants and there is no maternal wild-type gene product (-)(-) or fly Maternal effect gene is expressed during oogenesis. A. Dealing with pleiotropic phenotypes B. Studying maternal gene function

The entire cell lineage of a C. elegans hermaphrodite. From HHMI bulletin

Identify a defect in a specific cell lineage Zygote (-) Dead lava Zygote (+) (+)(-) Dead lava The gene is required in the A: “red” lineage B: “blue” lineage Zygote (+) (+) (-) Dead lava The gene is required in the “red” lineage Zygote (+) Live The gene is not required in the A: “red” lineage B” “blue” lineage (-)(+) Yochem et al used this method to determine the site of the essential Ras gene function.

1. Tissue, cell or nuclear transplantaiton 2. Chromosome loss Mosaic analysis in C. elegans. Examples. 3. Mitotic recombination - induced by radiation (Drosophila example) - induced by site specific recombinases (Drosophila, mice) Methods for generating genetic mosaics

MS mom-2(lf)

Mom-2 gene function Mom-2 acting site: EMS or P2? Thorpe et al Cell

Mosaics: issue transplantation EMS P2 mom MSE mom(-) mom(+) MSE mom(+) mom(-) MS Where is the action site of this mom gene? A: EMS. B: P2.

Methods for generating genetic mosaics 1. Tissue, cell or nuclear transplantaiton Example: Wnt action site in C. elegans early embryo. 2. Chromosome loss Mosaic analysis in C. elegans. Examples. 3. Mitotic recombination - induced by radiation (Drosophila example) - induced by site specific recombinases (Drosophila, mice)

Example #1: Determine the gene action site of Notch protein Mosatic in C. elegans Free duplication or Exchromosomal array Normal chromosomes Contain gene tested Contains a visible marker

WT Z1.pppZ4.aaa 50%ACVU 50% ACVU ablation AC100% AC100% Lin-12 mutants lfAC100% AC gfVU 100% VU Greenwald et al. Cell, 1983

lin-12 lin-12: A: promoting VU. B: inhibit AC. C: either. D: neither. lin-12 mutants lfAC 100% AC gfVU 100% VU Does lin-12 act as (A) part of the signal, or (B) part of receiving mechanism?

How do you determine whether lin-12 is a gene for the signal or receptor? Z1 Z4 Z1.pppZ4.aaa Lin-12(-) Lin-12(+) If AC VU Mosaic analysis A: Lin-12 is a receptor B: Lin-12 is a signal VU AC

Real experiment lin-12(+)ncl-1(+) ncl-1(-) lin-12(-) Chromosomal genotype free duplication (or extrachromosomal array) Mitotic division ncl-1(+) lin-12(+) ncl-1(-) enlarged nucleoli lin-12(-) Seydou and Greenwald, Cell 1989

Z1.aaZ1.ppp Z1 Z4.aaaZ4.pp Z4 Duplication near lin-12 Lin-12 nuc-1 Nuc-1 mutation cause nucleolus bigger

Indicating: lin-12 encodes a protein that act on (A) the signaling or (B) reception side ? (Adapted from the Hartwell Genetic book)

Methods for generating genetic mosaics 1. Tissue, cell or nuclear transplantaiton Example: Wnt action site in C. elegans early embryo. 2. Chromosome loss Mosaic analysis in C. elegans. Examples. 3. Mitotic recombination - induced by radiation (Drosohila example) - induced by site specific recombinases (Drosophila, mice)

WTsevenless

How do they know it is a receptor? - Structurally similar to receptor tyrosine kinase - Mosaic analysis determined that they act in R7 - The protein is expressed in the R7 membrane R8 Undifferentiated cellR7 photoreceptor cell ? Sev Sev receptor R7 differentiation

Somatic Recombination * * * * ** * * * *

Mitotic recombination: generating mosaic in Drosophila 1. Rare. Occur in G2 2. Enhanced by x-ray radiation. A B a b A B a b A B a b No recombination A B a b A B a b A B a b With recombination

Sevenless acts cell autonomously Sev receptor R7 to be Adopted from Hartwell et al, Genetics

Discovery of the Bride of Sevenless , BOSS was isolated by Larry Zipurski’s lab by the similar method - failure to response to UV. -It has exactly the same Sevenless phenotype

Mosaic analysis of Boss W-W- W-W- W+W+ W+W+ boss- boss+ W-W- boss- W-W- white boss- X-ray W+W+ boss+ W+W+ red Boss+ Mitotic cross over A: boss acts in R7 B: boss acts in R8

R8 Undifferentiated cellR7 photoreceptor cell Boss Sev Boss signal Sev receptor R7 differentiation RT fate is induced by RTK activation

Methods for generating genetic mosaics 1. Tissue, cell or nuclear transplantation Example: Wnt action site in C. elegans early embryo. 2. Chromosome loss Mosaic analysis in C. elegans. Examples. 3. Mitotic recombination - induced by radiation (Drosohila example) - induced by site specific recombinases (Drosophila, mice)

Mosaic genetic screens 1. Why do we need it? 2. Drosophila vs. C. elegans Principle: promote recombination in somatic cells using yeast FLP recombinase system. Screen in Drosophila: - create mosaic mutants - screen homozygous mutants after one cross

X-ray X TM * * X F1 * * X * * F3 homozygotes Traditional F2 screen FRT screen * X-ray X P(FRT) * Induction of mitotic recombination at the FRT site, e.g. HS- drive FLP in flies * * FRT: target for yeast FLP recombinase

* Induction of mitotic recombination at the FRT site, e.g. HS- drive FLP in flies * * FRT: target for yeast FLP recombinase * * S phase In some cells Xu and Rubin, 1995

Cell type specific gene knockouts using loxP-Cre recombination system LoxP mouse Exon 1 Exon 2 Exon 3 LoxP Cell-type-specific promoter Cre Cre mouse X Endogenous gene X with With LoxP sites flanking exon2 All cells carry cre transgene mouse is heterozygous for gene X knockout LoxP -Cre mouse: all cells carry one copy of loxP- modified gene X, one copy of gene X knockout, and cre genes Cells not expressing Cre Cells expressing Cre

Other methods to create mosaic genotypes: Tissue specific promoter driving RNAi Tissue specific promoter driving antisense Tissue specific promoter driving expression of wild type gene in mutant …. Somatic transposon excision.

Jim Priess’s screen lin-2(lf) “Bag of worms” (egg-laying defective) Po Treated with mutagen 1/4 Without maternal lethal mutation F1 lin-2(lf) F2 lin-2(lf) “Bag of worms” With a maternal lethal mutation lin-2(lf) ; mel + 1/4 Worms contain F3 dead eggs No viable progeny, but a maternal lethal mutation is identified lin-2(lf) ; mel “Bag of worms” 2/4 Keep for retaining the mutation lin-2(lf) ; mel +

Synthetic Hyperproliferation Model Cyclin levels Rb transcription stability FZR-1 Rb fzr-1 Relative Cyclin levels + + Very Low + – Low – + Low – – High, phenotype LIN-23