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TS4 - Development Flashcards

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1
Q

How can we analyze development? Give examples for each analysis type.

A
  1. Description (molecular and cellular)
    e.g., anatomy and fate mapping, RNA/protein profiling
  2. Physical manipulation
    e.g., cell isolation or grafting
  3. Genetic/molecular experimentation
    e.g., forward genetics and reverse genetics, conditional mutants, epistasis
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2
Q

What is the basic strategy of forward genetics?

A
  1. Define a developmental process in a suitable animal model
  2. Mutagenize a population of animals
  3. Screen for mutants
  4. Characterize mutant phenotype
  5. Identify mutated gene causing phenotype
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3
Q

What are the roles of the following proteins in Drosophila A-P formation:
- Gurken
- Oskar
- Nanos
- Bicoid
- Hunchback

A

Gurken: A maternal mRNA localised at the posterior intially. Signalling results in Bicoid mRNA localising to the anterior; Oskar mRNA to the posterior.

Oskar: Localized at the posterior pole; essential for organizing the posterior pole plasm and recruiting nanos mRNA, thereby promoting germ cell formation.

Nanos: A posterior determinant. Localized nanos protein inhibits Hunchback translation in the posterior, helping establish posterior cell fates.

Bicoid: A maternal mRNA localized at the anterior pole. It forms a gradient and activates Hunchback transcription in the anterior, contributing to anterior patterning.

Hunchback: A transcription factor whose expression is activated by Bicoid in the anterior and repressed by Nanos in the posterior. It helps define anterior structures and set up the embryonic body plan.

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4
Q

What are teratomas?

A

Mutated embryos that comprise all the cell types represented by the 3 germ layers, but with no positional information.

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5
Q

What are cytonemes and exosomes?

A

Cytonemes are thin, actin-based protrusions that extend from the surface of cells and allow for long-range signaling between cells. e.g., Notch signaling in nematodes

Exosomes are small, membrane-bound vesicles that are released by cells and contain a variety of signaling molecules for intercellular communication.

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6
Q

What are the two modes of division for cell fate choice in adult stem cells?

A
  1. Asymmetric - cell fate is determined by the asymmetry of the dividing stem cells
  2. Independent - determined randomly and/or by environment
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7
Q

Why are zebrafish used as a model organism?

A
  • Easy to raise and breed all year round
  • Short generation time
  • Large brood size
  • Rapid EXTERNAL development
  • Early embryo is transparent
  • Similar body plan to other vertebrates
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8
Q

Why is lateral inhibition important in C. elegans vulva induction, and what does it involve?

A

Prevents adjacent VPCs from all adopting the primary fate.

Primary fate of the P6p inhibits LIN-12 receptor expression, but upregulates the lin-12 ligand expression: DSL. DSL can then bind LIN-12 on the adjacent cells, inducing secondary fate allocation and inhibiting primary fate signals from LET-23.

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9
Q

What is the haematopoietic system?

A

The hematopoietic system is the organ system responsible for the production and development of blood cells, composed of haematopoietic stem cells. Whilst this occurs at different sites during development, eventually it concentrates to the bone marrow.

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10
Q

What are the two Hox complexes in Drosophila, and what do they control?

A

ANT-C (Antennapedia complex): Head/thorax segments. BX-C (Bithorax complex): Thorax/abdomen segments. Mutants: bithorax (T3 → T2, four wings), Antennapedia (antenna → legs).

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11
Q

What is the basic strategy behind reverse genetics?

A
  1. Choose a candidate gene.
  2. Create mutations in the chosen gene.
  3. Examine resulting phenotype.
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12
Q

What creates the stripe pattern on zebrafish?

A

The zebrafish stripe pattern is composed of melanophores (black pigment cells) and xanthophores (yellow pigment cells) that are distributed in a regular and reproducible pattern along the body axis.

These can either attract or repel one another to form the stripes.

Macrophages are also involved in patterning these pigments into stripes.

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13
Q

What is homeosis (or homeotic transformation)?

A

The development of one body part with the phenotype of another.

E.g., bithorax loss-of-function mutations that cause Drosophila to have an additional pair of wings, or the Antennapedia gain-of-function mutations that transform antenna into legs.

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14
Q

What are the roles of the following proteins:
- lin39
- lin-3
- LET-23
- LET-60
- lin-1

A

lin39: prevents cells from fusing with hypodermis to form VPCs
lin3: signal from anchor cell to VPCs
LET-23: EGF receptor for lin3
LET-60: Ras protein activated by LET-23
lin-1: TF upregulated by LET-23 signaling

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15
Q

How do muscle cells differentiate?

