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BIOL 3542 > Midterm 1 > Flashcards

Midterm 1 Flashcards

1
Q

What does differential gene expression control?

A 
Is to control development:
		○ Transcription
		○ Nuclear processing
		○ Cytoplasmic transport 
		○ mRNA Translation
		○ Protein modification
		○ Protein stability
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2
Q

What activates or represses transcription?

A

Control regions including enhancers, promoters, which are acted upon by transcription factors activate or repress transcription

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3
Q

A gene can have many different control regions that function to control expression in _______ (_________) and _______ (__________).

A
  • space (spatial)

- time (temporal)

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4
Q

Gene can respond to different combinations of _______ so expressed at specific _______ or ________/________.

A
  • inputs
  • times
  • cells/tissues
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5
Q

In the embryo, genes are responding to what kind of signals?

A

In embryo, they are responding to developmental signals

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6
Q

What comprises of the full complement that determines a cell’s property, behaviour, and state?

A

Transcription factors, signalling proteins, cytoskeleton proteins, cell cycle proteins and enzymes

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7
Q

Expression patterns

A
  • Are dynamic

- Past and current expression patterns define a cell’s fate

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8
Q

Tissue-specific proteins

A

Proteins that make cells different from one another

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9
Q

Housekeeping genes

A
  • These are common to most cells

- Required to keep cells alive

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10
Q

Eukaryotic mRNA:
A) synthesized by?
B) Enzyme requires what additional items to initiate transcription?
C) Once mRNA produced, what happens next?

A

A) Is synthesized by RNA Polymerase II: An mRNA molecule is produced when this enzyme initiates transcription at promoter
B) This enzyme then requires a series of additional proteins, general transcription factors, to initiate transcription
C) The mRNA then undergoes processing

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11
Q

What are some components of processing?

A
  • single stranded RNA template
  • 5’ end capping
  • splicing removes introns
  • 3’ end to polyadenylated tail (Poly-A tail, longer the tail the more stable the transcription process)
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12
Q

Promoters

*on exam

A
  • control region
  • Sequence where RNA polymerase II binds and initiates transcription
  • Recruiting required machinery
  • Close proximity to where transcription initiates (start site)
  • Has directionality
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13
Q

5’ untranslated region

A
  • The sequence intervening is called the 5’ untranslated region
  • 5’UTR=leader sequence
  • Can determine the rate of translation
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14
Q

Termination codon

A

-translation stops at the termination codon (TAA or stop codon)

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15
Q

3’ untranslated region

A
  • The mRNA sequence after the stop codon is called the 3’UTR polyadenylation sequence
  • Confers: Stability, nuclear exit, translation
  • 3’UTR contain controls for transcript stability and translational control
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16
Q

Activators

A
  • A typical eukaryotic gene has many activator proteins which together determine its rate and pattern of expression
  • These proteins help RNA polymerase and other general transcription factors to assemble at the promoter
  • attract ATP-dependent chromatin-remodeling complexes and histone acetylases
  • are the collection of proteins that can bind to enhancers
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17
Q

Enhancers

*on exam

A
  • control region
  • DNA elements that increase expression of when, where and how much
  • Small sequence that will promote the likelihood of transcription
  • Opposite of Repressors
  • Modular: can be moved around in the gene
  • Orientation independent
  • Tells you when, where and how much a gene is expressed
  • binding site for activated protein
  • critical in gene expression
  • major determinants of expression in space and time
  • A gene can have several enhancer sites
  • Each enhancer can be bound by multiple transcription factors
  • Interactions (probably direct) between enhancers and promoters
  • are the DNA sequence
  • are modular (can be moved around in the gene, they can regulate expression/transcription independent of distance and orientation to the promoter)
  • can be mixed and matched *key point to evolution where enhancers are the ones being changed, when and where things are expressed
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18
Q

Silencers

A
  • are a type of enhancer that inhibit transcription
  • “negative enhancers”
  • In Drosophila, sometimes the enhancers can become silencers, depending upon cell context and the transcription factors available
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19
Q

Transcription components

A
  • DNA-binding domain
  • Trans-activating domain (Often chromatin remodelers)
  • Protein-protein interaction domains
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20
Q

Chromatin

*on exam

A
  • DNA and its associated proteins
  • ATP-dependent Chromatin remodeling=Histones and DNA have high affinity for each other so the cell needs ATP to move them around, loosens them up
  • Histone modifications
  • Acetylation determines how tightly wound DNA will be
  • Chromatin is a Complex of DNA and associated with proteins
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21
Q

Nucleosome

A
  • Histones (H2A, H2B, H3, H4) form an octamer called nucleosome
  • DNA wraps around the nucleosome
  • 146bp of DNA in two wrap arounds
  • stabilized by histone H1
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22
Q

Histone H1

A
  • Nucleosomes are stabilized by histone H1
  • Is located on linker DNA
  • H1-dependent conformations inhibit transcription of genes
  • Prohibits access of RNA polymerase and transcription factors to control regions (like promoters and enhancers)
  • The default state of DNA is a closed configuration, tightly packaged by histone H1
  • are highly conserved
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23
Q

Gene expression is regulated by a reversible change in local chromatin structure (DNA compacting).
What is the role of ATP-driven/dependent chromatin remodeling complexes?

A
  • Protein machines that use energy, ATP-riven, to interfere with the DNA-histone interaction
  • histones and DNA have a high affinity fr each other so ATP is needed to move them around and loosen them up
  • DNA can become less tightly bound
  • alter and remodel nucleosomes
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24
Q

Gene expression is regulated by a reversible change in local chromatin structure (DNA compacting).
What are the roles of the covalent modifications of the four core histone proteins on the N-terminal tail?

A
  • The histone code, N-terminal tails are covalently modified with acetylation of lysines, methylation of lysines, and phosphorylation of serines
  • Acetyl groups inhibit the interaction between DNA and histones
  • Histone acetyl transferases (HATs) and histone deacetylases (HDACs)
  • Total sum of the modifications condense or loosen stretches of chromatin
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25
Q

What combination of inputs control final expression patterns?

A
  • Cellular signalling
  • Transcription factor level
  • Developmental history (History can determine regions of the genome that are open or closed)
  • Positive or negative feedback loops are linked to complex and interdependent transcriptional networks
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26
Q

What is transcription?

*on exam

A
  • RNA Polymerase II transcribes DNA sequence into mRNA

* Reading the genetic code to make an mRNA

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27
Q

What is translation?

*on exam

A
  • mRNA goes into the cytoplasm

* mRNA is translated into a polypeptide (string of amino acids) on a ribosome

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28
Q

Name two control regions.

*on exam

A

1) promoter

2) enhancer

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29
Q

Transcription factors

*on exam

A
  • Proteins that bind the promoter or enhancer (control elements) to control and modify the levels of gene expression (transcription)
  • Usually recruiting activators
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30
Q

A transducing signal can be transmitted from one cell to another in which three main ways?

A

1) secreted diffusible molecule
2) surface molecule receptor
3) gap junction

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31
Q

Summarize the steps involved in the production of B-globin and hemoglobin.

A

1) Transcription
2) Processing
3) Translation
4) Hemoglobin produced

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32
Q

What are four ways an animal cell’s dependence on multiple extracellular signals can affect it?

