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The Cell in Time and Space > TS4: Model Organisms > Flashcards

TS4: Model Organisms Flashcards

1
Q

What are the 7 ‘stages’ of development?

A

Embryogenesis (pattern formation)
Morphogenesis (development of form)
Differentiation (cell specificity)
Organogenesis (cell/tissue organisation)
Sex determination
[Regeneration and metamorphosis]
Ageing and senescence

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2
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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3
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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4
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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5
Q

What is epistasis?

A

The examination of double mutants to determine control hierarchy.

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6
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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7
Q

What is epigenesis?

A

the process by which plants, animals and fungi develop from a seed, spore or egg through a sequence of steps in which cells differentiate and organs form, where an organism’s development is influenced by both genetics and external factors that modify the DNA.

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8
Q

What is Waddington’s landscape?

A

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.

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9
Q

What is asymmetric cell division and why is it important in development?

At which point is this essential in C. elegans development?

A

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.

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10
Q

Why are epithelial sheets important in development?

A

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.

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11
Q

What are induction events in development?

A

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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12
Q

What are developmental fields and boundaries?

A

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

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13
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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14
Q

Why are stochastic choices important in development?

A

They break symmetry e.g., the point of sperm entry defines the polarization of the egg.

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15
Q

What cells are involved in C. elegans vulval specification?

A

7 cells: 1 inducing cell (gonad anchor cell) and 6 responding cells (vulval equivalence group - P3p-P8p)

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16
Q

Describe the process of vulval specification in C. elegans, starting from VPC differentiation.

A
  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.
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17
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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18
Q

How was the vulval induction signaling pathway discovered?

A

The analysis of vulvaless and multivulva mutants and subsequent epistasis analysis.

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19
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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20
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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21
Q

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

A

The maternal-effect mRNA Gurken is localized at the anterior-dorsal corner of the developing egg. This activates a receptor, Torso, that results in the expression of Dorsal.

Oskar mRNA is localized at the posterior pole of the embryo to promote localization of germ cell determinants and nanos mRNA.

Nanos mRNA is localized at the posterior pole, whereas Bicoid is at the anterior pole, defining the boundaries between these regions in the embryo. Bicoid actives Hunchback, whilst Nanos inhibits this protein, which is crucial for patterning.

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22
Q

What property of the Drosophila early embryo allows for easy diffusion of signaling molecules for patterning?

A

There are no cells formed meaning the egg is essentially a large cell containing a common cytoplasm with dividing nuclei.

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23
Q

Describe the Heidelberg screen.

A

A screen to identify genes involved in embryonic development of Drosophila.

Large populations of flies were mutagenized using EMS to induce random mutations into the DNA. These mutants were screened for abnormalities in development.

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24
Q

What is the role of 3’UTR in Drosophila A-P patterning?

A

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.

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25
Q

What are gap genes?

A

Zygotic genes coding for transcription factors expressed in early Drosophila development that subdivide the embryo into regions along the AP axis.

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26
Q

What are pair-rule genes?

A

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.

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27
Q

Describe the process of dorsal-ventral patterning in the Drosophila embryo.

A

The localization of Gurken at the anterior-dorsal corner of the embryo triggers expression of Dorsal. Initially, Dorsal expression is ubiquitous throughout the embryo.

Toll is a TM receptor activated by Spatzle (external signal). Spatzle is only located on the ventral side of the embryo, so only ventral Toll is activated.

Once activated, Toll activates the degradation of Dorsal inhibitor, Cactus, allowing Dorsal to translocate to the nucleus and activate expression of DV patterning genes.

Hence, Dorsal is only active in the ventral side of the embryo.

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28
Q

What are the Antennapedia and Bithorax complexes?

A

ANT: responsible for segmenting the head and anterior thorax

BX: responsible for segmenting the posterior thorax and abdomen

They are examples of Hox proteins.

29
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.

30
Q

What do homeotic genes encode? Describe the homeodomains found in homeotic genes.

A

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.

31
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.

32
Q

How are Hox expression patterns maintained at the level of chromatin?

A

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.

33
Q

What is the co-factor hypothesis of homeodomain specificity?

A

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.

34
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.

35
Q

What do we mean when we say ‘ground state’ of Hox gene expression?

A

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.

36
Q

What can cause changes in Hox gene expression?

A
  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
37
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
38
Q

Describe the pipeline approach used to identify hundreds of zebrafish mutant genes.

A

A genetic screen was set up, and the mutants were divided into groups depending on the mutation phenotype.

The groups were then assigned to different experts who specialized in each of the phenotypes.

To identify the genes, phenotype and mutant generation assays were used:
- phenotype: gross morphology, anatomy, behaviour, transgenic reporter
- mutant: ENU-based approaches, CRISPR, chemical/small molecules

39
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.

40
Q

What is so special about the zebrafish ‘caspar’ mutant?

A

It’s been made transparent to improve phenotypic readout.

41
Q

What is the ENU F2 screen?

A

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.

42
Q

What is the ENU F3 screen?

A

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.

43
Q

What are the advantages and disadvantages of both F2 and F3 ENU screens?

A

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

44
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.

45
Q

What is the embryonic neural crest? How can this be studied?

A

Vertebrate-specific tissue that produces neural crest cells. These go on to produce many other types of cells, such as melanocytes.

As the neural crest is vertebrate-specific, zebrafish are key to studying this system.

46
Q

Describe the steps in neural crest development.

A
  1. Gastrulation
    Interactions between neural plate and non-neural ectoderm forms the neural plate border.
  2. Induction
    Formation of the NPB is essential for cells that could become neural crest.
  3. Translocation
    Cells undergo an EMT to move from the neural tube to their target location throughout the body.
  4. Differentiation
    Neural crest cells differentiate e.g., sensory neurons, glia, pigment cells, etc.
47
Q

What is neurulation?

A

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.

48
Q

Give examples of genes that determine neural crest induction.

A

BMP (depends on Wnt signaling)
Retinoic acid
Notch
Endothelins

49
Q

What is the gene regulatory network in neural crest formation, and what is one of the main genes has been identified in this?

A

Multiple overlapping transcription factors that definitively specify the neural crest cell population.

Sox10 - a large number of upstream regulators converge on Sox10, and this gene largely controls EMT capability which is integral to neural crest identity.

50
Q

What is the cause of melanoma?

A

Overexpression of Sox10, a gene that controls melanocyte and melanin pigment formation.

51
Q

What are the 3 tissues in the germ layer of early mammalian embryos?

A

Ectoderm, mesoderm, endoderm

52
Q

What are master transcription factors?

A

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.

53
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.
54
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.

55
Q

Which transcription factors are associated with trophectoderm cells vs primitive ectoderm cells?

A

T: Cdx2
PE: Nanog and Oct4

56
Q

What are teratomas?

A

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

57
Q

How are embryonic stem cells maintained in culture?

A

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.

58
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.

59
Q

What is myostatin?

A

An inhibitor of muscle differentiation.

Mutations that block myostatin cause excessive muscle production, often seen in bulls.

60
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
61
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.

62
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.
63
Q

What are primordial germ cells?

A

Embryonic precursor cells that give rise to gametes.

64
Q

How can we reprogramme somatic cells?

A

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.

65
Q

What are heterokaryons?

A

Cells that contain two or more nuclei from different cells.

66
Q

How are fish able to regenerate some heart tissue?

A

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.

67
Q

How can iPSCs be used in different therapies and research?

A
  • 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.
68
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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