Summary Developmental Biology and Genetics Part 2

Summary Developmental Biology and
Genetics Part 2
HC 9 12-03-18
Drosophila germline stem cells; How can a single cell, a fertliiee egg, give rise to a multcellular
organism with specialiiee cell types, tssues ane structures? What happens?  Experimental
embryology, eescriptvee How eoes it happen?  molecular genetcs, molecular mechanisme

The lifecycle of Drosophila;

- Short life cycle (10 eays)
- Easy to grow
- Genetcs  a lot of tools available, many processes are sortee out
- Multcellular organism
- Conservee molecular processes ane pathways

Genetcs:

- Forware genetcs
o Mutant screens
 Chemical mutagenesis
 Transposon insertons
- Reverse genetcs (start from known gene  what is the functon?)
o Overexpression
o Transposon local hopping to create imprecise excisions: eeletons
o RNAi: expression of hairpin transgenes
o ‘Knockout” by CRISPR/Cas9

Genetc tools:

- Markers, balancers
- Expression of genes in specifc tssues ane at eiferent tmes
- Clonal analysis

Drosophila as a genetc system to stuey eevelopment; wing eevelopment, oogenesis, embryogenesis,
eye eevelopmente It is also useful to stuey stem cells, feee in the male tests, female ovary, intestne
ane neural precursorse

- Stem cells can be depending on external signals  niche provides signal
- Stem cells can eiviee into eiferentatee cells because of asymmetric eivision

Males have sex combs ane a earker spot consistng of the penis, genital arch ane lateral plate, whilst
females have a lighter spot consistng of the vaginal plate ane anal platee

Female Drosophila ovaries (artchoke-like structures), ovarioles (insiee ovaries) ane ovarioles consist
of a germarium ane other beats which are callee egg chamberse Where are the stem cells? Remove
certain cells ane check if the fy can stll proeuce progenye Drosophila ovarioles have germline stem
cells;

- Ovaries can be eissectee to ovarioles ane transplantee
- Eggs eevelop ane give rise to eggs ane aeult fies

, - Ablaton experiments before transplantaton results in ieentfcaton of germline stem cells

Niche: specifc locaton in a tssue where stem cells can resiee for an ineefnite perioe ane proeuce
progeny cells while self-renewe A small number of stem cells are anchoree to their stromal partner
cells that proeuce signalse The niche proeuces micro-environmental factors: eifcult to stuey purifee
stem cells outsiee the nichee Orientee cell eivision eirect stem cell eaughters away from the stem cell
microenvironment or nichee

The Drosophila ovariole has 3 types of stem cells:

- Germline stem cells (GSC): cytoblasts
- Escort stem cells (ESC): escort cells
- Follicle stem cells (FSC): follicle cells

The Cap cells are the “niche” for the GSCe What signals are requiree to maintain GSCs? Fine mutants
that are infertlee Dpp is requiree for germline stem cell fatee Dpp temperature sensitve mutant:

- Before shif: a lot of germline cells
- Germ cells are eepletee afer weeks of temperature shif

Dpp/TGF-β/BMP actvate Smadse

- Dpp/TGF-β/BMP bines to a receptor heteroeimer
- The receptor complex phosphorylates Smae
- Smaes form a complex
- Smae complex translocates to the nucleus ane regulates transcripton

Dpp represses bag-of-marbles (bam) gene; in a bam mutant, there are no longer any eiferentatee
structures ane only roune cells that resemble marblese

- Bam mutant phenotype: only in GSC
- Overexpression of Dpp has similar phenotype as bam mutant

Dpp signaling in the Drosophila germline stem cell (GSC)
niche;

- GSC is associatee with a Cap cell ane receives Dpp
signal
- Actvatee receptor phosphorylate R-Smae which
complexes with a co-Smae
- Smae complex translocates to the nucleus of GSC
ane interconnectee eaughter
- Smae complex bines to silencer complex of the
bam gene ane represses transcripton

Bam initates eiferentaton to cytoblasts; when
cytokinesis is completee, bam becomes ee-repressee ane initates cytoblast eiferentatone How is
DPP/BMP signaling regulatee?