A

1) survive
2) divide
3) differentiate
4) die

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33
Q

Name two types of intracelluar signaling proteins that act as molecular switches/signal integration.

A

1) signalling by phosphorylation

2) signalling by GTP-bindign protein

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34
Q

Cleavage (cell division)

*on exam

A
  • rapid cell divisions immediately following fertilization
  • no cell growth
  • After fertilization, a series of mitotic divisions occurs
  • Cell division occurs with cell growth in order to increase the number of cells in early embryo
  • Egg cytoplasm is divided into numerous cells and this is accomplished by abolishing the growth period (G1 an G2 phases of the cell cycle)
  • There is nuclear division (replication of DNA and mitosis)
  • The cyclins and their respective kinase (cyclin dependent kinases) are responsible for progression through the cell cycle
  • Is the result of two coordinated processes: Karrokinesis and Cytokinesis
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35
Q

Blastomeres (nucleated cells)

*on exam

A
  • the cells that result from the cleavage

- Volume of egg cytoplasm is divided into numerous cells (nucleated cells) and cells are called blastomeres

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36
Q

An egg cytoplasm division is accomplished how?

A

by abolishing the growth period, G1 and G2 phases of the cell cycle

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37
Q

How many divisions does a Xenopus larvis (frog) embryo undergo? The onset of gene expression is called what?

A
  • Undergoes a series of 12 divisions to create many cells before gene expression is initiated
  • The onset of gene expression is called the mid-blastula transition
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38
Q

What components are responsible for the progression through the cell cycle?

A

The cyclins and their respective kinase (cyclin dependent kinases) are responsible for progression through the cell cycle

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39
Q

Cleavage or cell division is the result of which two coordinated processes?

A

1) karrokinesis

2) cytokinesis

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40
Q

Karyokinesis

*on exam

A
  • division of the DNA (nucleus)
  • use of microtubules
  • mitotic division of the nucleus
  • the mitotic spindle with its microtubules are the driving force
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41
Q

How are microtubules created?

A

Microtubules are created through the polymerization of tubulin (protein)

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42
Q

Cytokinesis

*on exam

A
  • physical separation of the cytoplasm
  • using the actin cytoskeleton by microfilaments
  • physical separation of the cell
  • the contractile ring is the driving force
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43
Q

What is the contractile ring composed of?

A

The contractile ring is composed of microfilaments that result from the polymerization of actin

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44
Q

What is the importance of microtubules and microfilaments?

A

mircotubules and microfilaments are important for communication, transport and shape change of the cell

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45
Q

Cell-cell interactions

A
  • Allow for cells to communicate and respond to each other
  • In animals, there are hundreds of kinds or types of signal molecules
  • The target cell responds by means of a specific protein called a receptor, which can bind the signal molecule (also called a ligand) in the target cell
  • Signals can be close range or across a field of many cells
  • Or they can act via local mediators which help to transmit the signal
  • Another form of signalling is through direct cell contact
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46
Q

What are types of signal molecules?

A

-small peptides, steroids, gases, ions

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47
Q

Receptor

A

-binds the signal molecule (ligand) to the target cell

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48
Q

Local mediators

A

-help transmit the signal

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49
Q

Direct cell contact

A
  • another form of signalling
  • two cell surface molecules interact
  • An alternative form of direct cell communication is through gap junction
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50
Q

Gap junction

A
  • form of direct cell communication
  • These are specialized cell-cell junctions between plasma membranes
  • Directly connect the cytoplasms of the two cells
  • These narrow water-filled channels that allow exchange of small molecules (Ca2+, cyclic AMP)
  • But larger molecules such as proteins and nucleic acids cannot be exchanged
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51
Q

Cellular responses depends on what?

A

Depend on the state of the receiving cell such that the same signals can be used repeatedly throughout development and can elicit different responses depending upon the competence of the cell

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52
Q

Induction

A
  • signals from one group of cells (sometimes one or more cells) can influence the development of another
  • Often involves signals transmitted to its immediate neighbours
  • due to presence of component of a cell signalling pathway
  • Chromatin conformations can also influence the ability of a cell to respond to a signal
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53
Q

Competence (Potency)

A
  • the state of being able to respond to an inductive signal
  • What a cell has the potential to become
  • Is changing throughout development
  • Becomes more restricted as a cell makes decisions towards certain cell fates
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54
Q

Permissive signalling

A
  • Induction occurs when a cell makes only one kind of response and only makes this response when a given signal is produced
  • Binary choice (survive or die)
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55
Q

Instructive signalling

A
  • Cells can respond differently to the level of a particular signal
  • Low level signal=survive, slightly elevated signal=cell start to divide)
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56
Q

Antagonistic signalling

A
  • Molecules can block a signal transduction event
  • Can prevent a signal from reacting to its receptor
  • There is often cross-talk between signalling pathways which allows cells to respond to multiple inputs simultaneously.
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57
Q 
Intracellular signalling (signal transduction)
*on exam
A
  • relay of a signal within a cell
  • The process through which receptors in the membrane of the responding cell, upon binding of the ligand, elicits a cascade of interacting proteins that transmit the signal
  • These pathways are called signal transduction pathways and they mediate / transmit signals to the nucleus
  • Small intracellular mediators (second messengers) pass the signal by binding or altering the behaviour of target proteins
  • Responses can include:
    1. Change in gene expression
    2. Activate a signalling cascade (protein kinases)
  • These responses can regulate molecules to control cell division, cell death and/or cell migration
  • Many intracellular proteins behave like molecular switches
  • Such as upon receipt of a signal that can change cell response to an active or inactive state
  • Many signalling proteins are controlled by phosphorylation through kinases (protein kinase) that can modify protein through phosphorylation and these phosphorylations can be amplified through phosphorylation cascades
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58
Q

Signal transduction pathway mediate / transmit signals to where in the cell?

A

the nucleus

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59
Q

Small intracellular mediators (second messengers)

A

-pass the signal by binding or altering the behaviour of target proteins

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60
Q

When a small intracellular mediator (second messenger) passes the signal by binding and altering the behaviour of the target protein, what are the two responses possible?

A
  1. Change in gene expression
  2. Activate a signalling cascade (protein kinases)
    * These responses can regulate molecules to control cell division, cell death and/or cell migration
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61
Q

Protein kinase

*on exam

A
  • phosphorylate or modify proteins with the addition of a phosphate molecule (can cause change, receive a signal and send to another part of the cell, for stability)
  • control protein signalling by phosphorylation of proteins
  • phosphorylations can be amplified through phosphorylation cascades
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62
Q

Where on the ribosome does translation occur?

A

Translation occurs on the ribosomes at the ATG translation initiation start site

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63
Q

Cyclin

*on exam

A
  • proteins that are driving the mitotic cycle (one of the classes)
  • tightly regulated, if not can cause tumours and cancers
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64
Q

Cyclin-dependent protein kinases (CDK)

*on exam

A

*the kinases associated with the function of the cyclins (another class)

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65
Q

Phosphorylation cascades

*on exam

A
  • the signal activates a series of protein kinases that can be protein kinases themselves
  • allow for a quick cell response and amplification
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66
Q

Ligand

*on exam

A
  • secreted molecule

- can be receptors

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67
Q

What are two methods of direct cell-cell contact?

