6: C elegans Flashcards
What are some characteristics of C. Elegans (worms) that make it a good model organism for studying development?
1)Culture: agar plates covered with e.coli (bacterial plates) can also be grown in liquid broth with ecoli too
2) Size: just over 1mm, small enough and thin enough that all cells are visible by lught (phase or DIC) microscopy
3) Storage of Strain: can be frozen
4) Crosses: population is 99% “selfing” hermaphrodites (meaning that they have both male and female reproductive organs, so they can reproduce by selfing which is self reproduction: can use their own sperm to fertilize their own egg)
5) Generation time: 3 days (20 degrees)
6): Lifespan: reproduce for 3-4 days but live two more weeks: allwo researchers time to study all phases
7) # of progeny: 250-1000 per hermaphrodite (lower if herm selfed, higher if male cross)
8) Markers: ample visible markers (usually things like body shape and movement):
Genome size: 97 mB; total genes 19,000
# needed for development: 1700
Simple nervous system: 302 neurons exhibit limited behavior
What does being a selfing hermaphrodites allow them to create
genetically identical offspring since they are using their own sperm to fertilize their own eggs:
hermaphrodite means that they have both male and female reproductive organs
selfing: means self fertilization where they fertilize their owb sperm
what percentage of them are hermaphrodites: 99%
the remaining 1% are males
-result from X chromosome non-disjunction
(hermaphroditic female= XX
males: XO)
Why is the number of progeny less for hermaphrodite selfing
ebcause they are limited by the production of sperm in hthe hermaphrodite
Hermaphrodite and fertilization
-in early stages of development the herm produces sperm adn stores in the spermatheca: sperm is non-flagellate crawling amoeboid like cells
-Later in development, the herm switches to egg production (oogenesis): distal tip of ovary is syncytium and oocyte nuclei ar ebound by plasma membrane and bud off from the syncytium (oocyte develop in syncitium and undergo cellularization as it moves down the ovarhy)
-Oocytes arrested in prophase 1 meiosis: moves and are fertilized as they ar epushed through theSPERMATHECA:
essentially: the oocytes are rolled down the ovary until they reach the spermatheca where the sperm is and they are fertilized
where are the oocytes of c elegans fertilized
as they are pushed through the spermatheca:
triggers completion of meiosis since before hte ooxyte was arrested in prophase.1 of meiosis
How is polyspermy thiught to be blocked
by rapid deposition of chitin over the egg
What type of cleavage is observed in c elegans blastula?
Why can the lineage of any cell in the adult be traced back to the first division
Reproducible patterns of cell divison:
they have invariant embryonic development meaning that adult male or hermaphrodites display an EXACT NUMBER of cells (959 for herm, 1031 for male)
-since its invariant embryponic development: reproducible patterns of division occur meaning that it can be traced back to the first division that occured
What is type one embryogensis
-immediate activation of zygotic genes
-small number of cells at the start of gastruka
-rapid blastomere specfiication occurs in situ
Why is cell ablation possible in c elegans
because of invariaten lineage pattern of development and becaue all cells are visible, we can use a lasar to distry ceterain cells to understand it more
RNAi in c elegans
The process leverages the natural RNAi mechanism found in most eukaryotic organisms, where double-stranded RNA (dsRNA) induces the degradation of complementary messenger RNA (mRNA), effectively “turning off” the target gene.
how it owrks: can be introduced using e coli (as food source) that express dsRNA or by injecting it
-inside the cytosplasm, the enzyme DICER splices the dsRNA into short interfering RNAS
step 2: assembly of risc:
these are RNA-induced silencing complexes, the short interfering rnas are encorporated into this complex, the complex uses it as a guide to bind to complimentary mrna
step 3: now the RISC degrades the target mrna, preventing production of the corresponding protein
this is useful for researchers BECAUSE:
it allows them to silence certain genes in order to study their importance, fjunction, and effect on development , behaviour etc
What types of specification is used
Autonomous specification (assymetric divisions, so unequal distribution of cytoplasmic determinnats) and conditional (cell- cell interactions, so regulative developemtn(
is cell fate determined by cell to cell basis
yes, it is mosaic like and invariant embryonic development is caused by cell to cell basis
Cleavage is…
rotational holoblastic
holoblastic: complete cleavagel so the entirecell divides into seperate cells
rotational: meanins that at second division, one daughter cell divides longitudinally while the other divides transversley
for C elegans:
division are assymtric so resulting cells are different in size: anterior is larger than the posterior p cells in c elegans
Anterior founder cells: produce differentiated descendants
PosteriorP lineage: stem cells that become the germ line
Outline the model for how the anterior-posterior axis is
established in the C. elegans egg
LOOK AT card 27
unfertilized c elegans had no predetermined axis (unlike drosophilia)
-it is thought that the sperm specifies the initial assymetries by directing cytoplasmic rearrangements that cause determinants to become assymteirically localized
1) elongated egg shape: defines future anterior-posterior axis: decision for anterior end relates to position of sperm pronucleus
- the sperm centriole intiates cytoplasmic movement that pushes the male pronucleus to the nearest end- that will become the posterior pole
-one stage cell becomes polarized by a contraction of the actomyosin cortex away from the site of fertilization
-the fertilizing sperm has two cues for the assymetric contraction: centrsome and protein called CYK-4
SOOOOOO the different components of the cytoskeleton(ie the microfilaments: actin and myosin and the microtubules) are responsible for the assymetry (anterior and posterior sections)
-this assymetryic contraction is needed to bring the par proteins to the proper side to establish the axis
ESSENTIALLY: microfilaments and microtubles are needed for the assymetric contraction which brings the par proteins to the proper side that then establishes the axis (defines it)
-par 3, nd 6 in anterior
1 and 2 in posterior
Partition defective genes
-Maternal effect lethal mutations: occur in c elegans
-earliest defecgs observed as disturbances in te assymetry of the first mitotic division. Rather than making an unequal division, the mutants made the first division symmetrical
-all 6 par genes are required for the assymetric division of zygote
-these par genes have other functions yet they are incredibly important for polarity establishments.
