• Shuffle
    Toggle On
    Toggle Off
  • Alphabetize
    Toggle On
    Toggle Off
  • Front First
    Toggle On
    Toggle Off
  • Both Sides
    Toggle On
    Toggle Off
  • Read
    Toggle On
    Toggle Off
Reading...
Front

Card Range To Study

through

image

Play button

image

Play button

image

Progress

1/203

Click to flip

Use LEFT and RIGHT arrow keys to navigate between flashcards;

Use UP and DOWN arrow keys to flip the card;

H to show hint;

A reads text to speech;

203 Cards in this Set

  • Front
  • Back
What attaches to the polyA tail in mRNA?
PABP
What is Maskin?
A trxnal repressor
What happens when you repress maskin with an antibody?
There's an increase in expression of Cyclin B1, Map kinase, eIF4E.
What complex is made to silence mRNA?
It's the eIF4E-maskin-CPEB complex and CPSF is unable to bind.
What binds the Guanidine cap in active mRNA?
eIF4E, eIF4G, eIF3, 40S.
What is the complex on the CPE region of active mRNA?
Phosphorylated CPEB, maskin, and CPSF
Fertility concerns impact how many people?
6.1 million, about 10% of men and women of reproductive age.
How many babies in the US are born from IVF?
250K.
How are dairy cows bred in the US?
By artificial insemination using just a few bulls with sorted sperm to make over 80% of female calves.
What are blastomeres?
The large cells of the cleavage stage embryos.
What is MPF
maturation promoting factor. It's the complex of Cyclin B and cdc2. MPF must be activated to move from G to M phase.
How much bigger is the newly fertilized frog?
It has a volume over 1 million times larger than the average differentiated cell.
What dictates cleavage of the early embryo?
Centrosomes
Centriole:
barrel-shaped structure of microtubules
When do centrosomes (paired centrioles) replicate?
During S phase.
What is the Sand Dollar Experiment?
In a zygote, you put a glass ball which displaces the mitotic spindle. The cleavage furrow ends up being inhibited. It ends up making an extra cleavage furrow...
What are the steps of sea urchin cleavage?
The zygote has an interphase nucleus and two centrosomes appear on either side of the nucleus. Spindle fibers and metaphase chromosomes appear in between the centrosomes and then the cell divides.
What is the importance of axis orientation of a spindle positioned near the animal pole?
You end up making the polar bodies by having the centrosomes up near the top.
What is MZT?
Maternal zygotic transition - the clearing of maternal transcripts
What's the first part of early development?
1. Maternal zygotic transition: clearing of maternal transcripts
2. Activation of zygotic transcription
What's the second part of early development?
1. The concept of cell specification and cell fate
2. Sea urchin specification and Wnt signaling
What do miRNAs do?
They bind to mRNA and inactive them and are a signal for Dicer chop up the mRNA.
What is MiR-430?
It's a miRNA specifically transcribed by the zygote at the start of MZT. It leads to the degredation of maternal mRNAs.
Why is it impossible to knock out MiR-430 and how do you get around that?
MiR-430 has multiple family members so it's not possible to knock them all out. However, you can knock out Dicer in zebrafish. Dicer is an enzyme that is required for processing all miRNAs.
What happens to miRNAs if you don't have Dicer?
There are no active miRNAs.
What kind of gene is Dicer in early development?
It's a maternal effects gene.
Based on research with zebrafish Dicer mutants, what do we know about MiR-430?
It clears hundreds (but not all) maternal transcripts.
Name 3 of the first trxn factors to be translated from maternal mRNA.
1. Nanog
2. Sox19b
3. Pou5f1 (known as Oct4)
Name the trxn factors essential for zygotic trxn:
1. Nanog
2. Sox19b
3. Pou5f1
What are LOF's?
Zebrafish lacking the essential factors for zygotic transcription.
What is RPKM?
The standardized measurement for the amount of RNA expression.
4 features of autonomous specification:
1. Typical of invertebrates
2. Cytoplasmi determinants
3. Invariant cleavage and lineages
4. Specification precedes cell rearrangements
Trochoblasts:
Ciliated cells of the mollusc Patella.
What happens if you isolate trochoblasts?
They just develop into more trochoblasts.
4 features of conditional specification:
1. Cell rearrangements precede specification
2. Fates not invariant
3. Regulative development
4. Cell-cell interactions
Name two kinds of specification in early development:
1. Conditional specification
2. Autonomous specification
How do you test conditional specification?
You can take normal back cells and transplant them to the belly region and they back cells will turn into belly cells in the embryo due to cell-cell interactions.
What happens if you remove cells with a glass needle in a blastula?
You'll still have normal development of an embryo because they'll just make more cells and fix itself.
What is the Driesch experiment on conditional specification?
the embryo at the 4 cell stage and separate the four cells. You'll end up getting normal larvae from single cells at the 4-cell embryo stage.
What happens if you isolate the animal hemisphere alone of a sea urchin blastomere?
It ends up making a dauerblastula and has complete animalization.
What happens if you attach the animal hemisphere to the micromere (which is at the bottom) of a sea urchin blastomere?
You essentially rescue the larva a bit. You get endoderm from the animal layers.
What happens if you isolate the micromeres of a sea urchin blastomere?
They generate the skeleton in culture. They're autonomoulsy specified and act as an organizing center.
What's the canonical Wnt pathway?
Wnt --> Frizzles --> Disheveled --I GSK-3 --I beta-catenin --> trxn
What is the micromere specification signal?
Beta-catenin
How many fusion event in fertilization?
4 fusion events
2 kinds of polyspermy blocks
1. fast blocks
2. slow blocks
How does the first cleavage event after fertilization occur?
It involves synchronous cycles of M and S phases in the absense of RNA trxn.
What does fertilization mean?
Fusion of two gametes
What is the vitelline envelope?
Also known as the zona pellucida, it's the extracellular matrix involved in sperm recognition.
What is Resact?
It's a chemotactic peptide in sea urchins. The sperm follow a concentration gradient of Resact.
What lab study can you do with Resact?
You can place Resact in a plate and the sperm will migrate toward it.
What's the acrosome reaction?
1. Ca2+ mediates the fusion of acrosomal and cell membrare. I
2. t's the first membrane fusion event.
3. Release of the enzymes degrade egg extracellular.
4. Initiated by contact with egg jelly: species specific
What is immunostaining?
It's a method for detecting protein localization within the embryo.
What does bindin do?
It's a protein that localizes to the acrosome process.
What is the fertilization cone?
It's a cone formed in the egg by polymerization of actin. It allows for the entry of sperm. It's the 2nd membrane fusion event.
What's an example of fast block to polyspermy?
There's a change in the membrane potential of the egg which starts 1-3 seconds after fertilization.
How can you reverse fast block in frog eggs?
You prevent depolarization of the egg and it leads to increased polyspermy.