*on exam

A

1) by gap junctions

2) two cells touching each other

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68
Q

GTP-binding protein

*on exam

A
  • act as a molecular switch
  • turns protein in an on/off position
  • exchange of GDP and GTP result in change in active state
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69
Q

Transient extracellular signals

*on exam

A
  • can induce much more long-term responses in the cell

* for instance changes in gene expression can change the activity and response of cells in later development

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70
Q

Ubiquitin

*on exam

A
  • exists in cells either free or covalently linked to a protein
  • they “tag” the protein for destruction by the proteosome
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71
Q

Proteolytic pathways

*on exam

A

*confer short half lives on specific proteins whose concentrations change the state of the cell

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72
Q

How is whole cell movement (Crawl) achieved?

A
  • Shaping and restructuring of cells require the coordinate activities of the cytoskeleton
  • Influences organization and adhesion
  • For a cell to crawl it must maintain structural polarity through the microtubules (regulated through tubulin) and microfilaments (actin)
  • Protrusions at leading edge (via assembly of actin filaments)
  • Adhesion of the protrusion to a substratum (ie. The extracellular matrix), can involve focal adhesions
  • Traction via molecular motors (myosin) brings the cell body forward
  • Disassembly on the backend releases the cell contacts
  • Nucleated actin filaments are attached to preexisting filaments through the ARD (actin related proteins) complex
  • Filaments elongate which allow an anchored array to push the edges of the plasma membrane
  • Filaments become susceptible to depolymerization by cofilin
  • Resulting in a separation between net filament assembly at the front and rear of a cell
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73
Q

Microtubules and microfilaments are associated with which subunits: actin and tubulin?

A

Microtubules=tubulin

Microfilaments=actin

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74
Q

When a cell crawls, polarity is maintained through which structures?

A

Through the microtubules (regulated through tubulin) and microfilaments (actin)

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75
Q

When a cell crawls, _______ are at the leading edge (via assembly of _______ filaments.

A

Protrusions

Actin

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76
Q

When a cell moves, focal adhesions can occur how?

A

Adhesion of the protrusion to a substratum (ie. The extracellular matrix), can involve focal adhesions

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77
Q

What is the role of myosin in the movement of a cell?

A

Traction via molecular motors (myosin) brings the cell body forward

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78
Q

Actin related proteins complex (ARD)

A

Nucleated actin filaments are attached to preexisting filaments through the ARD (actin related proteins) complex

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79
Q

When a cell moves, how are the edges of the plasma membrane moved along as well?

A

Filaments elongate which allow an anchored array to push the edges of the plasma membrane

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80
Q

What is the role of cofilin in cell movement?

A
  • Filaments become susceptible to depolymerization by cofilin
  • Resulting in a separation between net filament assembly at the front and rear of a cell
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81
Q

Microtubules

A
  • polymers of tubulin subunits (+) and (-) ends
  • essential structure of cytoskeleton
  • Emanate from centrosome
  • Vesicles with plus end directed kinesin move outward
  • Minus end directed dynein move inward
  • In neurons, microtubule organization is complex
  • In axon, the microtubules are all in the same direction
  • Moving molecules toward the axon terminus
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82
Q

Microfilaments

A

-polymer of actin filaments subunits
-part of fibrous network under cell membrane
A) filopodia
B)lamelopodia
C) focal adhesions
-contraction (actinomyosin)
-essential structure of cytoskeleton

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83
Q

Fibroblast

A
  • derived from cells
  • migratory cells associated with dermis (connective tissues), part of dermis
  • easy to culture that’s why they’re most studied
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84
Q

In which direction do the plus and minus ends move for microtubules relative to the centrosome?

A
  • Vesicles with plus end directed kinesin move outward (away from centrosome)
  • Minus end directed dynein move inward toward centrosome
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85
Q

What is the directionality of microtubules in neurons?

A
  • In neurons, microtubule organization is complex
  • In axon, the microtubules are all in the same direction
  • Moving molecules toward the axon terminus
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86
Q

Cadherins

A
  • Boundaries between tissues can be created
    1. Cells have different types of adhesion molecules
    2. Different amounts of each molecule
  • Calcium-dependent adhesions
  • Through self-interaction are important in maintaining cell adhesion and intercellular connections
  • They link adhesion to the cytoskeleton via catenins
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87
Q

Epithelium

A
  • A layer of cells that are arranged in a cohesive sheet
  • Often to create a barrier between two environments
  • Ex) the cells that line our digestive tract
  • Epithelial cells have directionality (polarity)
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88
Q

Cell fate

A
  • Describes what a cell will normally develop into

- not a commitment

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89
Q

Cell differentiation

A

-Cells become structurally and functionally different from one another
-Differentiation during development is gradual
-Cells make decisions towards intermediate step in order to reach its final form
-The developmental process where cells start to take on structures and express genes (proteins) that are indicative of their function
Ex) muscle cell = contractile fibres

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90
Q

Undifferentiated

A

Describes a cell with NO structural features

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91
Q

Competence/potency

A
  • The totality of all structures, cell types that a cell can form
  • The potential of a cell can be tested by changing its environment
  • Experimentally, competency is determined based on transplantation of cells
  • Determining the potential of those cells to take on a new identity based on a new location
  • A cell’s competence slowly becomes restricted during development = cell’s slowly become committed to an identity
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92
Q

Specification

A
  • Is the first stage of commitment
  • Cells are considered specified if they develop according to their normal fate when isolated from its cellular context
  • This suggests that the cell fate is stable when isolated from its environment
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93
Q

Determined

A
  • The second stage of commitment
  • A cell or group of cells is considered determined if they develop according to their normal fate even when its environment is changed
  • Identity can be expressed by genes or proteins
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94
Q

Terminally Determined

A
  • Final step of differentiation
  • Cell will stop dividing
  • Cell cannot change fate/function
  • Stable fate
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95
Q

Give an example of how we would see if a cell is specified? What is the difference between determination?

A
  • Grab endoderm cell in neutral environment and isolate it in Petri dish
  • Can determine what cells they differentiate into
  • Indicates that fate has stabilized
  • But (difference with determination) it is still reversible
96
Q

Give an example of how we would see if a cell is differentiated?

A
  • The stage in development when fate is no longer reversible
  • If we take an intestine cell and put it with skin cell, and develops into intestine cell anyway, it was determined. If it developed into a skin cell, it was only specified (reversible)
  • Indicates cells cannot change their fate even when the environment has changed
97
Q

What does this mean if a cell has dedifferentiated?

A

They are cancerous cells

98
Q

Mosaic development (autonomous specification) vs Conditional specification (regulation specification)

A 
Mosaic development (Autonomous specification): Cells have all the information they need to develop according to their cell fate (independent)
Conditional specification ( Regulative development): Cells require additional signals in order to develop into particular fate. Sometimes cells of the early embryo can sense the entire embryo and missing cells and compensate for their loss. Twins can occur, organs can regenerate.
99
Q

What are stem cells?

A

Undifferentiated cells that divide/ replicate themselves and generate other cell types

100
Q

What are the properties of stem cells?

A

Properties:

a. Self-renewing: Upon division, one cell remains a stem cell and the other can give rise to a variety of cells. Some daughter cells can differentiate.
b. Niche: Stem cells are maintained in an environment that keeps them undifferentiated
c. Multipotent: Have the competence to develop into different cell types

101
Q

Germ cells vs Somatic cells

A

Germ cells: Give rise to the germ line
○ Ex) gametes of the egg or sperm
Somatic cells: The remaining cells of the organism

102
Q

What are the three levels of potency?

