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CMMB 403 > Final 2 > Flashcards

Final 2 Flashcards

1
Q

Vitelline envelope

A

An extracellular matrix envelope all around the Drosophila embryo. Vitellin proteins DO NOT get sulfated on the dorsal side

The proteins do get sulfated on the ventral side by Pipe *enzyme
Pipe is active ventrally.

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2
Q

What is the gastrulation defective (GD) protein and what does it bind to?

A

It is a protease in Drosophila embryos. It binds to sulfated vitellin proteins on the ventral side of the embryo.
Pipe enzyme sulfated the the vitellin proteins - these serve as docking sites for GD

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3
Q

What are the three proteases in the dorsal-ventral axis initiation pathway?

A

Gastrulation Defective (GD), Snake, and Easter

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4
Q

What protein cleaves the Snake protein?

A

GD will cleave the Snake protease to make it active. the cleavage will only happen when GD is bound to the sulfated vitellin proteins

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5
Q

What protein cleaves the Easter protein?

A

The activate Snake protease cleaves the Easter protease to make it active
- this is only happening in the ventral area because GD is bound to sulfated vitellin proteins here.

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6
Q

What does the Easter protein cleave?

A

When cleaved and activated, Easter will cleave the Spatzel protein, which can then function as a ligand for the Toll receptor on the embryo’s cell membrane (syncytium)

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7
Q

What ligand binds to the toll receptor?

A

the cleaved spatzel ligand. Easter cleaved the spatzel

The Toll receptor is present on the cell membrane of the embryo (syncytium)

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8
Q

What happens when the Toll receptor is activated?

A

The cleaved Spatzel ligand will bind the Toll receptor (which is present on the cell membrane of the embryo) and this will activate the protein kinase called Pelle within the embryo’s cytopasm

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9
Q

What is the Pelle protein?

A

A protein within the cytoplasm of the embryo’s syncytium in flies. It is a protein kinase
When the cleaved Spatzel ligand binds and activates Toll this will activate the Pelle Ser-Thr kinase which will then Phosphorylate Cactus - leading to the degradation of cactus.

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10
Q

What causes the degradation of Cactus? Where does this occur in the embryo?

A

The activation of Pelle protein kinase (initiated by Spatzel binding Toll receptor) which phosphorylated Cactus, resulting in its degradation.

Cactus is generally only degraded in the ventral part of the embryo (allows Dorsal to enter the nucleus)

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11
Q

What do mutations in proteins of the dorsal-ventral axis initiation pathway result in?

A

Mutation of: GD, Snake, Easter, Spatzel, Toll, Pelle, or Dorsal will all result in a dorsalized embryo because dorsal is not able to enter the nucleus and act as a TF
No cells will have dorsal entering into the nucleus

Mutation of Cactus: ventralized embryo because dorsal will always be allowed to enter the nucleus (anywhere in the embryo)

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12
Q

What causes the embryo to be dorsalized? ventralized?

A

Dorsalized - no dorsal entering the nucleus anywhere to act as a TF - This can result from mutation of numerus proteins (not including cactus)

Ventralized - dorsal entering the nucleus everywhere - This can result from mutation of cactus, which normally functions to keep dorsal out of the nucleus on the dorsal side of the embryo

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13
Q

What is IRAK protein in humans homologous to in flies?

A

Homologue to Pelle and Tube (adaptor that helps Pelle bind to the Toll receptor)

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14
Q

What is the vertebrate homolog of Dpp (decapentaplegic) ?

A

BMPs

Dpp is a BMP ligand for Ser-Thr kinase receptors
EXPRESSED DORSALLY

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15
Q

What is the vertebrate homolog of Sog (BMP inhibitor) found in Drosophila?

A

Chordin (a BMP inhibitor) in vertebrates

Sog is an inhibitor of Dpp in flies - keeps Dpp levels low ventrally by binding to them

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16
Q

What is the vertebrate homolog of MAD found in Drosophila?

