Drosophia practical session 1

Transcription

1 Drosophia practical session 1 Introduction to the adult ovary and midgut, and their highly active stem cells oviduct uterus Ovary midgut

2 Goals: 1. Isolate two major Drosophila stem cell based tissues 2. Identify stem cells, determine their lineage and study how they respond to changes in nutrition 3. Study several genes involved in stem cell maintenance and daughter specification: e.g. bam, piwi, Notch 4. Utilize some modern Drosophila genetic tools: transposon insertion mutations, protein trap, enhancer traps, site-specific recombination for clone induction, GAL4/UAS based transgene expression

3 Transposon insertions as genomic tools

4 Identifying the right flies w - ; P[w + ] CyO x P[w + ] w - ; CyO w - ; P[w + ] P[w + ] w - ; CyO w +

5 Lineage marking by site-specific recombination Site-specific recombination generates random marked cells Requirements: 1. Specific to mitotic cells 2. zero background 3. Rate controllable

6 Set up for lineage experiment Time of single heat shock 18 days ago 9 days ago 3 days ago 2 days ago Each group cultured in vials 1) with abundant wet yeast ( fed ) 2) with no added yeast ( limited ) We will dissect ovaries or midguts from each set set of flies and stain for lacz to analyze stem cell number and behavior

7 The Drosophila ovary GSCs ovariole ovary Germ cells Follicle cells germarium FSCs? Germ cell cysts GSC Cystoblast CB

8 Ovary dissection

9 The adult gut

10 Finding stem cell/niche control mechanisms GSC: stem cell loss 2 germline products BMP pathway nanos piwi pumilio differentiation block Genetic screens: Find genes that affect stem cell or niche function bam bgcn dpp overexpression Dots mark accumulating GSC-like cells

11 Genetic regultion experiments We will dissect 7 genotypes that show the roles of several genes important for ovarian and gut stem cell regulation Bag-of-marbles (bam) piwi c587-gal4/uas-egfp c768 GAL4 / UAS-EGFP Notch inhibition by feeding the drug DAPT Dl-GAL4 UAS-EGFP Notch reception reporter Fed or limited

12 Protein trapping frame 1 frame 2 frame 0

13 Plasma Membrane CB02038 CG31605

14 Enhancer traps report expression patterns Dorsal follicle cells stage 10

15 Regulated gene mis-expression

16 Drosophia practical session 2 Analyzing lineage data to reveal stem cell number, location and behavior oviduct uterus Ovary midgut

17 Set up for lineage experiment Time of single heat shock 18 days ago 9 days ago 3 days ago 2 days ago Each group cultured in vials 1) with abundant wet yeast ( fed ) 2) with no added yeast ( limited ) We will dissect ovaries or midguts from each set set of flies and stain for lacz to analyze stem cell number and behavior

18 The Drosophila ovary GSCs Drosophila ovary Germ cells Follicle cells germarium FSCs? ovariole GSC Germ cell cysts Cystoblast CB

19 QuickTime and a MPEG-4 Video decompressor are needed to see this picture.

20 Transient clones: t 1/2 = 10 hr 1.5d 2 d Number of stem cells = 2 2.5d 3 d

21 QuickTime and a MPEG-4 Video decompressor are needed to see this picture.

22 Unequivocally identifying individual stem cells by lineage marking Follicle stem cell (FSC) FSC GSC FSC Germline stem cell (GSC) FSC Still need to understand cell movements in tissue: movement

23 Identify transient and stem cell clones in the midgut as well What does a transient clone look like?

24 Todays tasks: Mount the tissue you dissected yesterday on microscope slides Seal with nail polish and examine using fluoresence microscope Identify transient and stem cell clones; count clone size Use this data to determine stem cell number, and rate of division What is the effect of nutrition on these parameters?

25 Protein trapping frame 1 frame 2 frame 0

26 An example: Insertions in CG31605 Give this pattern:

27 Drosophila practical session 3 Analyzing mutants and reporters to understand stem cell regulation oviduct uterus Ovary midgut

28 The fusome is a useful marker of GSCs and early germ cells Stain: monoclonal Ab 1B1 (red)

29 A round fusome GSC ( spectrosome ) labels GSCs GSC CB GSC GSC CB CB GSC GSC CB CB GSC GSC CB

30 Failsafe regulation of GSC vs CB fates BMP signal GSC bam 1. Local BMP signal 2. Activated Smads diffuse through open ring canal niche 3. Ring canal closure cuts of signal and initiates Bam expression

31 We will look at components of this process: Control Red = vasa Green = fusome White = fusome Bag-of-marbles (bam) hs-gal4:: UAS-dpp

32 wt Piwi mutants: piwi green = vasa red = fusome White = fusome Dark staining = piwi enhancer trap staining

33 A single cell in each nest expresses Delta Su(H) - Canonical pathway used Delta is functionally required: Dl - neu - neu -... and neuralized: EC ISC Ser - But not Serrate:

34 The ISC signals to its own daughter via Delta N reception reporter-lacz N* channel ISC Delta ISC Notch activation assay Summary: ISC Lineage (green) Delta Sending: Dl high N inactive ISC Notch signal EB Receiving: Dl low N active

35 High level Notch signaling is necessary and sufficient for EC differentiation N - N - Clone Delta Delta N - cells proliferate as ISCs MARCM UAS-N act We will use DAPT to block N reception Expressing N act induces ISCs to become ECs How are ee s specified?

36 ee s arise from ISCs with low Delta levels ISC ISC ISC... and are made in pairs 100% correlation; proved by effects of Notch manipulations

37 ISCs specify their daughters fates via Notch signals EC ee Notch High level EB ISC EB Low levels Regulatory signals

38 Genetic regultion experiments Dl-GAL4 UAS-EGFP Shows which gut cells express Dl Su(H)gbe-GAL4 UAS-EGFP Notch reception reporter Notch inhibition by feeding the drug DAPT Shows effect of disrupting Notch

39 Regulated gene mis-expression

40 We will look at: c768-gal4::uas-gfp c587-gal4::uas-gfp