in early zygotes:
-par 3 and 6: anterior cortex (become enriched here during one cell stage)
-par 1 and 2: enriched in posterior cortex
-par 4 and 5:remain symmetrically localized thru this period, both cortical and cytoplasmic
When do they adopt assymetric localization patters:
Post fertilization
When does blebbing occur
(pseudocleavage)
-before fusion of male pronucleus to female pronucleus
-cortical cytoplasm flows away from smooth domain at posterior
What does CYK-4 affect
the actin based cytoskeleton
Does actin mysoin network move away or toward the site of fertilization
away
After the cortical flow, where is the concenration of 1) f actin, b) myosin 2 c) par 3 d)par 2
a) anterior
b) anterior
c) anterior
d) posterior
Where does par 2 accumulate if there is is a meotic arrest phenotype (failure to complete meiosis after fertilization)
near the arrested meiortic microtuble spindle (on oocyte side)
What is PKC-3
-kinase found in the complex with par 3 and 6
-it can phosphorylate the lipid binding domain of par 1 and 2 meaning they can no longer bind to membrane
-prior to fertilization , the par 3 ,6 pkc-3 complex is found on the membrane THROUGHOUT the oocyte and can phosphorylate par 2 and mkae it fall off
What is a special feature of par 1
it is also a kinase (in complex with par 2) that similarily to pkc-3, can phosphoralte the lipid binding domain of Par 3
-meaning that par 3 can no longer can bind to membrane
If the posterior pars arent found what happens to the anterior pars
found all over the embryo, vice cersa occurs if anterior pars arent found
-meaning that each complex can prevent the other from binding to their membrane
Where does par 2 tend to accumulate
on the membrane closest to the microtubules around the sperm centrosome
-purified par 2 loves tobinf to microtubules themselves
because the microtubules protect the par 2 from phosphorylation
What type of mutation prevents par 2 from having its protection
3 AA change stops par 2 from binding to microtubules so protection is lost
SO what is the story with how the par 2 stays on posterior side
Since pkc3 can kick off par 2 from membrane,…
if par 2 stays with microtubules, it is protected so pkc-3 cannot phosphorylate and cause it to get kicked off, causing par 2 to recruit par 2 and then par 1 now can phosphoylate par 3, causing par 3 and its complex which is par 6 and pkc-3 to fall of the membrane, now more par 1 and 2 are localised to the membrane near the microtubles (which is posterior) and now the other complex is booted from it leading to the posterior end being determined.
-eventually there are microtubles thorughout embryo as first division spindle is established AND there is actin myosin flow and contraction twds anterior that carries the detached par 3 complex to the anterior, further assymetric distribution determinants also depends on this system
what does actin myosin cytoskeleton associate with? What about centrosome and microtubles
actin myosin cytoskeleton ( par 3 complex (3 6 and pkc-3)
micro: par2 and par 1 complex
COrtival flow moves par. but requires it
Male pronucleus defines what end
posterir
CYK-4
-RhoGAP
-enriched within sperm
monomeric small gtpases:
-gtp binding proteins
-one of two classes of g protiens
-molecular switches
binding of gtp or GDP rewuire GEF (guanine nucleotide exchange factors) and GAPS (gtpase activating proteins)
GEFS turn on
gaps turn off
so since it is a rho gap, this mea s that it can prevent rho1 activity in the posterior reguon since it is locally relaxing the activity in the posterior region with CYk4, causing the actomyosin contraction to move towards the anterior end idk chat it cuz its kinda confusing
What does activated rho do
binds to different targets known as effector proteins
-rho is known to influence the regulation of actin cytoskeleton :specficially non muscle myosin 2
What happens if embryos lack rho1
-myosin activation faisl to occur, actomyosin network doesnt work normally.