A

SMAD1,5

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17
Q

How do you get induction of the NS?

A

Inhibition of BMPs

Using Sog in flies, Chordin in vertebrates

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18
Q

Tolloid

A

A dorsally expressed protease that degrades Sog dorsally (prevents it from being active dorsally). Get more Dpp active in dorsal area because its inhibitor is being degraded)

Degrades inhibitor and thus increases Dpp expression

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19
Q

Is Sog active dorsally or ventrally (flies)?

A

Sog is active VENTRALLY - Need to inhibit Dpp ventrally for formation of the NS here. (want low Dpp ventrally)
Tolloid degrades Sog dorsally (so you get higher Dpp dorsally)

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20
Q

What is chordin?

A

A BMP inhibitor expressed in vertebrates. It is present dorsally to inhibit BMPs dorsally–> get low BMP dorsally which allows for NS induction.
Get the most phosphorylated SMAD1/5 where BMP signalling is the highest (ventrally)

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21
Q

BMP inhibitors in vertebrates?

A

Chordin, Noggin, Follistatin

Cells gain expression of these as they travel through the organizer during gastrulation

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22
Q

What do the cells that go through the organizer during gastrulation become?

A

pharyngeal endoderm, mesoderm (prechordal plate, notochord)
These cells will gain expression of BMP inhibitors as they travel through - expression of these molecules will induce the NS structures to form in the overlying ectoderm.

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23
Q

Where is the organizer formed in amphibians?

A

Dorsal lip of the blastopore

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24
Q

In what pole of the amphibian egg does the blastopore (and thus organizer) form?

A

Vegetal pole

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25
Q

When cells pass through the organizer, what molecules do they begin expressing?

A

BMP inhibitors (chordin, noggin, follistatin) as well as Wnt inhibitors that are necessary to induce the formation of anterior/head structures (such as the brain)

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26
Q

For amphibians, when is the location of the organizer determined?

A

At fertilization! The location depends on where the sperm enters the egg. The centriole brought in by the sperm is used to reorganize the disorganized arrangement of microtubules within the egg
- the organization of the microtubules then allows for cortical rotation

  • The organizer forms at the area opposite of sperm entry!
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27
Q

Cortical rotation

A

Rotation generated by now organized microtubules (the sperm brought in centrioles that could be used for organization)

  • 30 degree rotation at the cortex of the cytoplasm - important for establishing the axis of the embryo
  • in some species the rotation will reveal a lightly pigmented cytoplasm - the gray crescent

The cortical rotation relocates factors towards where the organizer will form
- required for the stabilization of B-catenin
The organizer forms at this gray crescent area!

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28
Q

What kind of cleavage does a xenopus egg undergo?

A

Holoblastic mesocithal cleavage (moderate amount of yolk)

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29
Q

Are vegetal pole cells smaller or larger than animal pole cells?

A

larger!

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30
Q

Which cells are the first to head inside during frog gastrulation?

A

The bottle cells (ingress) – these cells are constricted at their apical end which triggers their involution through the dorsal lip of the blastopore - These cells will later become the pharyngeal endoderm.
The next cells to involute become the mesoderm (prechordal plate (future head meso) and chordal meso (becomes the notochord), followed by the somitic meso)

  • anterior endoderm – followed by dorsal mesoderm
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31
Q

What does epiboly do during gastrulation?

A

Covers the entire endoderm and mesoderm with ectoderm. Spreading out of an epithelial layer.

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32
Q

What combination of molecules induces the amphibian organizer to form where it does?

A
  1. Stabilized B-catenin (Wnt signalling) in dorsal part of the blastula
  2. Cocktail of dorsal mesoderm-inducing signals - mostly NODAL-RELATED TGF-β superfamily members – which are secreted by the Nieuwkoop centre (located just below where the organizer will form). Thus, there will be phosphorylated Smad2,3 here
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33
Q

What signals do the Nieuwkoop centre cells secrete to help in forming the organizer at the dorsal lip of the blastopore?

