Midterm

what are the advantages of experimenting on smaller organisms?

• Life cycle is short

• Easier to study, the more simple the organism the smaller the genome

  • Diff mutations

  • Easier to screen

• Genetic similarities, conservation btwn them and the human genome

• Accessibility of the embryos

  • Frog will lay eggs vs mouse that grow a fetus inside

• Easier to induce genetic mutations in simpler organisms

what are the stages of animal embryonic development?

1. fertilization

2. cleavage

3. gastrulation

4. organogenesis

5. metamorphosis

6. gametogenesis

what happens during the stage of fertilization?

fusion of sperm and egg nuclei generates embryonic genome

what happens during the stage of cleavage?

Zygotes undergo many cell divisions

• Not to increase cell size but to increase the cell number

what happens during the stage of gastrulation?

formation of structure from cells. Cells are tuned into layers from the folding inwards into of the embryo

• Three germ layers

  • ectoderm

  • Mesoderm

  • Endoderm

• Precursor layer for all the diff tissue type and organs in the body

what happens during the stage of organogenesis?

formation of the organs themselves

what happens during the stage of metamorphosis?

not for all animal life cycles

• Conversion or animal form a simpler form to a mature adult that can reproduce

• Not all animals to this

what happens during the stage of gametogenesis?

development of sperm and egg

epigenesis

life starts in a simpler form and gains complexity, cell multiple and differentiate

germ vs somatic cells

Germ - sperm and egg, carry DNA that is used to recombine and generate a new organism, carry genetic information that in passed on.

Somatic - everything else

Mutations can happen in both lines

Can induce mutations in somatic cells but they are not passed on

Mutation in germ cells can be inherited, change phenotype not only of carrier but also their offspring

meiosis vs mitosis

Mitosis: full set of chromosomes, divide cell but maintain number of cells. No genetic diversity, just increasing cell number

Meiosis: daughter cells get half of the genetic info to create genetic variability when combined with the sperm. Two diff parents

nuclear determination, asymmetric assortment

• When a cell divides the genetic material is halved some of the factors are inherited in one cell and the other cell inherits two different factors. When the factors split into four they are once again differentially segregated.

• Each daughter cell will develop differently based on the difference in proteins they express = asymmetric assortment of factors

Is roux experiment consistent w/ nuclear determination?

Frog embryo, impaled one of the two embryos with a hot needle, killing one of the cells

• Die = didn’t give rise to anything

• One that survived continues to form structures that are consistent with the gastrulation phase  i.e the blastocoel and the neural tube which is the precursor for the brain, spinal cord and CNS

  • In the end he did not have a full embryo, half of an embryo

  • This is because one of the two cells died off earlier and the other cell was unable to compensate for the loss of the other cell

Consistent w/ nuclear determination bc only half of the embryo developed, kill off the factor that make up the left side than it will not develop

Is the Duriesch and Morgan experiment consistent with nuclear determination?

Sea urchin, embryo at two-cell stage

• Separated two cells and only one cell survived

• Developed into larvae and developed all its components

  • Smaller than the normal situation

Not compatible with nuclear determination

• In addition to intrinsic factors there is also a role for external cue the one surviving cells can compensate for the dead cell to from the missing half

 

Spemann and mangold

• Two embryos, carving out a piece embryonic tissue that sits close to the blastopore and will give rise to the neural cord and neural tissues

• Transplanting it to another embryo at a different site

• Two possible outcome:

  • Form and second blastopore

  • Alternative is nothing will happen and it will start blending with the surrounding tissue and take the fate of the neighbouring cells

• Outcome is:

  • Creates a second neural cord and tube

Why does this happen?

cells communicate and interact with each other to guide development

Morgan:

Fruit fly

Two diff strains w/ two diff coloured eyes

Gene = normal is red and mutant is white

Crosses and white eye female with a red eye male:

• When put into a punnet square we can conclude that he will end up with two females with red eyes and two males with red eyes

Gene is linked to the x chromosome

Why does this happen?

Females XX can compensate for the mutation and show red where as the males cannot and they are XY  and will show the mutation

what is a recessive mutation?

need both mutated alleles two see the mutated phenotype

Pattern of inheritance and severity of the phenotype we can decern the effect of these genes on the development of an animal.

Anterior to posterior vs Dorsal to ventral vs Left to right axis

Anterior to posterior  = head to tows

Dorsal to ventral = back to front

Left to right axis = left to right

Endoderm, Mesoderm, Ectoderm

Endoderm = gut liver and lungs

Mesoderm = bones, muscle, kidney and heart

Ectoderm = skin and nervous system

what is morphogenesis?

Morphogenesis: changes in cell position, folding event

• Evagination event where you have a single cell layer and a piece creeps up into the embryo and the loss of cells form one end or the other

Sea urchins

1. Hollow ball

2. Yellow = endoderm and forms a tube like structure, The tube is chopped off on one end by controlled cell death (apoptosis) this tube formation even generates the gastrointestinal tract and the apoptosis on one end opens up the tube and forms the sea urchin mouth

what can we take from this experiment

Morphogenic event shape the function and the structure of the organism

Cell differentiation

In one cell you get different germ layers and may differ cells type

The same genetic material in every cell but it is what gene is activated gives rise to only three cell types

Frog egg:

Zapped w/ UV light radiated egg and abolished genes inside

• No genetic material, hollow egg

• Take skin cell and sucks out the nucleus and transplants it into the egg

• Get a full from tag pole

why didn’t we get a skin cell?