What's an example of slow block to polyspermy?
It's the cortical granule reaction. This is the 3rd membrane fusion event.
What is the cortical granule reaction?
Serine proteases release the vitelline envelope. Glycoaminoglycans result in water intake in the perivitelline space, forming fertilization envelope. Peroxidase hardens the fertiliztion envelope by cross-linking Tyr residues. Hyalin is a glycoprotein that coasts the outer surface of the cell membrane, provides suppore during cleavage.
What are 4 molecules that aid in cortical granule reaction?
1. Serine proteases - release the vitelline envelope
2. Glycosaminoglycans - result in water intake forming a fertilization envelope
3. Peroxidase - hardens the fertilization envelope with Tyr cross-linkage
4. Hyalin - glycoprotein that coats the outer surface to provide support during cleavage.
What causes the cortical granule reaction in the first place?
A wave of Ca2+
What releases Ca2+ in the cortical granule reaction?
The ER.
2 events caused by sperm fusing to the egg membrane:
1. Na+ influx --> membrane potential change --> block
2. Kinase stimulation --> Ca release
5 downstream results of Ca2+ release from sperm-egg fusion:
1. Degradation of cyclin --> restoration of mitotic cell cycle
2. Membrane biosynthesis
3. Slow block of polyspermy
4. formation of hyaline layer
5. Stimulation of protein synthesis
4 steps of the union of haploid genomes
1. Chromatin decondenses
2. Microtubule aster forms
3. Pronuclei migrate towards each other and separately synthesize more DNA and fuse into zygotic nucleus
4. 4th membrane fusion event
What's the length of embryogenesis in C elegans?
16 hrs
How many somatic cells in c elegans adults?
959
What are Par proteins?
They are a series of maternal effects genes that affect the cleavage planes and assymetry of early cell divisions.
What happens to par mutants?
They have symmetrical early cleavages and arrest as a blob of cells.
What are p-granules in c elegans?
They are germ-line specific from the earliest stages and are though to be instructors of germ line development.
What do p-granule proteins bind to?
They bind to RNA and are thought to promote germ-cell specific transcription.
Where is Pie-1 expressed?
It's specifically expressed in P-cells and P-granules (maternal transcript)
What does Pie-2 do?
It represses all RNA pol 2 trxn in the early germ line (P2 and derivatives).
What happens to pie-1 mutants?
P2 becomes EMS.
What is Skn-1 required for?
It's required to make endoderm and mesoderm.
What represses Skn-1?
Pie-1
What happens to skn-1 mutants?
They lack endoderm and mesoderm. They make more of cell type C (body muscle and hypodermis).
What does POP-1 do?
It inhibits the formation of E fate (endoderm).
Skn-1 controls...
an endomesodermal gene regulatory network for EMS fate.
What does Skn-1 activate?
Med-1 and Med-2 which direct all EMS fates.
What can you use the bag of worms screen for?
To ID key regulators in early development.
When are D/V and A/P axis laid out in development?
Before fertilization.
When are the oocyte axes set up?
During oogenesis.
What genes initiate AP and DV patterning?
Gurken and Torpedo
Where can you find Gurken and Torpedo?
In drosophila.
What are torpedo and gurken and where are they located?
Torpedo is a receptor and gurken is the protein that binds to torpedo. You find them in the posterior side in the oocyte of the drosophila.
Where is gurken concentrated?
Dorsally
Where do you find pole cells in drosophila?
They're at the bottom...on the posterior side?
What do pole cells lead to in drosophila?
They lead to germ cells.
How can you make a germline chimera in drosophila?
You have an embryo from a wt mother and an embryo from a mother deficient in torpedo. You switch the pole cells. The torpedo-deficient cells will be in the wt female. The wt germ cells will be in the torpedo-deficient. The torp-deficient germ mom will have a torp-deficient oocyte in wt follicle. It will lead to normal DV patterning. The torp def female wil have wt germ cells with torp-def follicle which will lead to no patterning.
What does torpedo repress?
pipe expression.
Where is pipe expressed?
Ventrally.
What is pipe?
It's a sulfotransferase that activates a serine protease pathway.
What does Easter do?
It cleaves Spatzle.
What does Spatzle bind to?
It binds and activates Toll.
What does Toll do?
It activates Pelle kinase.
What does Pelle kinase do?
It phosphorylates Cactus causing it to be degraded
What happens when Cactus is degraded?
Dorsal is released and travels into the nucleus.
When is dorsal protein activated?
Only have 90 min post-fertilization.
What is the dorsal protein pathway?
Pipe --> Easter --> Spa --> Toll --> Pelle --I Cactus --> Dorsal
Where is dorsal located?
In the ventral nuclei.
What happened to nuclear dorsal in dorsalized embryos?
They lose their nuclear dorsal.
What happens to nuclear dorsal in ventralized embryos?
They have ectopic nuclear dorsal.
How is Spatzle activated?
It's cleaved by Easter
What happens to cells exposed to high nuclear dorsal?
They become mesoderm.
What represses Pipe and where is it repressed?
Gurken represses pipe and it's in the follicle cells.
Which cells express pipe?
Ventral follicle cells.
Where are the microtubules in AP distribution?
In the posterior side.
What happens in maternal bicoid loss?
The embryos lack anterior structures.
What does bicoid do?
It encodes a morphogen responsible for head structures.
What happens if you add bcd (bicoid) to a bcd mutant on the anterior side?
You get normal development.
What happens if you add bcd in the middle of a bcd mutant?
You get a head region in the middle.
What happens if you add bcd to the posterior of a wt embryo?
You get a two headed embryo.
Where is bcd mRNA located?
In the anterior portion of the oocyte.
What does Nanos pattern for?
The posterior
What do bicoid and nanos generate?
The hunchback/caudal gradient.
1 factor bicoid inhibits and 1 factor it activates:
1. inhibits caudal
2. bicoid is a trxn factor activating anterior genes like hunchback
What does bicoid activate?
Anterior genes such as hunchback.
What happens if bicoid mRNA reches the posterior?
There is a Nanos binding element in its 3'UTR that binds bicoid mRNA and degrades it.
How many bicoid-dependent enhancers are there?
66.
Changes in Bd concentration are not sufficient to...
change the AP location of Bd-enhancers.
What is Runt?
A txnal repressor.
How are Runt and bicoid related?
They have inverse gradients to each other.
Runt is sufficient to...
antagonize Bd-dependent activation.
How do you affect the precise localization of the gradient of anterior genes?
A bicoid gradient establishes only a rough framework of the downstream genes. The precise localization is shaped by a network of Runt repressors that integrate feedback from a few key Bd target genes.
Name 3 gap genes:
1. Kruppel
2. Hunchback
3. Knirps
What do gap genes?
They lead to the patterning of the embryo and produce inhibitory interactions between each other.
Name 2 pair rule genes:
1. ftz
2. eve
You would like to perform experiments that explore the role of the genes Sall1 and Pitx1 in zebrafish (Danio rerio) development.