A

Muscle cells are derived from myoblasts, which come from the dermomyotome in embryos.

Differentiation is driven by the master regulator MyoD which binds to promoters/enhancers to activate muscle-specific genes.

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16
Q

What is the co-linearity principle?

A

Describes the linear relationship between the linear sequence of genes on a chromosome and the spatial arrangement of the corresponding body structures during development.

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17
Q

How are transgenic embryos and adult zebrafish made? Why are these useful?

A

Embryos are injected with DNA (e.g., photo-convertable fluorophores) using transposons or viral vectors.
- monitor developmental processes

Adults use Tol2, a DNA transposon that uses a ‘cut and paste’ mechanism to insert randomly in the genome. TEAZ (electroporation) can also be used to get transgenes into adults.

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18
Q

What is the DNA sequence motif hypothesis of Hox proteins?

A

Different combinations of DNA modules can give different combinations of co-factors bound on the promoter of a gene and thus a different array of transcriptional interactions with each Hox protein.

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19
Q

What is the inside-out hypothesis of embryo development?

How does this potentially define epithelial polarity?

A

Cells of the early embryo sense their position as being outside or inside and use this as a cue to specify trophectoderm vs primitive ectoderm fate, respectively.

Epithelial cells are therefore either classed as ‘inside’ or ‘outside’, depending on whether they’re polar or not. Cell divisions produce either 2 polar or 1 polar and 1 apolar cell depending on the randomly determined plane of division.

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20
Q

What is a genetic mosaic? Why is it useful in experiments?

A

A genetic mosaic individual consists of cells of distinct genotypes, for example harboring homozygous mutant cells next to wild-type cells.

The controlled induction of genetic mosaicism in experimental animals allows to alter gene function at high spatiotemporal resolution.

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21
Q

What is the trophectoderm?

What are the 3 master transcription factors that control trophectoderm differentiation and what do they do?

A

A layer of cells that forms on the outside of the blastocyte, giving rise to the placenta and other extraembryonic tissues.

  1. Cdx2: homeodomain TF that is progressively restricted to outer cells.
  2. Oct4: TF that is progressively lost from the outer cells.
  3. Nanog: homeodomain TF that is progressively restricted to inside cells.
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22
Q

What 3 methods can generate different cells from a differentiated cell?

A
  1. Trans-differentiation:
    Production of one mature somatic cell type from a different mature somatic cell type, normally via a dedifferentiated progenitor. e.g., muscle to neuron.
  2. De-differentiation:
    Replenishment of a quiescent differentiated cell type via de-differentiation to a proliferating precursor.
  3. Reprogramming:
    Conversion of a somatic cell type into a multi-lineage embryonic progenitor that can give rise to other cell types.
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23
Q

What are the functions of SynMuv genes?

A

Act in the surrounding hypodermis to help ensure the Ras pathway doesn’t become hyperactive by targeting lin-3 for repression.

[Lin-3 is the signal released by anchor cells to activate VPC fate allocation]

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24
Q

What are the technical challenges of using iPSC cells in therapies?