A

a. Totipotent
- Highest level of potency
- Where the cell can give rise to all cell types of the embryo
- Ex) zygote or germ cells
b. Multipotent
- Can form more than one cell type but the fate is more limited
- Ex) stem cells required for tissue repair
c. Pluripotent
- Created for mammals
- Ex) embryonic stem cells (inner cell mass of the mammalian embryo)
- Can give rise to most cells of the embryo but not all
- In mammals, these cells give rise to the embryo proper but not the extra embryonic tissue (ex-placenta)

103
Q

Name the three germ layers and what they give rise to in the vertebrates vs insects.

A 
1) endoderm:
Vertebrates=gut, liver, lungs 
Insects=guy
2) mesoderm:
Vertebrates=skeleton, muscle, kidney, heart, blood
Insects=muscle, heart, blood 
3) ectoderm: 
Vertebrates=epidermis, nervous system 
Insects=cuticle, nervous system
104
Q

Pattern formation

A

Where cellular activity is organized in space and time

105
Q

How is the body plan of an organism organized?

A

a. Anterior vs Posterior = Head - tail
b. Dorsal vs Ventral=Back - belly
c. Left to right=Bilaterians
d. Radial axis: apical vs basal
- Gut tube inside is apical and outside is basal
- Associated with epithelial structures

106
Q

What does positional identity mean for the cell?

A
  • Cells can interpret their position within a coordinate system
  • Cells have intrinsic direction
  • Direction: direction in the embryo
  • Cells can have their own polarity
107
Q

Fate maps

A
  • The position of the cell is an indicator of its future cell fate
  • Experimentally performed by marking cells
  • Ex) injection of a dye into cells of the early embryo and determining what cells give rise to later in development
108
Q

Name some common model organisms used for studies in developmental biology.

A
  • Drosophila
  • C. Elegans
  • sea urchin
  • sea squirt
109
Q

Why do we study developmental biology and how?

A
  • Has been studied extensively using genetics such that mutation in genes (genetic loci), the genotype influence the phenotype (an expression of the genotype)
  • Phenotypes that are associated with developmental defects help us to identify the genes that are regulating development
  • A collection of genes with a similar defect may be acting in same genetic pathway
  • Ex) vestigial wings
110
Q

Forward genetic screens

A
  • Have become the basis of discovery for developmentally important genes
  • Organisms are exposed to a potent mutagen (mutant gene, EMS)
  • These generate mutation (random) in genome at expected frequency
  • If mutation occurs in cells that give rise to the germ line, they can be inherited by the offspring
  • The offspring are screened for defects
  • This approach is unbiased in that there is no preconception of the genes involved
  • Scientists screen for particular developmental defects
  • Most mutations are loss of function and recessive
111
Q

In situ hybridization

A
  • Detect gene and protein expression
  • Looks at where genes are transcribed
  • Where, when and how much mRNA is present
  • Transcribed genes are detected in the whole embryo
  • DNA probe is required
  • A DNA sequence that is complementary to the mRNA
  • Probe is labeled so that it can be visualized Ex) fluorescent dye
  • Proteins can be detected with immunohistochemistry (Immuno=using antibody that detects specific antigen, Ex) protein transcription factor)
  • Primary antibody binds to antigen Ex) protein
  • Secondary antibody against primary is labeled for visualization and amplification of the signal
112
Q

What are transgenic animals?

A
  • A genetically modified organism
  • Can be generated through the addition of exogenous sequence into the nucleus, even genome, of the organism
  • Modified piece of DNA that was introduced into the animal
113
Q

How are transgenic animals formed?

A
  • Can be generated through the addition of exogenous sequence into the nucleus, even genome, of the organism
  • Manipulated piece of DNA can be introduced into cells, which include the developing embryo
  • DNA, in the form of an oligonucleotide (A long chain of DNA sequence, Can be transient in nature)
  • DNA can be inherited by the germ cells and this results in DNA that can be inherited by the next generation = heritable
114
Q

What are the different methods of introduction of manipulated DNA into a transgenic organism?

A

i. Microinjection
ii. Electroporation
iii. Vesicle fusion
iv. Gold particle bombardment

115
Q

Oligonucleotide

A

A long chain of DNA sequence

Can be transient in nature

116
Q

What are the different reasons for transgenisis

A
  • Label cells or proteins
  • Engineer the genomic sequence
  • Ex) deletion of gene, knock out
117
Q

GFP? Role?

A

GFP=Green-fluorescent protein

  • Proteins / cells can be labelled with this protein
  • Allows detection of cells or proteins in the living organism or cell
  • A high energy light source (UV) excites a chromophore and this emits a visible light
118
Q

Hermaphrodite

A
  • It has female (oocytes) and male (sperm) sex organs
  • Undergo self-fertilization
  • Rare males can occur for sexual reproduction for genetic crossing with hermaphrodites
119
Q

What is an example of an organism who is hermaphrodite in the adult stage of their lives?

A

C. Elegans (Nematode)

120
Q

What is the lifecycle of C. Elegans (Nematode)?

A
  • After hatching, larvae undergo a series of molts to increase in size
  • The adult is a hermaphrodite=It has female (oocytes) and male (sperm) sex organs
  • Undergo self-fertilization
  • Rare males can occur for sexual reproduction for genetic crossing with hermaphrodites
121
Q

How many somatic cells does the adult C. Elegans have as a hermaphrodite

A

99 somatic cells

122
Q

Does C. Elegans have a gastro-intestinal tract or nervous system?

A
  • Yes, Simple gastro-intestinal tract and nervous system

- They’re small (1mm) and transparent

123
Q

Who are the three Nobel prize winners in Physiology or Medicine in 2002? Why did they win the Nobel Prize?

A

1) Sydney Brenner
2) H. Robert Horvitz
3) John E. Sulston
- For their discoveries concerning genetic regulation of organ development and programmed cell death

124
Q

What did John Sulston specifically discover to win the Nobel Prize?

A
  • Mapped the cell lineage where every cell division and differentiation could be followed during development
  • The first cell division of the fertilized egg is unequal
  • There is a larger AB cell and smaller P1 (This defines the anterior-posterior axis of the embryo)
  • The P1 afterwards will continue to undergo asymmetric cell division to maintain the germ line (P1, P2, P3, P4 = Four cell)
  • Actin microfilaments form at the anterior end of the one cell embryo due to sperm entry site = establishes polarity
  • P-granules (cytoplasmic bodies required for germ cell fate) are asymmetrically localized (segregate) at each subsequent cell division
  • An example of cytoplasmic determinants
125
Q

In C. Elegans, what initiates asymmetry?

A
  • during fertilization, when the sperm enters the egg

- entry will mark future POSTERIOR

126
Q

What are P-granules in C. Elegans development? Where are they localized

A
  • cytoplasmic bodies required for germ cell fate
  • a cytoplasmic determinant
  • are localized asymmetrically at each subsequent cell division
127
Q

What defines the anterior-posterior axis of the C. Elegans embryo?

A

Large AB cell and smaller P1 cell (resulting from unequal first cell division)

128
Q

What happens to the smaller P1 daughter cell after the first unequal cell division in C. Elegans development?