1. You decide to assay Sall1 expression by in situ hybridization. Unfortunately, you do not have a construct in your laboratory for the zebrafish Sall1 gene. However, a neighboring laboratory has an probe for the mouse Sall1 gene. Do you have enough information to determine if the probe against the mouse gene would work for in situ hybridization experiments in zebrafish? If so, describe why you think they would work. If not, what additional information do you need?
It is highly unlikely to work. However, you do not have enough information to say conclusively because you do not know how closely the fish and mouse genes resemble each other. If the probes extremely high conservation on the nucleic acid level (virtually identical), they experiment would work. On the other hand if the nucleic acid sequences are substantially different, the probe would not hybridize to the fish gene.
You would like to perform experiments that explore the role of the genes Sall1 and Pitx1 in zebrafish (Danio rerio) development. There are no probes available for Pitx1 so you need to make your own. You generate a plasmid that contains the entire cDNA sequence for the zebrafish Pitx1 gene. The first 10 bases of the "sense" strand are:5'-atggcctcat-3' What does "sense" mean?
The sense strand of DNA is the strand the corresponds to the mRNA strand (of course the mRNA will have U instead of T)
What would be the "antisense" RNA strand for the above sequence? 5'-atggcctcat-3'
5'augaggccau-3'
You prepare an in vitro transcription reaction to synthesize Digoxigenin-UTP labeled RNA for your probe. Which strand from above should be labeled? Why?
You must label the antisense strand so that it will hybridize to the mRNA (sense) within the embryo
You are deeply impressed with the Shapiro et al., 2004 study describing a likely role for Pitx1 in stickleback pelvic fin morphology. In those experiments, they showed that marine sticklebacks express Pitx1 in the pelvic region but freshwater sticklebacks have lost that expression. Is this a mutation in the gene or in a regulatory region? Explain your answer.
It is in a regulatory region. The gene was not mutated in freshwater sticklebacks. However, the gene not expressed in the pelvic fin region in the freshwater fish. Importantly, the gene is still expressed in other regions. This strongly suggests a mutation in a limb-specific regulatory module for the gene.
You are deeply impressed with the Shapiro et al., 2004 study describing a likely role for Pitx1 in stickleback pelvic fin morphology. In those experiments, they showed that marine sticklebacks express Pitx1 in the pelvic region but freshwater sticklebacks have lost that expression. What is the best evidence that this mutation specifically affects gene regulation (and the trait is not, for example caused by salt water)?
Remember that the F1 fish crossed from the marine and freshwater are all reared in captivity under identical conditions. They were then crossed with each other, generating F2 fish (again all under identical lab conditions). They then analyzed which portion of chromosomes are associated with pelvic fin reduction using SNPs specific to freshwater and marine alleles. They found that a region of the chromosome that contained Pitx1 accounted for a siginficant fraction of pelvic fin reduction. Since the fish are otherwise reared under identical condition, this strongly argues that something about the segment of DNA in that region confers pelvic fin reduction (it does not itself establish that it was Pitx1 - they inferred this by comparing with known mouse genes involved in hindlimb formation).
List the three germ layers. For each, give one example of one of the cell types that tissue will ultimately give rise to
Ectoderm (Brain/Skin), Mesoderm (Heart/Bone), Endoderm (Liver/Pancreas)
Many other correct examples for each (page 15 of your book)
Which of the following structure(s) is/are part of a typical vertebrate Bauplan? Notochord, malleus, pelvic spines, heart, pharyngeal arches, stapes
Notochord, Heart, Pharyngeal arches. The other structures are not embryonic. Moreover, the malleus is only found in mammals, the stapes is not found in sharks (rather a hypomandibular arch), pelvic spines are a highly derived structure found in sticklebacks
What is a general derivative of the first pharyngeal arch in all vertebrate animals?
The tongue, the jaw...
Hand2 is a highly conserved gene that plays essential roles in heart and pharyngeal arch development (embryos with a mutation in the Hand2 gene die early in development due to severe heart defects). Consistent with this role, the gene is expressed in both the heart and the pharyngeal arches. There is a well characterized cardiac enhancer and you wish to characterize a pharyngeal arch enhancer. You find a dozen candidate enhancer regions by performing genomic alignments of the Hand2 locus among multiple vertebrate species. How could you experimentally determine which (if any) of these regions actually functioned as pharyngeal arch enhancers?
Clone them into a DNA construct that contains a minimal promoter (ability to bind Pol2) and a reporter gene (LacZ or GFP). Generate transgenic embryos from these constructs. f the DNA construct causes the reporter gene to be expressed in the pharyngeal arch of the embryos, then it is acting as a pharyngeal arch enhancer.
In class, we talked about "deep homology" in Pax6, a transcription factor which is required for eye development in organisms ranging from flies to mammals. This pathway specifically targets photoreceptors. Photoreceptors transmit information to their targets - and, although we did not discuss, in class, this process to occurs with some homologous genes in both Drosophila and humans. One of these genes is another transcription factor, called Math5 in Drosophila and ATOH7 in humans. Recently, it was shown that a mutation in a long-range enhancer for ATOH7 underlies a form of human blindness caused nonsyndromic congenital retinal nonattachment (NCRNA) (Ghiasvand et al., Nat. Neuroscience 2011 vol. 14:578-86). This disease is autosomal recessive, and affects ~1% of the people in a remote village in Iran. The phenotype is nearly identical to that seen in Atoh7 null mouse embryos.