A
  1. Tumorigenicity
    Stem cells present a high risk of forming tumors.
  2. Immunogenicity
    Donor iPSCs can be rejected, so need of stem-cell banks or MHC-engineered iPSC.
  3. Heterogeneity
    iPSC show heterogeneity due to genetic differences in donor or acquirement of differences during expansion in vitro.
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25
What is the role of 3'UTR in Drosophila A-P patterning?
The 3' UTR is what defines the localization of the proteins. If the Bicoid 3' UTR is added to Nanos, the transgenic protein will be directed to the anterior, but inhibit translation of anterior proteins due to the Nanos portion of the protein.
26
What can cause changes in Hox gene expression?
1. Duplication of genes or whole gene clusters by unequal crossing over. 2. Diversification of coding and regulatory sequences after duplication. 3. Changes in coding sequences (rare due to selective pressures) 4. Changes in expression of Hox targets
27
How are embryonic stem cells maintained in culture?
Leukemia inhibitory factor - maintains expression of key TFs such as Oct4 and Nanog Fibroblast GF - promotes ESC self-renewal and prevents differentiation ECM to provide supportive microenvironment Small molecules that mimic the activity of signaling pathways, such as Wnt and BMP.
28
How was the vulval induction signaling pathway discovered?
The analysis of vulvaless and multivulva mutants and subsequent epistasis analysis.
29
What do we mean when we say 'ground state' of Hox gene expression?
Hox genes are conserved across species, and it's clear that throughout evolution the different patterns of Hox gene expression has allowed for the different body plans that give rise to all species. The 'ground state' is therefore the state where all Hox genes are expressed in all segments, theoretically at the time when there was a single body plan.
30
Which transcription factors are associated with trophectoderm cells vs primitive ectoderm cells?
T: Cdx2 PE: Nanog and Oct4
31
What are the 7 'stages' of development?
Embryogenesis (pattern formation) Morphogenesis (development of form) Differentiation (cell specificity) Organogenesis (cell/tissue organisation) Sex determination [Regeneration and metamorphosis] Ageing and senescence
32
Describe the process of vulval specification in C. elegans, starting from VPC differentiation.
1. lin-39 is expressed to prevent the 6 VPCs (P3p-P8p) from fusing with the hypodermis. 2. The anchor cell releases lin-3 in a graded manner to the VPCs. 3. lin-3 binds LET-23 at the highest concentration to P6p which adopts the primary fate through Notch pathways and let60 signaling. 4. P6p then laterally signals to P5p and P7p to inhibit LET-23 signaling in them, and hence a adopt secondary fates.
33
What are gap genes?
Zygotic genes coding for transcription factors expressed in early Drosophila development that subdivide the embryo into regions along the AP axis.
34
Why are stochastic choices important in development?
They break symmetry e.g., the point of sperm entry defines the polarization of the egg.
35
How are fish able to regenerate some heart tissue?
Cardiomyocytes are able to de-differentiate into proliferating precursor populations. This is also seen in amphibian limb regeneration and Schwann cell replacement after nerve damage.
36
What is epigenesis?
Epigenesis is the biological theory that an organism develops from an undifferentiated fertilized egg through a series of gradual, step-by-step processes, with new structures and functions forming over time
37
How is the anterior-posterior (AP) axis established in Drosophila?
Gurken mRNA localizes posteriorly → signals to follicle cells via Torpedo receptor. Follicle cells signal back, reorganizing microtubules to localize bicoid (anterior) and oskar (posterior) mRNAs. Bicoid protein gradient forms at the anterior, Nanos at the posterior.
38
What are pair-rule genes?
Any of a number of genes in Drosophila that are involved in delimiting parasegments. They're expressed in transverse strips in the blastoderm, each pair-rule gene being expressed in alternative parasegments, resulting in 7 transverse stripes.
39
What is the order of gene action in Drosophila segmentation?
Maternal genes (bicoid, nanos) → gradients. Gap genes (hunchback, Krüppel) → broad domains. Pair-rule genes (even-skipped, fushi tarazu) → 7 stripes. Segment polarity genes (engrailed, wingless) → 14 stripes.
40
What is the ENU F3 screen?
A large-scale screen that looks at the F3 progeny. A mutagenized male and WT female are crossed. The F1 progeny are outcrossed to generate a family at the F2 generation. Pairs of F2 animals are mated and if the family contains a mutation then about 1 mating out of 4 will be between 2 +/- individuals, and such matings will yield 25% -/- progeny, which should show an abnormal phenotype.
41
What is so special about the zebrafish 'caspar' mutant?
It's been made transparent to improve phenotypic readout.
42
What are heterokaryons?
Cells that contain two or more nuclei from different cells.
43
What is the ENU F2 gynogenetic screen?
Heterozygous females are treated with UV-inactivated sperm. The pseudo-fertilized eggs are heat-shocked to cause the haploid genome to become diploid. This allows for studying homozygous recessive mutants.
44
What is myostatin?
An inhibitor of muscle differentiation. Mutations that block myostatin cause excessive muscle production, often seen in bulls.
45
What is Waddington's landscape?