A

Nodal-like TGF - B superfamily members which function through Smad2,3
These are dorsal mesoderm-inducing signals!

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34
Q

Which maternal mRNAs are necessary for specification of the mesoderm?

A

vegT and Vg1

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35
Q

VegT

A

Maternal mRNA necessary for mesoderm specification

Located at the vegetal pole. Translated to form a TF that will turn on Nodal-related genes

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36
Q

Vg1

A

maternal mRNA that is necessary for specification of the mesoderm.
It is a nodal-like TGF-B superfamily member as a protein and therefore signals through Smad 2/3

Translated only in the dorsal region of the embryo (where the organizer forms)
-protein induces the future dorsal mesoderm to express Wnt inhibitors

.

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37
Q

GSK3 Binding protein (GBP)

A

binds GSK3 and inhibits it so that B-catenin can remain intact
Kinesin takes GBP and Disheveled toward the dorsal end where the organizer will form - allows for B-catenin stabilization here.

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38
Q

Stabilized B-catenin plus what other factors required for organizer formation? *

A

Association of B-catenin with a coactivator will get activation of target genes (siamois and twin gene) - these genes code for TFs that activate expression of BMP inhibitors.

Siamois and Twin transcription factors must bind in association with Smad2 to get transcription of BMP inhibitors activated

SO
1. TGF-B Nodal-like members (required to get phosphorylated Smad2
2. Stabilized B-catenin
are both needed to get organizer formation

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39
Q

What combination of factors gives ventral mesoderm induction?

A

low nodal related signalling and no stabilized B-catenin

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40
Q

What are involuting cells that form the pharyngeal endoderm expressing to specify the brain and anterior structures to form?

A

Head inducers = Wnt inhibitors - cerberus, frisbee, and dickkopf

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41
Q

What are examples of Wnt inhibitors?

A

Frisbee, cerberus, dikkopf, crescent (humans)
These molecules will bind Wnt ligands and prevent them from binding to Frizzled (even though ligand is present - no signalling)

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42
Q

What are two molecules needed to get formation of the head?

A

Chordin (BMP inhibitor) and frisbee/cerberus/dikkopf (Wnt inhibitor), both in the anterior!
See expression of BMP inhibitors and Wnt inhibitors
Both the prechordal plate and the pharyngeal endoderm express these

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43
Q

What molecules are expressed in the posterior of the embryo?

A 
BMP inhibitors (expressed in anterior and posterior)
No Wnt inhibitors - so there is Wnt signalling occurring.
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44
Q

What molecules are expressed to induce the epidermis?

A

Get both BMP and Wnt signalling to specify the epidermis.

No BMP inhibitors or Wnt inhibitors.

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45
Q

Where is the organizer in zebrafish gastrulation?

A

Called the embryonic shield
They do not undergo cortical rotation.

The same signals cause the organizer to form (Nodal signalling and stabilized B-catenin) but we don’t know why the specific location.

46
Q

What does the fish organizer originally secrete?

A

Wnt inhibitors and BMP inhibitors

The nieuwkoop centre does not become a part of the fish embryo

47
Q

What is bozozok?

A

An ortholog of the siamois (a TF gene activated by B-catenin in frogs).
Stabilized B-catenin helps this to form where the embryonic shield will form in zebrafish. It is a TF that cooperates with Smad2 to activate BMP inhibitor genes.

48
Q

area pellucida

A

Thin and transparent, becomes the body of the embryo. The primitive streak extends 2/3 of a way across this (from posterior to anterior)
Becomes the body of the embryo

49
Q

Area opaca

A 
Surrounds the area pellucida
Extraembryonic tissue (chick embryo)
50
Q

Henson’s node

A

In chick embryo. A thickening of cells at the anterior end of the primitive streak - Is the chick’s ORGANIZER

Once the primitive streak appears, the anterior to posterior axis of the embryo is defined.