A) In mutated individuals, a large deletion of 6523 base pairs removes an enhancer.
This occurs in humans - you can't make transgenic humans - but since there is a mouse model system, could do in mice or another vertebrate system (the actual study used zebrafish). Generate a minimal promoter::reporter construct (reporter can be GFP or LacZ, which encodes beta-galactosidase) and clone in the 6523bp region. Generate transgenic embryos (this construct is randomly inserted into their genome) and see if it drives expression in the eye during embryonic development.
In class, we talked about "deep homology" in Pax6, a transcription factor which is required for eye development in organisms ranging from flies to mammals. This pathway specifically targets photoreceptors. Photoreceptors transmit information to their targets - and, although we did not discuss, in class, this process to occurs with some homologous genes in both Drosophila and humans. One of these genes is another transcription factor, called Math5 in Drosophila and ATOH7 in humans. Recently, it was shown that a mutation in a long-range enhancer for ATOH7 underlies a form of human blindness caused nonsyndromic congenital retinal nonattachment. Confusingly, there is already a well defined enhancer much closer to ATOH7 in mice (and conserved in humans) that drives enhancer activity in the exact same cell population at the same developmental time. What do you think this second enhancer could be doing?
Since the phenotype (in humans) mirrors a null phenotype (in a mouse model system), it provides strong genetic evidence suggesting that the NCRNA enhancer is critical for driving sufficient levels of ATOH7 expression for proper eye formation. Since the more proximal element also drives expression, perhaps both enhancers are needed to drive sufficient transcription of the gene.
You assay primary oocytes that are still in their primary cell cycle arrest. How many chromosomes are present at this stage?
The are 4 copies of each chromosome.
How many chromosomal copies are extruded in the first polar body? How many in the second polar body?
2, 1
You divide Xenopus oocytes into two groups. You do not treat the control group and you treat the second group with progesterone. What visible change would you expect to see after progesterone treatment?
Nuclear envelope breakdown (also called germinal vesicle).
You divide Xenopus oocytes into two groups. You do not treat the control group and you treat the second group with progesterone. You perform in situ hybridization on both of the above groups with an antisense probe for mos. What is this technique measuring? Which samples do you anticipate detecting a signal?
In situ hybridization measures gene expression. Mos mRNA is present throughout oogenesis so you expect to see staining in cells from both groups.
How does Maskin repress translation? How is this repression relieved? What in your view is the most important mRNA that must be translated upon progesterone translation? Justify your opinion.
Maskin binds to eIF4E, preventing it from being bound by eIF4G, which is the critical protein required to recruit the 40S ribosomal subunit for translational initiation. The repression is relieved when CPEB is phosphorylated, resulting in the extension of a longer poly-A tail, which then stabilized interactions between eIF4G and eIF4E, outcompeting interactions between eIF4E and Maskin. Mos is the most important protein because experiments showed that in the absence of new protein synthesis, the injection of Mos protein is sufficient to cause germinal vesicle breakdown.
The table below indicates the S-phase and M-phase for zygotes immediately upon fertilization and in the next two complete cell cycles are listed. Indicate the presence (+) or absence (-) of each of the component during each stage.
Fertilization (1-2 Cells) 2-4 Cells
S-Phase M-Phase
Cyclin B mRNA + +
Cyclin B Protein -** +
Cdc2 mRNA + +
Cdc2 Protein + +