-anterior par proteins remain uniformly distributed along the anteroposterior axis of embryo SOOOOOO anterior par proteins remaind EVERYWHERE
What is the difference between rhogef and rhogap
rho-gef: activates rho signalling by triggering gdp/gtp exchange
rho-gap: inactivates rho signalling by catalyzing gtp hydrolysis
What are P granules
Ribonucleoprotein complexes containing translation regulators that specifiy the germ cells and are localized by par
-initially all over then become localized in posterior after fertilization
-inherited by p1 cell
-when p1 divides, granules remain in posterior and inherited by only p2
-then associatd with nuclues of p3 and p4 cells form gernline
what does p cell behave as
stem cell
what does p granule localiation need
microfilaments but not microtubules
what drug prevents p granule posterior localization
cytochalasin D: disrupts microfilaments
COLCHICINE DOES NOT because it disrupts microtubules
How does p cells have open cell fate like stem
they have pie-1
-it is from mom
-needed for germ cell fate
-it represses rna pol 11 which keeps it open
If the p1 cell is isolated from ab cell what occurs
it can generate all cell types normally seen in the posterior
-therefore, the p1 lineage and all of te founder cells of the p cell lineage are specified autonomously (meaning that intrinsic factors are what cause a cell fate) not by external factors
If the AB cell is isolated from p1… what happens
it can only generate a small fraction of the cell types that it would normally make
therefore the specifications of some of the ab blastomere descendants is conditional and requires interactions with descendants of the p1 cell in order to develop normally
What does the dorsal ventral axis relate to
division of the ab cell, because of space, one ab cell is forced anterior and one posterior, making ABa and ABp
-as it divides, the egg shell becomes longer than wide
AB divides before p1?
Yes
Where does ABp lie and define
above ems cell
-ems cell results from division of P1
Defines: the future dorsal side of the embryo
What does the ems cell mark and define
defines the future ventral surface of the embryo
ems is precursor of muscle and gut cells
Conditional specification
-evident at 4 cell stage
-ems cell divides to give MS cell (future mesoderm) and E cell (future endoderm)
-WITHOUT P2 cell, ems will divide into two ms cells and no e cell with form so no endoderm cell
-ABa and ABp are specified by relative position to P2 cell NOT to eachother
IF notch signalling embryo doesnt exist, what happens
lack dorsal structure
-APX-1 (delta) signals to glp-1 (notch) specifies dorsal fate of ABp: and bc of spatial constraint and notch/delta being juxtacrien signalling Aba does not recieve this signal
AB cell cleavage means ABa is more anterior to wat
ABp
What does ema moving do
ABa and ABp reversal does not disrupt development (ABa and ABp are equivalent), but the displaced EMS cell inverts the normal D/V axis.\
If ABp is prevented frm contacting p2 what happens
an ABa cell is formed instead
this is because the induction of abp by p2 involves the glp-1protein in the abp cell and APX-1 protein in the p2 cell
GLP-1= notch
APX-1 = Delta
if…
ems is removed at the end of 4 cell stage: it can produce gut structures
ems is removed at earlier: it cannot
P2 is removed at early stage: ems cannot form gut structures
P2 signals to EMS is MOM-2 (WNT) and receptor in ems is MOM-5 (frizzeled)
p2 signal results in polarization of the ems cell
Does c elegans have strong left right symmetry
yes: specification of left right occurs after A/p and D/V and occurs at the third cleavage division
CELL CELL interactions and positions influence specification
SKN-1
Skinhead 1
-maternally expressed transcriptoon factor that controls the fate of the ems blastomere
-ems cell is the progenitor of the posterior pharynx and intestine
-maternal skin hebaves as a determinant of pharynx and intestine = autonomous specification
IF U LOSE IT (skn-1):
it (ems blastomere) makes hypodermal and muscle cells instead of pharynx and intestines so these have extra hypodermal cells if skn-1 is lost/ mutant
Why is ems called ems
gives rise to endoderm, mesoderm and stromodeum (part of pharynx)
SOOOO what does each cell type give rise to
ABa: anterior end (buccal cavity area)
EMS: gives rise to MS and E
e: midgut intestines
MS; pharynx
ABp: rectum
look at slide 90
Gastrulation in c elegans
cells move small distance
blastocoel spaceis small and transiet
starts after p4 cell made in 24 cell stage embryo
Ea and Ep cells migrate from ventral side to centre of embryo – here they divide to form 20 cells that form the gut – migration does form a small blastopore
P4 cell migrates through blastopore and positions itself beneath the gut primordium
