Tetrad Genetics

1. Tetrad Genetics Todd Nystul, Ph.D. UCSF Depts. of Anatomy & OBGYN-RS Center for Reproductive Sciences Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research

2. In Class Question #1 The paralytic shellfish toxin, saxitoxin, is a deadly toxin that is both a human health risk and a potentially useful insecticide*. You decide to use Drosophila to study how saxitoxin works at the cellular level. First, you show that Drosophila grown on food with saxitoxin (10 mg/ml) die as larvae. Starting with an EMS mutagenized population of males, design a screen to isolate mutants that are resistant to saxitoxin. *The details have been contrived for this problem set

3. Chr II balancer Culture in 10 mg/ml saxitoxin

4. Transposon mutagenesis in Drosophila • To mutagenize with P-elements: • Cross a source of transposase to a line with a P-element (that lacks transposase) so it will hop around the genome randomly in germ cells of the P0 generation • Cross P0 to a balancer stock • F1 progeny that did not inherit the transposase gene have stable, novel insertions • P-elements usually land in 5’ region of gene, and often disrupt the promoter rather than the ORF. • Flanking genomic sequence can be cloned by inverse PCR, allowing for rapid identification of mutated gene • P-element excision can lead to reversion (precise) or new alleles (imprecise) • Mutations are publicly available for approximately 2/3 of all known or predicted genes.

5. C. eleganstransgenesis Generation of lines with heritable extrachromosomal arrays Mariner/CMOS transposon system

6. Cell-type specific expression • In C. elegans, it is common to clone specific promoters upstream of a transgenic gene of interest and introduce DNA by transgenesis • In Drosophila,it is more common to screen for random P-element insertions • P-elements have a strong bias for inserting near the 5’ end of genes, but otherwise transposition is somewhat random. • Enhancer traps are generated by P-elements carrying a reporter gene with a minimal promoter that lands in the regulatory region of an endogenous gene • usually an approximation of the cellular expression pattern of the gene, but not the subcellular localization of the protein LacZ LacZ Typical enhancer trap construct

7. Tagging proteins • In C. elegans, transgenesis is also used to express tagged proteins • In Drosophila,protein traps are generated by P-elements carrying a reporter gene that is flanked by splice acceptors and donors are hopped around the genome. • Must land within the transcriptional unit of a gene to be expressed • Must be in frame to form fusion protein (otherwise, it disrupts normal protein translation • Fusion proteins are accurate reports of both cellular transcription pattern and subcellular protein localization patterns. Typical protein trap construct

8. Tagging proteins

9. ϕC-31 integrase attB insertion sites in the original collection

10. Gal4/UAS transcription system • Gal4 is a transcription factor that activates the UAS promoter. • Gal4 P-element can use endogenous enhancers (enhancer trap) or can include a promoter. • Gal4 can be driven by • a ubiquitous promoter (e.g. Tub-Gal4 or Ubi-Gal4) • a tissue-specific promoter (e.g. elav-Gal4 is expressed in all neurons and MHC-Gal4 is expressed in all muscle cells) • Variations to provide temporal control: • Gal80 inhibits Gal4; Gal80ts is only active at permissive temp. (18° - 22°C) • Gal4ER is only active in the presence of an estrogen analog

11. In Class Question #2 Your screen is successful! You find that one mutation maps to the paralysis gene. para1 homozygous mutant flies are viable and fertile (though they move slowly), and para1 larvae can survive a 10-fold higher dose (100 mg/ml) of saxitoxin than their wildtype siblings. Design an experiment to determine whether resistance to saxitoxin in para1/1 flies is, in fact, due to the mutation in the para gene (and not to some other unknown factor such as a background mutation in this strain). You do not need to describe how to do the crosses, just state what genotype you want to use and what you would do with it.

12. RNAi and CRISPR • RNAi and CRISPR can be used for either forward genetics (i.e. screens) or reverse genetics (i.e. targeted knockdowns) • For C. elegans, there are libraries of E. coli that express RNAi for each gene • In C. elegans, RNAi spreads from cell to cell, so uptake by gut cells causes knockdown throughout the entire organism • For Drosophila, there are libraries of stocks with UAS-RNAi constructs • In more modern collections UAS-RNAi transgene inserted using the ϕC-31 system • No spreading of RNAi, so cross to ubiquitous Gal4 for whole animal knockdown or tissue/cell type-specific Gal4 for limited knockdown • For CRISPR • Co-inject construct with Cas9 gene driven by a germline promoter or Cas9 protein and construct with a guide RNA driven with a U6 promoter (Pol III) • In Drosophila, there are stocks with vasa-Cas9 on a P-element • Coming soon in Drosophila are libraries with guide RNAs on P-elements • Allows for screening with CRISPR or tissue-specific CRISPR

13. In Class Question #3 Design an experiment to determine whether expression of para in muscle cells is necessary for sensitivity to saxitoxin. Why wouldn’t a similar approach work with C. elegans? What could you do instead?