**Immediately at fertilization, cyclin B protein is destroyed but they are quickly translated and cyclin B protein starts to build up. In this sense, this first S-phase is not so very different than the others.
You treat Xenopus oocytes with A23817, a calcium ionophore. What will be the first major visual change in the eggs?
It will cause the cortical granule reaction (visible fertilization envelope)
You assay primary oocytes that are still in their primary cell cycle arrest. How many chromosomes are present at this stage?
The are 4 copies of each chromosome.
What causes the vitelline envelope to swell after the cortical granule reaction?
The release of glycosaminoglycans
What are the four membrane fusion events during fertilization?
Acrosome fuses with sperm plasma membrane; sperm plasma membrane fuses with egg plasma membrane (fertilization cone); cortical granules fuse with egg plasma membrane; male and female pronuclei fuse (in some species the nuclear membranes surrounding the pronuclei break down before fusion).
You have used molecular biological techniques to generate a fusion protein of Cyclin B and GFP (CyclinB::GFP). You perform extensive tests that show that the fusion protein is indeed as active as regular Cyclin B and that it also glows green under the appropriate fluorescent filters. You inject large (but not toxic) amount of this mRNA into oocytes immediately before fertilization.

A. What do you anticipate seeing as you watch these cells under a fluorescent microscope over the next several hours?
Cyclin B protein is degraded at the end of mitosis. Early cell cleavage is successive cycles of M-S-M-S….The cells should glow green, turn off, glow green, turn off, etc. This would only stop at the onset of zygotic transcription (or earlier if the mRNAs degraded before then).
You have used molecular biological techniques to generate a fusion protein of Cyclin B and GFP (CyclinB::GFP). You perform extensive tests that show that the fusion protein is indeed as active as regular Cyclin B and that it also glows green under the appropriate fluorescent filters. You inject large (but not toxic) amount of this mRNA into oocytes immediately before fertilization. What would you expect to see if you also injected these cells with cycloheximide and why?
The Newport and Kirschner experiment showed that CHX injection causes cells to quickly arrest in S-phase. Cyclin B protein levels are absent (or very low) during S-phase even in a normal cell. However, CHX blocks protein translation so none of the RNA in the cell will be translated and you will not see any green protein.
Sketch a just-fertilized C. elegans egg at the single cell stage. Indicate that following components: sperm binding site, centrosome, anterior, posterior.
Sketch should show sperm at future posterior of eggs (which is dictated by the centrosome from the sperm)
Sketch the same egg as above - but this egg is derived from a Par-2 null mutant mother. Can you predict the future anterior and posterior regions? Why or why not?
Sketch should show sperm binding site and centrosome - but Par-2 mutants are symmetrical with no anterior or posterior axis. So you could not label these regions.
Which of the following crosses in an organism having a recessive mutant allele in a hypothetical maternal effects gene called 'Maternal' would result in an embryonic phenotype? Here, '-' refers to the mutant allele while '+' is the wild-type allele.
a) Mat +/- eggs x Mat +/- sperm
b) Mat +/- eggs x Mat -/- sperm
c) Mat -/- eggs x Mat -/- sperm
d) Mat -/- eggs x Mat +/- sperm
e) Mat +/+ eggs x Mat -/- sperm
C,D. Maternal effects phenotypes depend on the genotype of the mom (eggs) while the genotype of the embryo itself or the paternal chromosome is irrelevant.
For each cross(es) above generating a maternal effects embryonic phenotype, what fraction of embryos should contain a phenotype?
For all crosses, maternal effects mutations should be 100% penetrant.
The Rappaport sand dollar experiment presented in class support a model where cleavage furrows are generated by the centrosomes and do not require chromosomes or a mitotic spindle. If those components had been required, what would have been the final outcome of the experiment?
Only three cells.
Once present, are the micromeres of a sea urchin specified or determined? Justify your answer.
Determined. When transplanted to the animal side of a sea urchin embryo, they still retain their identity (indeed they respecify the animal cells into endoderm and mesoderm, creating a mirror-image embryo).
Sketch a cell indicating where you would expect to find following components of the Wnt pathway in a cell actively receiving a Wnt signal (membrane, cytoplasm, nucleus, outside the cell): Wnt, Frizzled, B-catenin.
Wnt is a secreted ligand (synthesized in cells and secreted outside the cell - so present in the cytoplasm and mostly extracellularly). Frizzled is a receptor (membrane), Beta-catenin is in the cytoplasm in the absence of Wnt. In the presence of Wnt signaling, B-catenin moves to the nucleus.For more info., see pages 92-93 of the book.
You remove the EMS blastomere from a C. elegans embryo and culture in isolation:
A. What will be the identity of the two daughter cells? What cell type(s) would they give rise to?
Both cells will be MS. MS cells give rise to mesoderm
Sandwich the EMS blastomere between two P2 blastomeres (obviously derived from two different embryos). What do the EMS blastomores give rise to? How could you experimentally show these are descendants of EMS and not P2?
Both EMS daughter cells will become E. These give rise to endoderm. You could look for the presence of endodermal markers, but would also have to have some type of lineage tracing strategy (injecting a fluorescent dye, mRNA encoding GFP….) since in an experiment you don’t know ahead of time what will happen.
What happens to microRNAs if there is no Dicer around?
MicroRNAs will not be processed into the short single-stranded 22nt form of an active miRNA. This will have the effect of making all miRNAs inactive.