A metaphorical representation of the developmental pathway of a cell or organism, used to explain how cells differentiate into specific cell types during embryonic development.
46
How are Hox expression patterns maintained at the level of chromatin?
Polycomb and Trithorax complexes. Genes not being expressed will be bound by Polycomb proteins PRC1 and PRC2 which will deposit H3K27me3 and recruit more Polycomb complexes. Trithorax proteins counteract Polycomb by adding H3K4me3 to open up the chromatin through binding of other chromatin remodelers, thus upregulating Hox gene expression.
47
How do Hox proteins achieve precise DNA binding despite low in vitro specificity?
Co-factors: Extradenticle (Exd), Homothorax (Hth). DNA motif combinations: Unique promoter interactions.
48
What property of the Drosophila early embryo allows for easy diffusion of signaling molecules for patterning?
There are no cells formed meaning the egg is essentially a large cell containing a common cytoplasm with dividing nuclei.
49
What is epistasis?
The examination of double mutants to determine control hierarchy.
50
What are developmental fields and boundaries?
Fields: regions of embryos that are capable of producing a specific set of tissues/organs Boundary: the regions separating two fields that plays an important role in further patterning
51
How can iPSCs be used in different therapies and research?
- Generate dopaminergic neurons to study Parkinson's disease, etc. - Organoids for toxicology/drug screening - Gene therapy to replace damaged cells, such as in AMD and following cardiac arrest.
52
What is the co-factor hypothesis of homeodomain specificity?
In vitro, homeodomains have very broad binding specificity, yet in vivo it's highly refined. There's a possibility this refinement is caused by specific co-factors, such as Exd.
53
What are the 3 tissues in the germ layer of early mammalian embryos?
Ectoderm, mesoderm, endoderm
54
What is the colinearity principle in Hox genes?
Gene order on chromosome matches anterior-posterior expression (e.g., labial → Abd-B).
55
What cells are involved in C. elegans vulval specification?
7 cells: 1 inducing cell (gonad anchor cell) and 6 responding cells (vulval equivalence group - P3p-P8p)
56
What is the cause of melanoma?
Overexpression of Sox10, a gene that controls melanocyte and melanin pigment formation.
57
How can we reprogramme somatic cells?
Introduce Yamanaka factors to generate iPSCs. This involves maintaining a pluripotency signal, providing an opportunity for Yamanaka factors to bind, activating a TF feedback circuit, and erasing stabilizing epigenetic chromatin states.
58
How is even-skipped stripe 2 formed?
Activators (Bicoid, Hunchback) and repressors (Giant, Krüppel) bind the eve stripe 2 enhancer. Sharp boundaries created by mutual repression (e.g., eve vs. ftz).
59
What is neurulation?
The process by which the neural plate, a flat sheet or cells, transforms into the neural tube which gives rise to the brain and spinal cord.
60
What are the advantages and disadvantages of both F2 and F3 ENU screens?
F2: + homozygous recessive mutants + less space required than F3 - not all loci are homozygous F3: + homozygous recessive mutants + all chromosomal loci are tested - requires large amounts of space - labor intensive - mainly only useful for recessive alleles
61
What is asymmetric cell division and why is it important in development? At which point is this essential in C. elegans development?
The process where a single cell divides to produce two daughter cells with distinct identities and fates. This is essential for development as it allows the generation of specialized cell types with specific functions. e.g., when a nematode zygote divides unequally to give the large anterior and small posterior cells.
62
What do homeotic genes encode? Describe the homeodomains found in homeotic genes.
The homeotic genes encode transcription factors called homeodomain proteins. The homeodomain is a 60aa protein domain that binds DNA in specific combinations to cause unique expression patterns in each segment in development.
63
What are primordial germ cells?
Embryonic precursor cells that give rise to gametes.
64
How do changes in Hox expression explain arthropod diversity?
Ubx expression shifts correlate with limb type (e.g., crustacean maxillipeds). Duplication and diversification of Hox clusters (e.g., insects vs. mammals).
65
What are master transcription factors?
TFs that bind to the promoters of hundreds of target genes to establish regulatory circuits. Cells will typically have more than on master TF for combinatorial responses.
66
How does the Bicoid gradient pattern the embryo?
Bicoid concentration thresholds activate genes (e.g., high → hunchback, medium → Krüppel). Syncytial blastoderm allows diffusion of Bicoid to nuclei.
67
What are the Antennapedia and Bithorax complexes?
ANT: responsible for segmenting the head and anterior thorax BX: responsible for segmenting the posterior thorax and abdomen They are examples of Hox proteins.
68
Why are epithelial sheets important in development?
They're the basis of many tissues and can serve as physical barriers to protect internal tissues. Cell divisions create these sheets and allow for 3D order to be created by folding them.
69
What are induction events in development?
When cells are brought close together, they can signal to one another and induce fate and behaviour of one another e.g., nematode vulval induction by the gonad anchor cell.

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