14. Clonal analysis • Two types of clonal analysis: • Lineage analysis: analysis of the hierarchy, composition, etc. of a cell lineage • Mosaic analysis: analysis of the effects of eliminating or mutating some (but not all) cells in a tissue • Allows for studying somatic genetics (the genetics of cells rather than organisms) • Can study mutations that are organism lethal (but not cell lethal) • Can assay for phenotypes at any developmental stage • Can study cell-cell interactions within a tissue (cell autonomous versus non-autonomous interactions)

15. Lineage analysis: the classical approach

16. Mosaic analysis using laser ablation The AC/VU Decision Screen to identify mutants

17. Mosaic analysis in C. elegans The NMDA and AMPA classes of glutamatergic receptors play critical roles in learning nkat-1:gpf expression

18. Learning correlates with activity of a single NMDAR-expressing neuron, (RIM) reconstitutions Activating and inactivating RIM

19. Lineage/mosaic analysis using site-specific recombination • Flipase (FLP) is an enzyme that catalyzes recombination between two FRT sites. • FRT sites can be on the same chromosome (e.g. tub-FRT-STOP-FRT-Gal4) or homologous chromosomes. • Flp can be controlled by a cell-type specific promoter (e.g. eyeless), or it can be inducible (e.g. heat-shock) • The result is a genetically heritable rearrangement that positively or negatively labels cells. • When labeled cells divide they form a “clone” that is distinguishable from surrounding unlabeled cells. • Cells in clone can become homozygous mutant for or overexpress a gene of interest. FRT FRT Tub Tub Stop Gal4 or GFP, etc Gal4 or GFP, etc

20. LacZ hs-Flp hs-Flp Flp/FRT recombination Positive marking, all cells are wildtype Negative marking, labeled cells are mutant FRT Tubulin FRT GFP FRT Tubulin FRT GFP FRT * FRT LacZ mutation FRT * FRT mutation

21. 19A I 40A 42D II 80B 82B III The Bruin Fly Collection • FRT sites have been integrated at the proximal end of every chromosome arm: • ...and UCLA undergraduates have put all publicly available lethal mutations onto an FRT chromosome (1200 stocks total)

22. Planar cell polarity • Cell autonomous: only cells within the clone are affected. • Cell non-autonomous: cells within the clone affect cells outside the clone (and/or visa-versa) In class Q3

23. Tumor suppressor screens • 1967: Gateff and Schneiderman identify lethal giant larvae, the first in vivo example of a tumor suppressor. • 1995: Xu et al., identify the first component of the hippo pathway by generating clones in larval imaginal discs and screening adults for mutations.

24. Lineage analysis to study stem cells Lineage analysis has become the “gold standard” for identification of stem cells in Drosophila and mammals.

25. 15% Percent of ovarioles with a fully labeled follicle cell population FSCs 10% 5% 0% Day 7 Day 14 Day 21 Identifying stem cells Fully labeled follicle cell population 50% labeled follicle cell population

26. Analyzing the lineage of a stem cell population The FSC lineage The ISC lineage compare

27. Mosaic Analysis with a Repressible Cell Marker (MARCM) • Labeled cells in the clone are positively marked. • Cells in a clone can be homozygous mutant and/or overexpress a transgene from a UAS • Equivalent system in mice is called MADM

28. Multicolor clone technologies

29. FSCs FSCs DNA GFP B-gal Dual-marked clones Tracking multiple lineages simultaneously Differentially labeling single cells Stem cell niche cells

30. A review of concepts covered • The C. elegansand Drosophila lifecycle and the advantages that the different stages offer as a model. • Karyotypes, nomenclature and Mendelian genetics with C. elegans and Drosophila • Working with hermaphrodites • Balancers: what advantages they provide and how to use them in a cross. • Methods for interrogating gene function: • Forward screens (EMS and P-elements), and reverse genetics. • Allele types—mutations in promoter vs. coding regions. • Sensitized backgrounds • Epistasis • Somatic genetics: Gal4/UAS, enhancer traps and protein traps, clonal analysis.