You have identified a maternal transcript that is very efficiently cleared during MZT in zebrafish embryos. You hypothesize that it is controlled by MiR-430 as we discussed in class. Design an experiment that will allow you to compellingly demonstrate that this transcript is SPECIFICALLY controlled by MiR430. Your experiment should include experimental reporter constructs and controls and you should indicate what portion of the transcript is being tested in your constructs.
miRNAs bind to the 3'UTR so the first thing to do is to look at the sequence of the 3'UTR (available in public databases) and see if there is homology to the MiR430 seed region. If no homology, it suggests this is being regulated in an MiR430-independent mechanism. If, as expected, there is one or more seed regions, you clone the 3'UTR of your gene downstream of a construct containing the coding region for a reporter gene e.g. GFP - you call this construct A. You also clone the same 3'UTR into the reporter but first generate point mutants in the MiR430 binding regions (construct B). If GFP expression in construct A goes away at MZT but persists at MZT in construct B, the experiment supports your hypothesis. A nice control to ensure your RNA construct isn't simply being degraded is to con-inject a second mRNA construct encoding a reporter that does not quickly degrade (has no mIR-430 binding regions and is known to have strong expression for a long itme - could be the dsRed control.
You wish to identify the set of mRNAs that are activated zygotic transcription in Xenopus embryos. Describe two experimental strategies that would allow you to do this.
Divide a pool of early embryos into two pools. One fraction (Fraction A) is untreated. Treat the other fraction (Fraction B) with a transcriptional inhibitor (e.g. alpha-amanatin). Extract total RNA from both fractions and process it for massively high-throughput RNA-sequencing (RNA-seq). After you get the data, which contains tens of millions of independent reads, you look for reads corresponding to intronic sequences. Such reads represent nascent, still unspliced transcripts that are indicative of zygotic transcription (maternal transcripts were spliced before fertilization). An alternative strategy would be to look for transcripts present in Fraction A that are either reduced no longer present in Fraction B.
Do you think MiR430 is processed from a maternal or zygotic transcript? Why
It is zygotically transcribed. If this were a maternal transcript, it would degrade maternal transcripts way too early (before zygotic transcription).
Skn-1 is a master regulator of EMS fate. It activates the transcription factors Med1 and Med2, which in turn activate End1 and End3. Both E and MS have Med1, 2 present but only E activates End1 and End3. Why?
Because these genes are also regulated by the transcriptional repressor Pop-1. If Pop-1 is around it prevents End1,3 transcription even in Med1,2 are also present. MOM (Wnt) activity secreted by P2 only cause a depression of POP1 in the EMS region directly adjacent to P2. As a result when EMS divides, the blastomere closest to P2 becomes E while the other cell (MS) represses Endoderm fate and becomes mesoderm (driven by Tbx35).
Skn-1 mRNAs are maternal transcripts that are present in oocytes and then translated only in a subset of cells after fertilization. SKN-1 protein is present in both EMS and P2.
A. Why doesn't SKN-1 protein cause P2 to become E/MS in wild-type embryos?
Because PIE-1 prevents Pol2 transcription - Skn1 is not active.
You are interested in understanding how Skn-1 mRNA is selectively translated in only two of the four blastomeres.You decide to test two ideas. One is that Skn1 mRNA is localized to the P1 blastomere during the transition from 1-2 cell stage (this could in principle be by active recruitment of the mRNA in P1 or destruction of the mRNA in AB). The second is that the mRNA is present everywhere but is translationally repressed in AB. Design experiments to test the ideas in A and B.
You could do in situ hybridization with an antisense probe against Skn1 to detect mRNA expression in WT 1 and 2 cell stage embryos. If mRNA is localized to the posterior, it would support idea #1. If mRNA is localized throughout the embryo, it indicates idea #1 is wrong (in fact in the literature mRNA is localized throughout the embryo). To test translational represssion, you could take the Skn1 3'UTR and clone it downstream of a GFP reporter. Then use this to make transgenic embryos and see of GFP co-localizes to P1. The actual mechanism by which Skn1 is translationally repressed is unknown.
Cactus is a maternal effects gene that is essential for dorsal-ventral patterning. Describe its function?
Cactus is present in the cytoplasm and binds to the Dorsal transcription factor, preventing it from entering the nucleus by blocking the nuclear localization signal. Upon Toll receptor activation, the Pelle kinase phosphorlyates Cactus, causing it to be degraded. Dorsal then enters the nucleus
. Predict the phenotype of a Cactus null mutation on the embryo.
Dorsal expression in all nuclei -> complete ventralization
Generate germline mosaic adult flies by grafting Cactus mutant pole cells onto wild-type embryos, whose own pole cells have been removed. What would be the phenotype, if any, for the resulting embryos derived from a cross of these mosaic females with wild-type males?
Will make a mosaic fly that has produces Cactus -/- oocyte with wild-type (Cactus +/+) follicle cells. All fertilized eggs from this mosaic fly will produce ventralized embryos because Cactus is required in the egg only.
Generate germline mosaic adult flies by grafting wild-type pole cells onto Cactus mutant embryos, whose own pole cells have been removed. What would be the phenotype, if any, for the resulting embryos derived from a cross of these mosaic females with wild-type males?