A

The P1 afterwards will continue to undergo asymmetric cell division to maintain the germ line
○ P1, P2, P3, P4
○ Four cell

129
Q

What is the purpose of genetic screens? Which proteins are used?

A
  • Looking for defects in asymmetric cell division

- PAR proteins (partitioning-defective) provided a foundation for understanding asymmetric cell division

130
Q

Why is C. elegans one of the best studied models of autonomous specification (Mosaic) development?

A
  • The divisions of the cell of C. Elegans embryo is invarient (does not change between individual embryos)
  • A cell lineage was created
  • The fate of the cell could be tracked at each cell division
131
Q

What is the purpose of the E cell in C. elegans development?

A
  • An 8-cell embryo is specified upon its formation to form the intestine (20 cells in adult that form a tube-like structure)
  • Experiments that destroyed the E-cell determined it is required for the intestine
  • Ex) when missing no intestine formation so E-cell is necessary for intestine
  • Removal of the E-cell by culturing in isolation determined that it is specified to become intestine (autonomous specification)
132
Q

What is Apoptosis?

A
  • Programmed cell death
  • Cells can undergo cell death as a part of their fate
  • Is a cell fate for some cells
133
Q

Why is apoptosis important?

A
  • It is required to maintain appropriate number of cells during development
  • There is a fine balance between cell death and division
  • It shapes tissues, maintains size
  • In nervous system such as the development of the brain, apoptosis is an important aspect of neural circuitry
  • Ex) networks =neural pruning
134
Q

What was used to identify genes regulating apoptosis? What was discovered?

A
  • Genetic screens were performed to identify genes regulating this process
  • Defects in apoptosis were detected due to low genetic redundancy
  • Many genes have homologues in flies and vertebrates
135
Q

EgL-1 , BID , BIM

A
  • are homologues in flies and vertebrates
  • Important promoter of apoptosis
  • Lies at the top of the genetic / molecular pathway
  • This is associated with the mitochondria
136
Q

CED-9 , BCL-2

A
  • are homologues
  • Is a key inhibitor of apoptosis
  • Inhibits caspase action
137
Q

CED-3

A
  • The key mediators of apoptosis
  • are capsases
  • Represented by CED-3
  • These initiated a proteolytic cascade or degradation that amplifies a signal that now cannot be stopped
  • that results in an irreversible cell death fate
138
Q

What three steps does an apoptotic cell go through in C. elegans development (an overview)

A

1) cell commits suicide
2) dying cell is engulfed by neighbour
3) corpse is digested, leaving no trace

139
Q

Organ Development in C. elegans: Vulva

A
  • The vulva is the opening through which eggs are laid by the hermaphrodite
  • Within the hypodermis (skin-like precursors) there are a group of cells
  • Vulva precursor cells (VCPs or Pnp)
  • Located on the ventral side
  • Pnp cells undergo division and differentiation to form distinct parts of the vulva
  • Mature vulva has 22 cells, all competent* to form the vulva
140
Q

RTK signal transduction pathway

**TO MEMORIZE

A
  1. Ligand binding
  2. RTK receptors dimerize and causes autophosphorylation of receptor
  3. Adaptor proteins recognize phosphotyrosine and bind
  4. GNRP (a mediator) binds
  5. GNRP recognizes and activates Ras G
    (G protein)
  6. Ras G protein activated through GTP / GDP exchange (Ras­GDP → Ras­GTP)
  7. GAP recycles Ras
  8. Ras G activates Raf and initiates a phosphorylation cascade (kinases)
  9. Activates downstream effectors like PKC (protein kinase C)
  10. PKC phosphorylates
    MEK (a kinase)
  11. MEK phosphorylates
    ERK (a kinase)
  12. ERK enters nucleus
  13. It is now an activated transcription factor
  14. Transcription modulation
141
Q

Receptor tyrosine kinases (RTK) is a receptor for which other pathways other than RTK signal transduction pathway?

A
  • Fibroblast growth factor (FGF)
  • Epidermal growth factor (EGF)
  • Stem cell factor
142
Q

In the RTK signal transduction pathway, what is the role of Ras G and what happens if it mutates?

A
  • Huge marker in mutations of Ras G in cancerous cells
  • RAS is a like a molecular switch to begin activation of kinases
  • important in cancer progression
143
Q

How are apoptotic cells removed from the cell?

A

phagocytosis

144
Q

Inductive signalling/induction

A

one cell is signalling to another group of cells to influence or instruct their fate

145
Q

Lateral inhibition

A

the mechanism by which cells inhibit neighbouring cells from developing in a similar way to themselves (primary cells inhibits neighbouring cell from same fate)

146
Q

Hypodermis

A
  • Protective outer layer of vulva of C. elegans
  • not as complex as epidermis
  • VPCs or PnPs are a group of cells within hypodermis of vulva, on ventral side of worm
147
Q

What is the default state of cells forming the vulva in C. elegans?

A
  • The default state is 3^o (tertiary)

- These are fated to become hypodermis

148
Q

What is the primary (1^o) and secondary (2^o) cell fates of the vulva in C. elegans?

A
  • There is a cell present called the anchor cell that provides an inductive signal for the VPCs to take on a 1^o or 2^o cell fate
  • The 1^o primary fate makes up the central part of the vulva
  • The 2^o form a mirror image on the periphery (either side of the 1^o cell)
149
Q

Genetisists used a forward genetic screen to isolate genes required for vulva induction in C. elegans. What happened when they destroyed the anchor cell?

A
  • caused no vulva to form
  • so this cell may be providing the signal
  • Isolated mutants the (vul) vulvaless or (Muv) multivulva
  • Vul resulted in no vulva formation and embryos hatched inside the mother, creating a “bag of worms” phenotype
150
Q

Genetisists used a forward genetic screen to isolate genes required for vulva induction in C. elegans. How were they able to prevent the vulva from developing in C. elegans?

A
  • Lin-3 EGF ligand produced by the anchor cell
  • Let-23 EGF receptor was expressed in the VPCs to receive the signal
  • Let-60 RAS molecule initiated a signal cascade within the VPC
  • Loss of function (reduction or complete loss)
  • Alleles of Lin-3, Let-23 and Let-60 resulted in no vulva development
  • Let-60 had gain of function alleles isolated that created a pathway that was consistently active (always on)
  • The cell closest to the anchor cell receives the strongest signal to become primary
  • All cells are competent because they express the receptor
151
Q

Notch signalling pathway: Why is it important?

**TO MEMORIZE!

A
  • responsible for lateral inhibition (cells in contact with primary cell are inhibited from taking on same fate)
  • maintains constant patterning
  • involved in direct cell-cell contact
152
Q

Notch signalling pathway: What is the role of Notch?

**TO MEMORIZE!

A
  • Is a receptor that binds to a ligand delta that receptor in the receiving cell
  • Ligand binding activates proteolytic cleavage of the notch, which releases a protein that can enter the nucleus
153
Q

Notch signalling pathway: What are the steps in C. elegans?

**TO MEMORIZE!

A

1) Lin-12 is a notch receptor in C. Elegans and binds to the ligand lag-2
2) All VPCs initially express Lin-12 and lag-2 at low levels
3) The inductive signal sways the balance for Lin-12 to become more stable in one cell
4) Lin-12 bind lag-2 on the adjacent cells to inhibit its own production (an example of a negative feedback loop or pathway)
5) The result is that the neighbouring cells cannot take on the 1^o and take on the secondary instead

154
Q

Notch signalling pathway: Mutations that reduce Lin-12 causes what?
**TO MEMORIZE!

