Cytoplasmic Determinants

Question 1

what is a localized cytoplasmic determinant?

Answer 1

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.

Question 2

what was the earliest experimental evidence for these?

give an example.

Answer 2

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.

Question 3

what is an example of cytoplasmic determinant sequestration? describe the first step of this process.

Answer 3

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

Question 4

what happens after first cleavage?

Answer 4

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

Question 5

what happens to polar lobe after 2nd cleavage?

Answer 5

resorbed into D blastomere, now larger than A,B, and C blastomeres.

Question 6

what does the polar lobe likely contain?

Answer 6

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

Question 7

what is evidence for the polar lobe containing cytoplasmic determinants of mesoderm-derived structures?

Answer 7

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

Question 8

what is the function then of the polar lobe?

Answer 8

formation is method for sequestering determinants such that they are selective allocated to D blastomere + descendents

this allocation is necessary for mesodermal organ formation

Question 9

what happens after cleavage of the D blastomere (especially with one specific descendent)?

Answer 9

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.

Question 10

what is the D blastomere called? what does the original one contain?

Answer 10

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)

Question 11

what are the primordial germ cells in flies? what is a distinguishing feature of them?

Answer 11

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.

Question 12

do we know if determinants are present in the pole cells? if yes, how?

Answer 12

UV irriadiation of posterior end of embryo prior to cellularization prevents gamete formation, suggesting cytoplasmic determinants reside there (in the so-called pole plasm)

Question 13

what does UV-irradiation do to the cytoplasm? can irradiated embryos be rescued?

Answer 13

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)

Question 14

is pole plasm sufficient to induce pole cell formation? give a brief overview of the experiment that confirmed this

Answer 14

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

Question 15

what is oskar+?

Answer 15

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

Question 16

where is oskar normally localized? how is movement performed?

Answer 16

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

Question 17

what is Vasa+? what is its role in pole cell formation?

Answer 17

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)

Question 18

bicoid is another example of a localized cytoplasmic determinant. what is the phenotype of bicoid- (bicoid mutant) embryos?

Answer 18

missing head and thorax, instead have posterior structures where anterior structures should be. can be mimicked by “bleeding” out of anterior cyto.

Question 19

can bicoid- embryos be rescued? what happens if bicoid is added in the wrong position?

Answer 19

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.

Question 20

where is WT bicoid+ localized in anterior end of cell? how does it get there?

Answer 20

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.

Question 21

what are other maternal effect genes important for bicoid localization?

Answer 21

exuperantia (or Exu)+, swallow+, staufen+.

Exu may help in initial recruitment of bicoid mRNA into complexes w/ Swallow and Staufen (mediates transport).

Question 22

what else is necessary and sufficient for bicoid localization?

Answer 22

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

Question 23

what is one of the first events in A-P axis determination in Drosophila? what is vital to this process?

Answer 23

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.

Question 24

what is myoplasm (Ascidian eggs)? where is it found?

Answer 24

yellow-pigmented cytoplasm that gets segregated into cells that will form tail muscle, initially found in layer just beneath PM

Question 25

what is the ectoplasm and gray cytoplasm? characteristics of each?

Answer 25

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

Question 26

egg activation involves 2 phases of cytoplasmic rearrangements. describe phase 1

Answer 26

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

Question 27

describe phase 2 of egg activation

Answer 27

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

Question 28

how are the A-V and A-P axes of polarity been established?

Answer 28

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)

Question 29

what do cells that inherit myoplasm differentiate into? can this be done artificially?

Answer 29

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

Question 30

what is myoplasm important for, and why? what is the default state of the embryo?

Answer 30

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

Question 31

what do the myoplasm experiments suggest about interaction between anterior + posterior states?

Answer 31

posterior is inhibitory towards anterior end

Question 32

what are yellow pigment granules associated with? what is this also associated with?

Answer 32

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)

Question 33

what happens with the PML after fertilization?

Answer 33

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.

Question 34

what does cytochalasin B do? what does this suggest?

Answer 34

treatment w/ cytochalasin B inhibits phase 1, not phase 2 of cytoplasmic rearrangements.

suggests cytochalasin depolymerizes actin

Question 35

what does treatment of colcemid or nocodazole do? what does this suggest?

Answer 35

treatment w/ either of these 2 inhibits second phase, but not first

suggests they depolymerize MTs

Question 36

what is the determinant for muscle formation? describe its general characteristics

Answer 36

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

Question 37

how does macho-1 depletion/insertion affect cells?

Answer 37

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