Will make a mosaic fly that has a wild-type oocyte (Cactus +/+) surrounded by Cactus -/- follicle cells. All fertilized eggs from this mosaic fly will be normal because Cactus is not required in the follicle cells.
You intercross Gurken +/- male and female flies to produce Gurken null mutant embryos. What is the predicted phenotype? Why?
The resulting Gurken null embryos will have a wild-type phenotype because the mothers were Gurken +/-. Gurken is a maternal effects gene, and so the genotype of an embryos mother gives the phenotype of all of its progeny.
What would be the phenotype of progeny generated by crossing Gurken -/- female flies with wild-type males?
All embryos would be ventralized because a lack of Gurken will cause a lack of Torpedo receptor activation in dorso-anterior follicle cells. Without Torpedo, the follicle cells will not repress Pipe, and they will therefore express Pipe. Expressing Pipe causing the initiation of a protease cascade that is activates the Spatzle ligand, allowing it to activate the Toll receptor, causing Cactus to be degraded and Dorsal to then translocate to the nucleus. N.B. The Dorsal protein is not translated until about 90 minutes after fertilization. So the D-V gradient is already in place before fertilization - but in an mRNA-fashion.
What is the dorsal mutant phenotype? What is dorsal? What is the germ layer composition of the region of cells receiving the highest dose of dorsal?
The dorsal mutant phenotype is a dorsalized embryo. Dorsal is a transcription factor that acts as a morphogen to activate ventral cell populations in a dose-dependent fashion on the future ventral side of the embryo. The cell receiving the highest dose of dorsal become mesoderm.
How is bicoid mRNA localized anteriorly? If mRNA is restricted anteriorly, how would you predict a protein gradient would be established?
The 3'UTR of Bicoid mRNA attached to dyneins (microtubule "motor" proteins that carry things towards the minus end of microtubules). It also requires the “escort’ proteins Expuerantia and Swallow. Recall that Gurken signaling to Torpedo in posterior of the cell causes microtubule orientation to be arranged such that the minus ends are all at the anterior end of the oocyte. Not specifically applying to this answer but related is that non-anterior Bd mRNA is translationally repressed by Nanos.
You identify a previously unknown gene, Drosophillin, that is expressed several vertical stripes along the A-P axis of a syncitial stage Drosophila embryo. Zygotic mutants for this gene exhibit a novel phenotype, and you decide to undertake a research project that examines how the gene is transcriptionally activated. You examine gene expression (using which technique?) and determine that the one of the stripes of gene expression (stripe 1) occurs in the anterior embryo in a domain overlapping Hunchback (Hb) and Biocid (Bd) protein expression domains (which technique measures this?). The gene is not expressed until several hours after the onset of Hb and Bd protein expression, and based on the correlation in expression patterns, you hypothesize that stripe 1 of Drosophillin is a directly activated Hb, Bd (or both). Describe a genetic experiment that would allow you to determine if Drosophillin is genetically downstream of either of these?
You examine gene expression using in situ hybridization. You use immunostaining to detect Hb and Bd protein expression using antibodies against Hb and Bd with fluorescent-conjugated secondaries. Examine the expression of Drosophilin mRNA in Hb and Bd mutant embryos using in situ hybridization. If there was a loss of the imaginary Drosophilin stripe 1 gene expression in 1 or both of the mutants, it indicates that it is genetically downstream of that gene. For example if Stripe 1 is absent in Bd mutants, then it is genetically downstream of Bd.
What is the best technique for determining if an early embryonic cell is in the S-phase at a given timepoint?
Labeling DNA with radioactive nucleotides.
What kind of cells are nurse cells?
Germ cells
What is Par-2 involved in?
Regulating AP polarity in c elegans
The P blastomere in c elegans is ______ specified. The E blastomere in c elegans is ____ specified.
Autonomously; conditionally
The acrosome reaction and the cortical granule reaction are derived forms of...
exocytosis
You fertilize sea urchin eggs then immediately inject them with a calcium chelator (something that strongly binds all free calcium). You do this quickly and the injection is completed about 5 seconds after fertilization. What do you think happens?
The embryos will likely have polyspermy because this disrupts the cortical granule reaction.
A morphogen is...
a substance that can determine cell fates in a concentration-dependent manner.
4 factors that participate in the bicoid gradient:
1. Exuperantia
2. Vasa
3. Nanos
4. Dynein
What protein directly binds to the cap of the mRNAs?
eIF4E
What molecule acts as a direct trxnal activator in reponse to a canonical Wnt ligand?
Beta-catenin
Scientists believe that limbs are evolutionarily derived from fins. What evidence would you need in order to be convinced that this was consistent with Von Baer's Laws?
The embryonic origin of both fins and limbs would need to be the same.
Based on what we discussed in class, do you think MiR-430 is maternally or zygotically transcribed? Why?
It is zygotically transcribed because it clears out maternal transcripts. If expressed maternally, it would likely clear out maternal transcripts even before fertilization.
You identify Drosophila embryos with a distinct phenotype. You determine that they have a mutation resulting in constitutive activation of the Toll receptor (it is always on). What is the phenotype and why?
These embryos will be completely ventralized because activating the Toll receptor (normally in the ventral region by activated spatzle) causes nuclear Dorsal accumulation.
You identify Drosophila embryos with a distinct phenotype. You determine that they have a mutation resulting in constitutive activation of the Toll receptor (it is always on). Do you think this would be a recessive mutation? Why?
