Cytoplasmic Determinants Flashcards
what is a localized cytoplasmic determinant?
any component of the cytoplasm that is unevenly distributed in egg or embryo AND affects fate of the cells that will contain it.
could theoretically refer to organelle(s) but usually refers to mRNA or proteins.
what was the earliest experimental evidence for these?
give an example.
ligation experiments
ex: in sea urchin, if egg is divided such that each half retains some animal + vegetal cytoplasm, 2 normal embryos result.
if egg is cleaved equatorially, then the animal half develops into a “dauer” blastula (abnormal ciliated blastula that doesnt develop further), vegetal half forms very abnormal embryo.
showed that inheritance of both cytoplasms is necessary for embryo development.
what is an example of cytoplasmic determinant sequestration? describe the first step of this process.
polar lobe formation in the snail Ilyanassa (spiral cleavage)
1) an anucleate lobe of cytoplasm is formed at vegetal pole of zygote just before first cleavage
what happens after first cleavage?
lobe remains attached to the CD blastomere (resulting 3 lobed structure is called trefoil-stage embryo)
lobe is resorbed back into CD blastomere subsequently, now significantly larger than AB blastomere
before 2nd cleavage, another polar lobe is extruded at vegetal pole of CD blastomere
what happens to polar lobe after 2nd cleavage?
resorbed into D blastomere, now larger than A,B, and C blastomeres.
what does the polar lobe likely contain?
appears to contain cytoplasmic determinants (possibly anchored to cytoskeleton in lobe), and are vital for formation of mesodermally derived (or partially-mesodermally-derived) organs such as muscles, intestine, heart, mouth, and food
what is evidence for the polar lobe containing cytoplasmic determinants of mesoderm-derived structures?
removal of polar lobe at trefoil stage results in embryos lacking in muscles, intestines, heart, etc
similar embryos occur if D blastomere is removed
if free-flowing part of cytoplasm is removed, cytoplasm from other blastomeres is used to replace it, then a reasonably normal embryo results
what is the function then of the polar lobe?
formation is method for sequestering determinants such that they are selective allocated to D blastomere + descendents
this allocation is necessary for mesodermal organ formation
what happens after cleavage of the D blastomere (especially with one specific descendent)?
descendent of D called the 4d blastomere (mesentoblast) inherits B-catenin, enters nucleus (regulates gene expression) w/ translational suppressor Nanos
due to determinant inheritance, 4d can undergo autonomous mesodermal/intestinal differentiation. Notch signaling is important for 4d to play inductive roles.
what is the D blastomere called? what does the original one contain?
set of D blastomeres are the “organizers” of snail embryos. original D blastomere contained determinants required for proper cleavage rhythm and orientation (+ organizers for 4d to autonomously differentiate into mesoderm and intestine)
what are the primordial germ cells in flies? what is a distinguishing feature of them?
primordial germ cells of fly are pole cells (if pole cells are removed/damaged, resulting adults have no gametes)
disting. feature of pole cells = polar granules - dark in colo, contain RNAs necessary for pole cell formation as well as for formation of some abdominal structures.
do we know if determinants are present in the pole cells? if yes, how?
UV irriadiation of posterior end of embryo prior to cellularization prevents gamete formation, suggesting cytoplasmic determinants reside there (in the so-called pole plasm)
what does UV-irradiation do to the cytoplasm? can irradiated embryos be rescued?
UV irradiation crosslinks RNAs + proteins, effectively neutralizing them in terms of function
UV-irradiated embryos can be saved (in terms of gamete formation) by injecting donor posterior cytoplasm (note that anterior cytoplasm does not work)
is pole plasm sufficient to induce pole cell formation? give a brief overview of the experiment that confirmed this
yes
a primary host with recessive alleles for some genes at blastoderm stage had pole cells ectopically transplanted to a secondary host with other recessive alleles (homozygous)
the mosaic adult flies with recessive alleles was crossed with flies of the genotype of the original primary host, found that a small amount of WT flies were born - could not happen if pole plasm was sufficient
what is oskar+?
oskar is a maternally-encoded protein that is necessary + sufficient for pole cell formation (injection @ other sites can cause ectopic pole cell formation).
protein product functions by causing localization of other proteins + RNAs nec. for germ cell formation
where is oskar normally localized? how is movement performed?
mRNA normally localized to posterior pole (after transfer from nurse cells) by MTs (localization prevented on treatment w/ colchicine)
movement of oskar mRNA requires Staufen protein, appears to couple RNA to kinesin
what is Vasa+? what is its role in pole cell formation?
encodes a protein that interacts w/ oskar protein, once oskar has been produced by tln.