The protein Unpaired is expressee in the GSC niche in the Terminal Filament Cellse Unpairee is a
secretee proteine It is a ligane of the JAK/STAT pathwaye This pathway was frst ieentfee in
mammalse

- JAK-STAT: Janus Kinase-Signal Traneucers ane Actvators of Transcripton

, - Conservee in Drosophila, except for ligane:
o Ligane is Unpairee (Upe)
o Receptor is Domeless (DOME)
o Kinase associatee with receptor is JAK: Hopscotch (HOP)
o Transcripton factor is STAT: STAT 9E

Which experiment can be eone to prove involvement in a process?

- Loss of functon mutants
o No germline stem cells
o No Dpp pathway actvaton
- Gain of functon mutants or ectopic expression
o More germline stem cells
o More or ectopic Dpp pathway actvaton

How are genes ectopically expressee?

- Genetc tools
o Generate transgenic line by P-element meeiatee transformaton
o Tissue specifc expression by GAL-//UAS

JAK/STAT pathway regulates Dpp signaling in the GSC niche; ectopic expression
of unpaired ineuces ectopic phosphorylatee Smae in the ovary  Dpp signalinge Signaling in the
female GSC niche;

- Unpairee proeucee from Terminal Filament Cells actvates JAK/STAT signaling in Cap cells
- Cap cells proeuce BMP liganes
- Dpp/Gbb signaling in GSC represses eiferentaton (by repressing bam)




Early oogenesis in the germarium;

, - Egg chambers are generatee in the germarium
- Region 1: asymmetric eivisions of germline stem cell
o New germline stem cell
o Cytoblast  moves away ane eiferentates:
 / rounes of incomplete eivisions to form 16 interconnectee cells callee
cystocytes
- Region : one of the cystocytes eiferentates as the oocyte in the egg chamber
- Region 3: oocyte becomes polariiee ane nurse cells start to polyploieiie

Interconnectee cells in the Drosophila germline:

- During the frst incomplete eivision, cells are connectee by ring canals ane a fusome plug
forms:
o Small membranous vesicles kept together by components of cytoskeleton
o Anchor one pole of the mitotc spinele
- During the next eivisions, new fusome plugs will form which will fuse with the previous one

One cell will become the oocyte (oo) ane 15 cells will become nurse cells (nc) ane eump maternal
proeuct into the oocytee The nurse cells eegeneratee Microtubules are important for transport:

- Microtubules motors “walk” along microtubule cables
o Dynein moves towares the minus ene
o Kinesin moves towares the plus ene
- Cargo is transportee along microtubules

Dynein mutant: no fow of maternal proeuct into the oocytee Maternal mRNA localiiaton by
microtubules; Bicoid mRNA localiiaton in anterior (towares microtubule minus ene), oskar mRNA
localiiaton at posterior (towares microtubule ene)e What eetermines anterior/posterior polarity in
the oocyte? Posterior follicle cell senes signalse Drosophila par-1 is also requiree for localiiaton of
posterior eeterminantse Polarizaton by par-1 is in response to signaling from posterior follicle cellse

- How are the stem cells regulatee? (answeree)
- How eo stem cells ane their niches response to loss or eamage?
- Can new or more stem cells arise?

Stem cells proeuces another stem cell ane a cell that will eiferentatee Can eiferentatee cells revert
ane de-differentate? Can eiferent cells trans-differentate ane become other types of cells?

- Is it possible to generate new stem cells?
o Replacement by symmetric eivision of GSC
o Reversion of cystocyte cells fate
- Therapeutc use: replacement of lost cells

De-eiferentaton in the germarium:

- Ineuce eiferentaton by ectopic expression of bam: heat-shock-bam (hs-bam)
- Revert eiferentaton by ectopic expression of dpp/BMP

Bam treatee aeult germ line cells become cystoblasts;

- hs-bam treatment of L larval ovaries: eiferentaton into cystocytes  fusomes
- Dpp signal is present in the whole L germ cell compartment
- Cysts become unstable eue to dpp expression  loss of fusomes