No - it will not. If it is turning a receptor on (instead of getting rid of activity), it would be dominant.
You cross pipe +/- females with pipe +/- male flies. What will the resulting embryos look like? Why?
All embryos will look normal. This is because pipe is a maternal effects gene (once the embryos grow up, the progeny of pipe -/- embryos will all be dorsalized.
You wish to ID the set of mRNAs that are activated zygotic trxn in xenopus embryos. Describe a specific experimental strategy that would allow you to specifically ID these mRNAs while excluding maternal RNAs?
Option 1: Treat embryos with an inhibitor of trxn and then perform RNA-seq on these embryos and compare the sequences to control, untreated embryos. Transcripts absent in the inhibitor but present in the control group are likely to be zygotic transcripts.
Option 2: Perform RNA-seq on oocytes and specifically look into intronic sequences (will occur only transiently since they are spliced out of primary transcripts but will only occur in newly transcribed, zygotic mRNAs.
What is the biggest difference in the specification of the AP axis between c elegans and drosophila? Think big picture only.
The posterior axis of c elegans is defined by the sperm (which will go to the nearest oblong end from its entry point). In contrast, the AP axis of flies is specified during oogenesis.
Polar bodies are extremely small compared to the oocyte. Which organelle/structure is directly responsible for defining the size of the polar body?
The centrosome.
You generate oocytes that only contain a mutated form of CPEB that cannot be phosphorylated but is otherwise normal. You then treat them with progesterone. You come back a few hours later and look under the microscope. What do you see and why?
No change - the oocytes will fail to undergo NEB. This is because if CPEB can't be phosphorylated, Mos will remain trxnally repressed. This will prevent the activation of the downstream events that ultimately generate an active MPF that drives NEB.
You are interested in understanding how Dicer is regulated in zebrafish. List the genotype of the crosses and only the informative genotypes that would allow you to determine if embryonic development requries maternal transcripts only zygotic transcripts only or materal and zygotic transcripts. How would you analyze the data to make your conclusion?
Maternal only:
Zygotic only:
Maternal and zygotic:
Maternal only: Female Dicer -/-
(don’t need to specify that she was generated by
transplantation) x Male Dicer +/+;
Zygotic only: Dicer +/-
males x and Dicer +/-
females to generate zygotic nulls (Dicer -/-
)
Maternal and zygotic: Female Dicer -/-
x Male Dicer +/-
to generate Dicer -/-
embryos.
The key would be looking at the severity of the phenotypes. If maternal only, the
maternal and zygotic mutation should be no worse. Likewise, if zygotic only, the
maternal zygotic mutation should be no worse.
You identify a Bicoid-responsive enhancer and confirm that it is expressed in
Drosophila embryos by generating transgenic embryos containing an enhancer::LacZ
reporter.
A) What do you think would happen to reporter gene expression if you crossed the
transgenic into a mutant embryo lacking any Biocid?
Reduced or absent reporter activity
You identify a Bicoid-responsive enhancer and confirm that it is expressed in
Drosophila embryos by generating transgenic embryos containing an enhancer::LacZ
reporter.
What do you think would happen to reporter gene expression if you crossed the
transgenic into a mutant embryo lacking any Runt?
The reporter activity would be expanded
You identify a Bicoid-responsive enhancer and confirm that it is expressed in
Drosophila embryos by generating transgenic embryos containing an enhancer::LacZ
reporter. What do you think would happen to reporter gene expression if you crossed the
transgenic into a mutant embryo expressing high levels of Runt everywhere?
The reporter activity would be reduced or absent
When P2 divides in a Pie1-/-
embryo, the C blastomere adopts an MS fate while P3 becomes another
E cell.
A. This should be slightly surprising to you (or at least you should be able to think of a plausible
alternative fate). If I hadn’t told you this, what fate do you think P2 might have adopted?
Explain what you think might be going on.
In the absence of Pie-1, Skn1 activity will not be inhibited. Therefore, Skn1 will convert P2 into
another EMS-like cell. Without a P2 blastomere, it would seem likely that there might not be MOM1
around to induce an E fate. Therefore, I have predicted the P2 would divide to form two MS cells. The
phenotype suggests that the EMS-like cell retains MOM1 expression and further suggests that MOM1
can self-induce an E fate.
When P2 divides in a Pie1-/-
embryo, the C blastomere adopts an MS fate while P3 becomes another
E cell. Propose a detailed experiment that would allow you to test this
Several credible experiments. One would be to dissect the EMS-like cell and make an explant containing it as well as a wildtype EMA. If the EMS-like cells retains MOM1, it could cause the wild type EMS cell to produce an E cell (MOM1 would self-induce this fate). You would need to use a marker of E cell identity (eg End1) and describe how you would detect it. (eg, in situ hybridization. Lots of variations on this.
When P2 divides in a Pie1-/-
embryo, the C blastomere adopts an MS fate while P3 becomes another
E cell.
You decide that you need a faster way of interpreting data. Describe the features of a
transgenic worm that would allow you to visually identify the E cell in living worms?
You could generate a minimal promoter GFP (or some other fluorescent protein to allow for detection
in living worms) construct that also included an endoderm enhancer from an endodermal gene (such
as End1). You received partial credit if marking it with POP1 even though POP1 is actually not unique
to that cell type and is an inductive signal.