Oskar/Vasa/Staufen complexes are thought to interact w/ posterior pole cytoskeleton, trap other RNAs (important for pole cell formation) that are brought into proximity via. cyto. stream. (forms mature polar granules)
bicoid is another example of a localized cytoplasmic determinant. what is the phenotype of bicoid- (bicoid mutant) embryos?
missing head and thorax, instead have posterior structures where anterior structures should be. can be mimicked by “bleeding” out of anterior cyto.
can bicoid- embryos be rescued? what happens if bicoid is added in the wrong position?
yes, introduction of bicoid+ anterior cyto. @ anterior end of mutant
injection of bicoid+ laterally (in middle, basically) causes embryo to form head structure in middle flanked by throacic structures
injection at posterior end results in head structures at both ends.
where is WT bicoid+ localized in anterior end of cell? how does it get there?
in WT, bicoid RNA is localized to anterior end
after synthesized in nurse cells, it is transported to oocyte by a MT-dependent mechanism, localized to anterior end.
what are other maternal effect genes important for bicoid localization?
exuperantia (or Exu)+, swallow+, staufen+.
Exu may help in initial recruitment of bicoid mRNA into complexes w/ Swallow and Staufen (mediates transport).
what else is necessary and sufficient for bicoid localization?
3’ UTR of bicoid mRNA. region has elaborate 2ndary structure, mediates interaction w/ swallow proteins. if 3’ end is spliced onto a diff. mRNA, it will be localized to anterior end.
such 3’ UTRs are called zipcodes
what is one of the first events in A-P axis determination in Drosophila? what is vital to this process?
oocyte polarization - bicoid mRNA being present at anterior pole, oskar + nanos (w/ others) present at posterior pole, leads to formation of important protein gradients.
formation of RNPs (ribonucleoprotein particles) is also vital to process.
what is myoplasm (Ascidian eggs)? where is it found?
yellow-pigmented cytoplasm that gets segregated into cells that will form tail muscle, initially found in layer just beneath PM
what is the ectoplasm and gray cytoplasm? characteristics of each?
ectoplasm = clear cytoplasm, derived from germinal vesicle. cells that inherit it give rise to ectoderm, starts out in animal half
gray cytoplasm - yolky, contributes to endoderm
egg activation involves 2 phases of cytoplasmic rearrangements. describe phase 1
occurs while egg is completing meiotic divisions
a) myoplasm streams down egg periphery, accumulates as yellow cap at V pole
b) ectoplasm flows towards V pole, forms clear layer above yellow cap
c) in process, gray cyto. is displaced towards A pole
d) by end of phase 1, egg looks like a sand-art thing
describe phase 2 of egg activation
begins after meiotic divisions are complete
a) male pronuc. migrates towards A pole, myoplasm + ectoplasm somehow move w/ it
b) much of gray cyto. moves back into V hemisphere
c) 4th region of cytoplasm (dark chordoplasm) forms roughly opposite of myoplasm - will contribute to notochord
d) 2 axes of polarity have been established - animal-vegetal, and anterior-posterior
how are the A-V and A-P axes of polarity been established?
A-V: marked by ectoplasm, polar bodies, and gray cytoplasm, V will be future dorsal side of embryo
A-P: marked by yellow myoplasm (future posterior end), dark chrodoplasm (future anterior end)
what do cells that inherit myoplasm differentiate into? can this be done artificially?
cells that inherit myoplasm (myoblasts) contribute to muscle
blastomeres (from animal pole) that normally contribute to epidermis can become muscle cells if infused w/ anucleate “cyto. frags” containing myoplasm
what is myoplasm important for, and why? what is the default state of the embryo?
myoplasm important for posterior dev:
1) removal results in formation of anterior structures all around embryo
2) ectopic transplantation of myoplasm to ant. pole of eggs lacking myo. results in “reverse-polarity” tadpoles
thus, anterior state is default
what do the myoplasm experiments suggest about interaction between anterior + posterior states?
posterior is inhibitory towards anterior end
what are yellow pigment granules associated with? what is this also associated with?
yellow pigment granules of myoplasm are associated w/ lattice (believed to be composed of intermed. filaments)
above lattice is associated w/ actin-rich layer underlying the PM. actin-rich layer is called PML (PM lamina)
what happens with the PML after fertilization?
after fert., PML moves to vegetal pole and is hypothesized to be driving force for concomitant movement of myoplasm to V pole.
meanwhile, male pronucleus gets swept down w/ it towards V pole.
what does cytochalasin B do? what does this suggest?
treatment w/ cytochalasin B inhibits phase 1, not phase 2 of cytoplasmic rearrangements.
suggests cytochalasin depolymerizes actin
what does treatment of colcemid or nocodazole do? what does this suggest?
treatment w/ either of these 2 inhibits second phase, but not first
suggests they depolymerize MTs
what is the determinant for muscle formation? describe its general characteristics
macho-1+, a maternally-derived mRNA
encodes a Zn-finger transcription factor (think DNA-binding, allows for dimerization)
can be depleted by injection of antisense RNAs - bind to sense strands, hybrids destroyed by cell
how does macho-1 depletion/insertion affect cells?
depletion of macho-1 via treatment w/ anti-sense RNAs from fert. eggs results in muscle-deficient embryos
microinjection of macho-1 mRNAs into other parts of embryo results in muscle formation