A
  • causes multivulva phenotype
  • More than one VPC takes on the primary fate
  • The multivulva (Muv phenotype) results from lack of lateral inhibition and the other cells VPCs can become 1^o in fate
155
Q

What are microRNAs?

A
  • Are RNA molecules (a gene) that are transcribed into poly-adenylated primary miRNA molecules
  • Similar to genes that control expression to genes that code for proteins
  • But miRNA do not produce proteins
  • They produce short double-stranded RNAs as their final product (not present in the cell, otherwise would indicate to the cell that there might be something foreign in the cell)
156
Q

How are the miRNAs used?

A

1) The pre miRNA forms a hairpin structure that is processed by a protein called Drosha (endonuclease)
2) This is exported from the nucleus and further cut into 20-twenty-five nucleotide pieces by Dicer 3) small dsRNA pieces become incorporated into the RISC complex
4) one strand is used as a template to recognize mRNAs with base pair complement with 3’UTRs of target genes = inhibition of target genes
4) Gene expression is inhibited through inhibition of mRNA translation
5) Translation is inhibited through RISC complex

157
Q

What is the main difference and similarity between RNAi (siRNA) and mRNA?

A
  • Difference with RNAi is that target mRNA is not degraded

- Similarity is that Dicer is common to miRNA and siRNA (RNAi) for double stranded processing

158
Q

Heterochronic genes

A
  • discovery of the first miRNA pathway
  • development of cells with defective in timing of cellular differentiation
  • regulate timing and sequence of developmental events
159
Q

Heterochronic mutations in lin-14 during C. elegans development has which two different phenotypes?

A

1) Precocious: skips larval stage 1 and goes to stage 2, differentiation occurred ahead of time = loss of function mutation
2) Retarded: larval stage is repeated multiple times and never continues into the next larval stages, behind in development = gain of function mutation

160
Q

What is RNAi?

A
  • RNA interference

- double stranded RNA used to knock down gene function miRNAs

161
Q

How are the two different phenotypes of Lin-14 mutations created?

A
  • Lin-14 loss of function and gain of function mutants shifted to differentiation pattern
  • Lin-4 (miRNA) seems to be regulating a transcription factor Lin-14
  • Lin-4 is regulating levels of the Lin-14 protein such that high levels are associated with L1 identity and later stages associated with little or no protein
  • Lin-4 miRNA is binding the Lin-14 3’UTR to inhibit translation
  • Lin-4 loss of function and Lin-4 gain of function fail to turn Lin-14 off
  • Lin-14 mRNA has Lin-4 consensus binding sites
  • Lin-14 gain of function: Prevent Lin-4 from repressing Lin-14, Have a duplication
162
Q

What are the four stages of the life cycle of Drosophila melanogaster?

A

1) Embryonic stage: Major axes are set up, Gastrulation, germ-band extension(long), Segmentation
2) Larval stage: Feeding and growth, Larval molts
3) Metamorphosis: Almost complete shedding of larval tissue
4) Adult stages: reproduction through gametes

163
Q

Gastrulation

A
  • Internalization of endoderm and mesoderm

- during embryonic stage of Drosophila development

164
Q

Segmentation

A
  • segment positional identity is established

- during embryonic stage of Drosophila development

165
Q

Metamorphosis

A
  • almost complete shedding of larval tissue

- Stage 3 of Drosophila development

166
Q

What happens in reproduction of Drosophila?

A
  • The sperm enters the oocyte through the micropyle (anterior end)
  • After fertilization, a syncytium is formed
  • Rapid nuclear divisions with cytokinesis
  • Multiple nuclei share a common cytoplasm
  • 12 divisions every 90 minutes
  • Results in 6000 nuclei
167
Q

Why would cells want to share the same cytoplasm?

A

So cells don’t have to communicate amongst each other

168
Q

What happens after fertilization in Drosophila embryo development?

A
  • Pole cells, a subset of nuclei migrate to the posterior pole before the others and are fated to become or give rise to the germ cells
  • The first to be surrounded by a cell membrane
  • Setting aside a group of cells to continue the germ line
  • This protects the cells from developmental cue that somatic cells are subject to
  • Help them retain totipotency
169
Q

What are the differences between insect and vertebrate development?

A
  • Insects are inverted with regards to their Dorsal-Ventral axis
  • Such that Gastrulation initiates on the ventral side
  • Nerve cord is ventral (NOT dorsal)
  • In evolution, their was an inversion of D-V axis in chordates
170
Q

Following cellularization of the syncythium, what is the continuation of embryonic development of Drosophila?

A

1) the cellular blastoderm forms
2) The embryo consists of a single cell epithelial layer on the periphery of the embryo
3) Gastrulation initiated with ventral furrow
4) Germ-band extension (makes up the trunk of the embryo)
5) This temporarily drives the posterior onto the dorsal surface
6) Creation of evenly spaced grooves that are the precursor to segments = parasegments

171
Q

Parasegments

A
  • Creation of evenly spaced grooves that are the precursor to segments
  • Temporary intermediate form before the segments
  • A function of how the pattern sets out
  • Established and used to make segments
  • Slightly out of register compared to the segments
172
Q

What are the three segments (broad domains) of the Drosophila larva?

A

1) head
2) thorax
3) abdomen
* first signs of segmented patterns

173
Q

Parasegments vs segments

A
  • The relationship between parasegments and the final segments are that they are shifted with regards to their apparent border
  • parasegement boundaries: “out of register”compared to the segment boundaries
  • segment: posterior region of one parasegment and the anterior region of the adjacent parasegment
174
Q

Initial segment

A

Initial segment identity is read out through denticles patterns

175
Q

Denticles

A

small tooth-like outgrowths of the epidermis

176
Q

Imaginal discs

A
  • Are a sac/pouch of tissue that will give rise to the adult structures
  • Have distinct characters
  • Small sheets (when flattened) of epidermal cells that are set aside from the cellular blastoderm stage
  • Set aside about 40 cells
  • These 40 cells are associated with each future segment
  • They grow during larval molts (stages) by cell division (proliferation) and replace larval tissue and metamorphosis
  • Provides continuity in pattern formation (positional identity)
177
Q

Segment identity of organs in Drosophila

A
  • Don’t have lungs but have an elaborate trachea to provide oxygen to the body
  • Tracheal system is highly branched tubular system required for respiration
  • Forms an air pocket
  • Similar branching patterns as our lungs
178
Q

Peripheral nervous system in Drosophila

A
  • Each segment has similar template of nerve cell precursor that have distinct function and growth patterns
  • Start as Neuroblasts then will take distinct identities
179
Q

In the anterior-posterior patterning of the body axis in Drosophila, what is the transcription factor hierarchy?