51
Q

Epiblast

A

all of the cells of the embryo come from the epiblast. The epiblast cells ingress through the primitive streak to form the endoderm and mesoderm. Those cells of the epiblast that do not ingress form the ectoderm (those that do not gastrulate)

52
Q

How might space and time of ingression during gastrulation predict which structures will form as a result?

A

Cells ingressing through the streak more anteriorly (toward the node) will form more DORSAL structures
Cells entering the streak first tend to form more ANTERIOR structures. (intermediate and lateral plate)

The cells that enter through the middle of the streak will form more ventral mesoderm structures like heart, blood, kidneys

53
Q

Difference between movement of cells through the organizer in amphibians vs chicks?

A

Cells that go though the node/streak INGRESS - they undergo a epithelial to mesenchymal transition whereas cells in amphibian gastrulation go inwards via involution.

54
Q

What is Koller’s sickle?

A

Thickening of cells at the posterior end of the embryo where the primitive streak extends out from

55
Q

What is the posterior marginal zone?

A

At the junction between the area pellucida and opaca
The Nieuwkoop centre!

Vg1 is a Niewkoop centre gene

56
Q

What induces formation of the organizer in the chick embryo?

A

Vg1 (Nodal-like signalling - gives Smad2/3-P)
chordin (BMP inhibitor)
and stabilized B-catenin

57
Q

Hypoblast

A

Forms only the embryonic membranes

The yolk sac comes from the hypoblast

58
Q

When does the human embryo transition from a bilaminar germ disc to a trilaminar germ disc?

A

14-15 days of development

59
Q

What type of cleavage do mammals have? Amphibians?

A

Mammals - rotational cleavage

Amphibians and Echinoderms - radial cleavage

60
Q

Mater

A

The first mammalian maternal effect gene discovered.
Haplosufficient
Needed for symmetric divisions of the early embryo
If the mother of the embryo is homozyg null for Mater - she will be sterile and the embryo will soon die.

Mater forms a complex just beneath the plasma membrane of egg –> subcortical maternal complex - required for early divisions

This complex is required for symmetric divisions in the zygote

61
Q

Is cactus haploinsufficient?

Most MEG are haplosufficient

A

Yes

62
Q

What proteins form the subcortical maternal complex?

A

Mater, Filia, FLOPED, TLE6

All of these are required for early symmetric divisions of the zygote. symmetric mitotic spindle.

TLE6 mutation - causes earliest known human embryonic lethality

63
Q

what does the trophectoderm(trophoblast) form?

A

The embryonic part of the placenta
2 types of trophoblast, the syncytia- trophoblast and the cyto-trophoblast, together form the chorion

Chorion - forms the embryonic portion of the placenta
- involved in gas exchange

64
Q

What is the hatching of a mammal?

A

When the embryo arrives in the uterus to implant, it hatches out of the zona pellucida, which causes it to become ‘sticky’ and implant in the uterus.

65
Q

What is happening at day 10-12 of human development?

A

Embryo is at bilaminar disc stage with chorion

66
Q

How is the amnion formed?

A

Through delamination of the epiblast (epithelial layer forming from another epithelial layer)

67
Q

What day does human gastrulation begin?

A

~Day 14 (ie. bilaminar to trilaminar germ disc)

Endoderm and mesoderm ingress through the primitive streak

68
Q

What does a blastocyst consist of?

A

Inner cell mass (becomes the epiblast and hypoblast) and trophoblast (chorion)

69
Q

Where does the gut form from?

A

Cells which ingress through the primitive streak. They form the endoderm and then the hind gut later. Primordial germ cells also ingress through the streak - they end up near the hindgut

70
Q

Kartagener Syndrome

A

These people have reversed left-right asymmetry (situs inversus, chronic bronchitis, male sterility.
Result of a mutation/defect in the motor protein that is required for movement of cilia.

71
Q

How is left-right asymmetry in your body set up during gastrulation?

A

~100 cells on the ventral surface of the node that have cilia (motile). The movement of these cilia is responsible for left-right asymmetry. Clockwise rotation generates flow to the left

cerberus - gene that is expressed on the left side
Nodal - gets expressed at the node and on the left side (beating cilia push it left) - Nodal activates Pitx2 - heart develops on the right side
Pitx2 - a TF also on the left side

Need functional motile cilia AND asymmetrical gene expression

72
Q

What is the normal location (L vs R) of common body parts?

A 
Liver - right
heart - left
spleen - left 
3 lobed lung - left 
2 lobed lung - right
ascending part of the large intestine - right 
appendix - right
descending colon - left
73
Q

What are the more 5’ hox genes?

A

The posterior ones! These genes are expressed more posteriorly in the organism and are turned on later in development than more 3’ or anterior genes.

74
Q

Which vertebrate hox gene paralogs share the same ancestral origin as AbdB homeotic genes?

A

The 9 paralogs! Hoxa9, Hoxb9, Hoxc9, Hoxd9

75
Q

What are homeobox genes?

A

They encompass ALL Hox genes.
They encode TFs with a 60 amino acid. 3α helices, DNA-binding homeodomain

The human genome codes for over 200 of these proteins

The homeodomain sequence is highly conserved between paralogs, but there will be slight differences.

76
Q

Hox genes

A

A subset of Homeobox genes with the same evolutionary origin as the Drosophila homeotic complexes.
So they have a 60 a.a. region with 3 α helices that is a DNA-binding homeodomain (MOST CONSERVED REGION)
39/200

77
Q

What amino acids are conserved in the 5th position of the homeodomain part of the homeobox genes?

A

Arginine or Glycine

The DNA binding function may be impaired if the conserved amino acids are changed (in the TF)

78
Q

How many thoracic vertebrae do humans have?

A

12 (12 pairs of ribs)

Mice have 13

79
Q

Which Hox paralogs pattern the cervical vertebrae?

A

The 4 paralogs! And the 5 paralogs helps too

80
Q

What are the Hox6 paralogs responsible for patterning?

A

Expression limit of the Hox6 paralogs is at the most anterior part of the thoracic vertebrae
ie. pattern the somites that will form the anterior thoracic vertebrae

81
Q

What are the most posteriorly expressed Hox genes that pattern the thoracic vertebrae?

A

The Hox9 paralogs!

82
Q

Where is the anterior expression limit of the Hox10 paralogs?

A

The most anterior part of the lumbar region (the somites that form the lumbar vertebrae)

83
Q

What is the anterior expression domain of the Hox11 paralogs?

A

The somites that go on to form the sacral vertebrae

84
Q

What is the function of the most posterior Hox genes expression in a given region?

A

The Hox gene that is the most posterior (along the chromosome) expressed in a give area will have dominance in patterning. Determines which structures will form - posterior prevalence.

85
Q

Posterior prevalence

A

The most 5’ or the most posterior Hox genes expressed in a tissue usually have the dominant patterning function over the more 3’ or anterior genes expressed in that same area.

** The most anterior region where a Hox gene is expressed is usually where its patterning function is most important.

86
Q

What is the most anterior region that Hox genes are expressed?

A

At the base of the skull, hindbrain, ears, occipital bone

Hox genes cannot function where they are not expressed

87
Q

Which Hox genes pattern the base of the skull?

A

Hox1 and Hox2 paralogs

88
Q

Reverse genetics

A

Knockout a gene –> indirectly determine function by observing changes in the phenotype

89
Q

Forward genetics

A

mutant phenotype is first generated and then the gene is cloned/characterized/resultant genotype is screened later.

90
Q

What is Cas9?

A

A nuclease that is guided by an RNA to a specific region. Here, it induces a double stranded break in the DNA

91
Q

Non-homologous end joining (NHEJ)

A

The cell’s mechanism of repairs a double-stranded DNA break. When it does this, however, indels (insertions and deletions of base pairs) are introduced.
This can cause mutations and truncations (via introducing stop codons), altering the gene’s function

92
Q

Homology-directed pair

A

Another mechanism besides NHEJ to repair double stranded breaks in the DNA
The cell uses inserted homologous DNA (ie has regions of homology) that may have specific gene edits to repair the double stranded breaks
- introduced specific changes in the genome

93
Q

Tetra-amelia disease

A

Total absence of four limbs.