A

1) maternal factors
2) gap genes
3) pair-rule genes
4) segmentation genes
5) selector genes
- Starts off as simple, but as you go down the hierarchy, things get more and more complex

180
Q

Maternal vs Zygotic genes

A
  • Early development relies on genes (mRNAs, proteins) contributed by the mother
  • More important are the mother’s genes than the zygote at the early stages
181
Q

Segments

A
  • repeated section of the body plan
  • block units that repeat many times throughout the body of the animal
  • In millipedes, centipedes, fly
  • Have ability to develop independently = diversify function
  • Ex) In thorax, can have wing present or absent, different number of legs
182
Q

Maternal factors

A

Molecules (mRNA and Proteins) that are deposited in the egg prior to fertilization

183
Q

Compare the initiation of asymmetry in Drosophila and C. elegans.
*On exam

A

In Drosophila:

  • mRNA deposition initiates asymmetry
  • Bicoid mRNA is localized to the far anterior of the egg
  • Fertilization signals for translation of the protein
  • A Bicoid protein gradient is established
  • initiation of asymmetry AFTER fertilization

In C. Elegans:

  • fertilization, when the sperm enters the egg, this initiates asymmetry
  • entry will mark future POSTERIOR
  • But in the fly, the sperm has a slightly different function
  • Asymmetry is initiated during fertilization
  • mRNA are already asymmetrically localized
184
Q

What initiates Drosophila asymmetry?

A
  • mRNA deposition initiates asymmetry
  • Bicoid mRNA is localized to the far anterior of the egg
  • Bicoid protein gradient is established
  • asymmetry initiated AFTER fertilization
185
Q

Bicoid protein

A
  • localized to far anterior of the egg
  • establishes a gradient
  • initiates asymmetry after fertilization
  • determines position in the embryo in a concentration-dependent manner
  • a morphogen
186
Q

What is the French Flag Model?

A

1) each cell has the potential to develop as blue, white, or red stripes = fate/type of cell
2) position of each cell is defined by the concentration of morphogen
3) depth of the gradient provides positional information
4) positional value is interpreted by the cells which differentiate to form a pattern
5) threshold concentrations instruct cell identity

187
Q

Morphogen

A
  • any substance that has a variable concentration and is involved in pattern formation
  • Needs to be able to form a gradient
188
Q

Threshold concentrations

A
  • The thresholds relate to a boundary
  • There is a mechanism to maintain the gradient
  • Threshold concentrations: where different fates are specified by different concentrations
  • Threshold: the magnitude or intensity that must be exceeded for a certain reaction to occur
  • different fates are specified by different threshold concentrations of a morphogen
189
Q

Necessary vs Sufficient

A

Necessary: A factor is necessary for development of a structure or tissue, when that factor is absent and that structure /tissue fails to form

Sufficient: A factor is sufficient for development of a structure/tissue when that factor is expressed in a region where it is not normally present and can allow for formation of that structure/tissue in that new location
(Sometimes called Ectopic expression: expressing a factor where it is not normally present (space or time)

190
Q

Give examples of why Bicoid mRNA is both necessary and sufficient for development in Drosophila.

A
  • important in anterior structures
  • loss of head structures happens without Bicoid mRNA=necessary
  • head structure began to develop in the centre of the embryo due to Bicoid mRNA gradient=sufficient
191
Q

Which protein was the first identified morphogen? Why is it a morphogen?

A
  • bicoid mRNA
  • The protein forms a gradient and concentrations dictate identity along the A-P axis
  • High Bicoid concentrations = anterior / head structures
  • responsible for head structures in Drosophila
  • Specific to Drosophila and insects
192
Q

Caudal

A
  • A maternal factor that specifies posterior in Drosophila

- It’s translation is repressed by Bicoid protein

193
Q

Cdx (Caudal-type homeobox)

A
  • Homologues throughout the animal kingdom
  • Homeobox: in plants, animals, transcription factor homeobox evolved a long time ago, a particular motif, a DNA sequence involved in regulation of patterns of anatomical development, bind to and control activities of other genes=act as transcription factors
194
Q

Homologue

A

has similar evolutionary or ancestral history, also implies similar function

195
Q

Homeobox

A
  • in plants, animals
  • transcription factor homeobox evolved a long time ago
  • a particular motif
  • a DNA sequence involved in regulation of patterns of anatomical development
  • bind to and control activities of other genes
  • act as transcription factors
196
Q

Name some examples of the phenotypes of mutations affecting the three types of segmentation genes.

A

1) gap gene (Kruppel)
2) pair-rule gene (even skipped)
3) segment polarity gene (gooseberry)

197
Q

Gap genes

A
  • Determine broad domains in the embryo
  • Maternal factors such as Bicoid control expression of gap genes
  • Gap genes respond to the Bicoid gradient
  • gap genes regulate expression of other gap genes
198
Q

What are some examples of gap genes?

A
  • hunchback
  • giant
  • Kruppel
  • Knirps
199
Q

Hunchback

A
  • gap gene
  • Responds to the Bicoid gradient
  • High Bicoid concentrations = high hunchback concentrations
  • Mutations that reduce Bicoid levels, hunchback responds with lower expression
200
Q

Krupple

A
  • gap gene that regulates other gap genes
  • Krupple is responding to hunchback (regulated by concentration of hunchback)
  • Krupple is normally expressing stripes (white stripes mentioned before)
  • If can manipulate the levels of hunchback, high concentrations of hunchback = repression of krupple
  • Hunchback intermediate levels of concentration=activation of krupple protein expression
  • Decreased concentrations of Hunchback = no expression
  • This is an example of a gene responding to threshold concentrations and regulating cell fate
201
Q

Is hunchback behaving like a morphogen?

***on exam

A
  • YES

- has a variable concentration and is involved in pattern formation = morphogen

202
Q

Give two examples of morphogens.

A

1) bicoid

2) Hunchback

203
Q

Pair-rule genes

A
  • each pair-rule gene is expressed in seven transverse stripes (every second parasegment)
  • delimit the boundaries of parasegments
  • They are regulated by complex transcriptional control Bicoid and the gap gene
  • These are expressed just before the nuclei of the syncytial blastoderm become surrounded by membrane
204
Q

What are two examples of pair-rule genes?

A
  • fushi tarazu (ftz)

- even-skipped (eve)

205
Q

Eve gene

A
  • Modular organization of regulatory DNA
  • Fragment of DNA were linked to a reporter
  • Segments of the regualtory DNA could drive expression in particular patterns
  • Two segments in tandem drive expression in a pattern that is the sum of the two patterns generated by each
  • Each segment was called a module controlling eve expression
206
Q

Even-skipped

A

-specific promoter regions of the even-skipped gene control specific transcription bands in the embryo

207
Q

Complex transcriptional control region: Explain how to get a stripe.
*on past exams

A
  • One control region of even-skipped (eve) controls expression in the third stripe
  • Activators of the third striped element Bicoid and Hunchback
  • Repressors are Krupple and Giant
  • These help to define boundary of the stripe
  • This repression dominates activation of bicoid and hunchback
  • Each eve stripe is under unique control region with different binding sites and different concentrations/combinations result in the final pattern
208
Q

Segmentation genes

A
  • Define boundary of parasegment and later the segment

- segment polarity is linked to boundary formation

209
Q

How do the pair-rule genes and segmentation genes work together?

A
  • Pair-rule genes define the anterior boundaries of all 14 parasegments
  • But this activity is temporary
  • After cellularization, the segmentation genes respond to the pair-rule genes
  • With segmentation genes, the boundaries of parasegments become fixed
  • At first, the boundaries were fuzzy, but now fixed and clear
210
Q

Engrailed

A
  • a segment-polarity gene
  • *creation of segment and parasegment compartments
  • a transcription factor
  • Can be both an activator and strong repressor
  • Most important role: marks the interior boundary of the parasegments and later become the posterior compartment of the segment
  • Is expressed throughout the life of the animal
  • Expression of engrailed marks a compartment
  • Mutatant clones of cell can be generated in Drosophila where small patches of cells can be missing engrailed expression (and function)
  • Result: Mutant patches lacked compartment boundaries
211
Q

Compartment

A
  • cell lineage and/or cell fate/type restricted by a boundary
  • cells are unable to move into an adjacent compartment
212
Q

How is engrailed involved with parasegments?

A
  • Earlier in development, engrailed is first expressed in a single line of cells at the parasegment boundary (anterior margin)
  • Expresssion is the result of the combinatorial regulation by pair-rule genes fushi tarazu and even-skipped
213
Q

Canonical Wnt Signalling Pathway: Components

*ON EXAM

A

1) Wnt
- Diffusible ligand
- is acting like a morphogen
2) Frizzled
- Receptor
3) Dishevelled
- Adapter/co-receptor
4) APC/Axin/GSK-3B
- B-catenin destruction complex
5) TCF/LEF1
- Transcription factor
6) B-catenin
- Transcriptional co-factor
- Transforms transcription factor TCF/LEF1 into enhancer

214
Q

Canonical Wnt Signalling Pathway: Steps when Wnt absent

*ON EXAM

A

1) nothing binds to the receptor Frizzled
2) Dishevelled does not act as a co-receptor (no role)
3) APC/Axin/GSK-3B complex does B-catenin degradation
4) nothing enters the nucleus and there is no transcription

215
Q

Canonical Wnt Signalling Pathway: Steps when Wnt present

*ON EXAM

A

1) Wnt (diffusible ligand) binds to Frizzled receptor
2) Dishevelled arrives (an adapter/co-receptor) and phosphorylates
3) APC/Axin/GSK-3B complex also binds to Dishvelled
4) B-catenin degradation is inhibited
5) free B-catenin accumulates
6) stabilization of the transcription co-factor B-catenin (involved in polarity) and enters the nucleus and binds to TCF/LEF1 (a transcription factor)
7) transcription begins

216
Q

How are segment boundaries related to polarity?

A

Segmentation genes stabilize the parasegment boundary and patterns the segment along the A/P axis

217
Q

What are two examples of segment polarity genes?

A
  • engrailed (en)

- wingless (wg)

218
Q

Wingless/Wnt

A
  • initiated by pair-rule genes
  • The boundaries are maintained by interactions between engrailed Wnt/wingless (wg) and hedgehog (Hh)
  • Hedgehog signalling forms a positive regulatory loop to maintain parasegment boundary and establish engrailed expression
  • Wingless and engrailed are initiated by the pair-rule genes
  • High concentrations of even-skipped or fushi tarazu activate engrailed
  • Wingless is only expressed when fushi tarazu and even-skipped are absent
  • The continued expression of engrailed and wingless are maintained by interactions between each other
  • The wingless protein is secreted and diffuses to surrounding cells
  • In those cells competent to express engrailed (ex-increase in eve) wingless binds to frizzled and enables activation of engrailed
219
Q

Hedgehog

A
  • Transcription is activated by engrailed
  • Engrailed activates its own expression
  • Hedgehog then diffuses which enables the transcription of wingless
220
Q

What is the model for the transcription of the segment polarity genes engrailed (en) and wingless (wg)?

A
  • Wingless (Wg) and Hedgehog (Hh) feedback on each other
  • Wg promotes expression of Engrailed (En)
  • In the absence of Wg activity, expression of Hh and En is lost, leading to a loss of segment polarity and pattern
221
Q

Let-7

A
  • An miRNA
  • More highly conserved
  • Has a conserved function or sequence creating a family of miRNA
222
Q

Which noble prize winners did forward genetic screening?

A

Edward Lewis

223
Q

Who are the three noble prize winners in 1995 in Physiology and Medicine for Genetic Control of Patterning of the Drosophila Embryo?

A

1) Edward Lewis
2) Christiane Nusslein-Volhard
3) Eric Wieschaus

224
Q

Mutations in Wnt (wingless) and Hedgehog (Hh) does what in Drosophila development?

A

Mutations in either of these genes Wg/Hh resulting in defects in segment polarity

225
Q

What is similar with Hedgehog and Wingless gradients.

A

Hedgehog and wingless gradient run opposite directions of each other

226
Q

Selector Genes/Homeotic/Hox genes

A
  • All refer to the same family founded by Edward Lewis

- Controls segment identity along anterior-posterior axis

227
Q

Which body segment is most difficult to study?

A

-head is more difficult than thoracic and abdominal segments

228
Q

Homeotic selector genes

A
  • A series of master regulatory genes
  • Set the future fate of each segment
  • Transcription factors
  • Homeodomain containing (protein motif) and bind to homeodomain (DNA sequence in control element)
  • They control the activity of genes, differentiation of cells in each segment
  • They are required throughout development to maintain this pattern of gene expression
  • Highly conserved in animal development collectively called the Hox Genes
229
Q

Which two Homeotic selector gene complexes exist?

A
  • Two complexes exist called the Antennapedia and Bithorax

- Named after initial fly mutations

230
Q

Homeotic transformation

A

Mutations cause transformation of one segment identity into another

231
Q

Bithorax mutation

A
  • Transforms anterior haltere to anterior wng
  • T3 to T2 transition in anterior
  • Bithorax complex is made of Ubx, Ultra Bithorax, Abd-A (Abdominal A) and Abd-B (abdominal B)
232
Q

The following homeotic selector genes are expressed where?
A) Ubx
B) Abd-A
C) Abd-B

A

A) Ubx: Expression in segments five to 12
B) Abd-A: 7-13
C) Abd-B: 10-14

233
Q

Removing one or several homeotic selector genes results in what kind of defects?

A
  • H all are removed parasegments five onward are missing and take on parasegment identity
  • Parasegment four was a default identity
  • Ubx only resulted in identity of parasegment five and six were restored, and parasegment seven onward took a parasegment six identity
  • Ubx was responsible for parasegment five and six
  • Parasegment four, five, six, seven, eight and nine restored, nine onward were repeated
  • Abd-A is responsible for seven, eight and nine identity
  • Abd-B is required for parasegment 10,11,12,13 identity
  • With Ubx alone missing, parasegment five changes to a parasegment four identity
  • Loss of Ubx was determined to be the cause of the Bithorax mutant parasegment five to parasegment four identity anterior haltere (in anterior T3 segment) to anterior wing (anterior T2 segment)
234
Q

Which homeotic selector gene mutation results in posterior domination?

A
  • Where ultrabithorax is only one absent after being readded

- Ubx is only one missing

235
Q

What are three key features of Hox genes?

A

1) Homeotic transformations
- Where mutation in the two complexes result in change of identity of segment along the A/P axis of the fly
- Example) Antennapedia where antennae take on the fate of the legs
- Bithorax: halter becomes a wing
2) Co-linearity
- The placement of the gene with the complex correlated to their expression pattern in the animal
3) Posterior dominance:
- Hox genes expressed in more anterior regions are repressed by more posterior ones

BIOL 3542 (10 decks)

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