Associated with given mutations in the Rspo2 region (truncated protein due to introduced STOP codons)

94
Q

What is the mutation that causes sickle cell anemia?

A

Single bp change causes an amino acid to change from Glu to Val (which is hydrophobic) in the β hemoglobin gene - causes blood cells to sickle. This causes clumping and blockage of RBC and destroys them as well.

95
Q

What Hb do normal adults have in their blood?

A

Tetramers with 2 alpha and 2 beta chains

96
Q

What causes repression of Hb genes?

A

Changes in DNA methylation are what changes the particular Hb genes being expressed. Methylation of the promoter represses gene expression.

Proteins will bind to the methylated promoter and further repress gene expression

97
Q

BCL11A

A

A TF that represses the expression of the gamma globin gene. This helps transition the embryo into producing B globin (in adult Hb)

98
Q

Which subunits make up fetal Hb tetramers?

A

2 alpha and 2 gamma

99
Q

How has CRISPR been used to fix sickle-cell anemia patients?

A

You don’t target the globin itself but instead target an enhancer for the TF that normally represses the expression of non-mutated globin gene.

-Design a guide RNA that targets the enhancer for the BCL11A gene. Introducing a double strand break here with the nuclease, allowing the cell to ‘repair’ the break with indels- at the GATA binding site - will make the GATA protein unable to bind the enhancer to activate the expression of BCL11A.

No enhancer activated - no gene expression - don’t get the BCL11A transcription factor repressing the expression of gamma globin - so gamma globin can continue to be expressed.

100
Q

What day is the neural tube closed in human development?

A

Day 28

101
Q

How long is embryonic development in humans?

A

8 weeks. After this, fetal period begins.

Fetus is 3 cm long at 56 days.

102
Q

Anencaphaly

A

Neural Tube closure defect
Caused by a failure of the anterior neural pore to close
Exposed the spinal cord to amniotic fluid during development. This is fatal!

103
Q

craniorachischisis

A

neural tube close defect caused by the failure of the entire neural tube to close (FATAL)

104
Q

spina bifida

A

neural tube closure defect where the posterior neuropore of the neural tube fails to close (in the sacral region)

105
Q

What are some of the derivatives of the surface ectoderm and what induces this to form?

A 
High BMPs (low BMP inhibitors)
Lens, cornea, nails, hair, bulk=epidermis
106
Q

What are some derivatives of the neural crest cells and what levels of BMP are present where these are specified?

A

Moderate BMP levels
These cells come from cells between the surface ectoderm and the neural plate
Derivatives: majority of the peripheral nervous system, adrenal medulla, melanocytes

107
Q

What are some derivatives of the neural plate/neural tube and what levels of BMP are present where these are specified?

A

LOW BMPS present here because cells of the underlying mesoderm (prechordal plate, notochord) an pharyngeal endoderm release lots of BMP inhibitors
Derivatives: part of the pituitary, brain, spinal cord, motor neurons, retina

108
Q

primary neurulation

A

When the neural tube is formed by invagination of an epithelial layer and pinching off and internalizing (this happens in the anterior and dorsal part of the middle during the formation of the neural tube)

109
Q

secondary neurulation

A

When the neural tube is formed by coalescence of mesenchymal cells to form a rod followed by cavitation to form a tube.
This only occurs at the posterior end of the animal and the ventral side of the middle.

The inside of the tube is then cleared out to form a hollow rod.

110
Q

rostral

A

anterior (head region)

111
Q

What are the effects of dorsal on Noggin and dpp and twisted?

A

Dorsal represses Dpp ventrally
Dorsal activates Sog at intermediate levels of Dorsal
Dorsal activates Twisted at high levels of Dorsal

CMMB 403 (7 decks)

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