• The skin cell has the same DNA but the skin cell is programmed to create a skin cell, but when you put the skin cell into the form of the egg it can develop into the organism

• Skin cell has the genetic potential to become anything but must be placed in that particular environment

central dogma

 Dna transcribed into rna translated into protein

Explain the Diagram

Control region: promoter, transcription factor binding modules to turn a gene on/off

Coding region = instruction for making a particular region (red)

Upstream we have a promoter region:

Rna polymerase binds to promoter to synthesize rna using the coding region of DNA as a template

• RNA gets helps form transcription factors

• Aren't always the same in every cell type

 

More upstream regulator sites

Epigenetic regulation: The configuration of the DNA can either be tightly wounded or loosely wound to make it more of less accessible to the expression the genes 

 

Two levels of control

• Diff. specific transcription factors

• Epigenetics

mRNA has been transcribed:

Exons vs Introns:

Exons code for protein

Alternative splicing = putting different exons together

• From a single gene we can create diff. parts of proteins

 

mRNA leaves the cell and attaches to ribosome and ribosome synthesis  the correspond peptide

• Sometime peptide are not ready to be processed yet, they are biological inactive, post translational modification

• Chemical group that attach to the protein to make them more active

epigenetics

no change in dna sequence change in dna structure (tightens or losens it that makes it more or less accesible)

transcription factor expression

diff transcription factors in diff cell types

alternative splicing

Piecing diff exons together to get diff variants of a protein

post-translational modification

Post translational modification to either activate or  leave a protein in its inactive form

cell fate, determination, specification

Cell fate: undefined cell is destined to become

Determination: whether or not the cell realises that fate

Specification: Cell achieves is fate

when putting green cell and stick them into yellow cell they to not adopt a hexagonal shape and stay square

are the green cells determined?

it is determined because despite the fate of the cells around it, it stay as what it is fated to be

Green into yellow still become square they are determined - doesn’t change their fate

Take a portion of green cells and stick them into yellow cell they adopt the hexagonal shape of the yellow cells

are the green cells determined?

their fate is change by the fate of the surrounding cells

they are not determined

Green absence from any external environmental influence still becomes square

is the green cell specified?

it is specified because it develops according to its when isolated

what do we learn from the “determination of the eye region with time in amphibian development”?

Yellow tissue in diff. embryo - fated to become eye tissue and when transplanted into embryo it does not become and eye

It become a trunk as it is in that area during early development

• Later on in development in eye area is develop into an eye in the trunk region

• Extrinsic factors that influence cell after can change during the phase of developmen

Changes in cell fate can change whether they are induced early or later

what is induction?

 a group of cells can secrete a signal that can influence an external group of cells to have that fate

permissive vs instructive

Permissive: all-in-one response

• Cell will respond to a signal or not

• cells make only one kind of response to a signal once a given concentration of signal is reached

Instructive: means depending on the concentration of that signal the cell can respond differently

• cells respond differently to different concentrations of signal

how do we transmit inducing signals?

1. Diffusion: Neuron consist of two diff sides, pre and postsynaptic terminal.

   • In order to communicate with each other the presynaptic terminal has to release neuro transmitter which then diffuse across the cleft and bind to neuro transmitter receptors.

2. Direct contact: receptors that fit together like puzzle pieces

3. Gap junctions: ion channels that connect one cell membrane to another to allow ions to flow through

three signal transduction pathways

Signal transduction usually starts at the cell surface w/ the receptor  and then culminates in change in gene expression

• Receptor is activated by binding to some kind of ligand which may be secreted by a neighbouring cell

At rest the receptor, not active is not binding to anything inside the cytosol of this cell and there is no transcription of the gene

First mechanism

• When the ligand binds is may induce phosphorylate and activation of the intercellular receptors,

• Chain rxn phosphorylate more enzymes that active transcription. 

Second mechanism:

• Ligand binds to a receptor and induce a conformational change in some downstream effector complex that will release a portion of it t get used into the nucleus and act like a transcription factor to activate gene expression

Third mechanism

• Direct contact

• A receptor that in impaled in one cell will attached to a receptor from another cell and will induce a conformational change in the second receptor which induced gene expression

morphogens

any substance active in pattern formation whose spatial concentration varies and to which cells respond differently at certain threshold concentrations

threshold

can represent the amount of morphogen that must bind receptors and activate intracellular signalling or express specific genes

patterning by morphogen gradients

• Arrange cells spatially, and put the inside an organism next to a source of a secreted factor

• 1 closest to the source and 6 being farthest

• Concentration = higher to the source

•  Concentration threshold of the secreted factors that a cell will sense, perhaps by the virtues of the receptor on the surface and cause diff changes in genes expression depending how far the cells are from the source

why does this signify?

• A cells fate is not only dependent by the genes it has but that it position relative to other cells is important

• The further it is from some inducing factor the more impact it will have on sed cell

The cells in the diagram are turning red at random

How do they find each other?

Cellular contact, the expression cellular receptors that are compatible with/ each other and can bind

requires different cell-adhesion molecules or differing amount of the same adhesion molecules to be expressed on cell surfaces

what are embryonic stem cells?

ES cells: have the potential to become any cell in the body

• They are pluripotent

• Don’t divide identically

• Able to from completely diff daughter cells that take on diff functions and structures from the source cell.

what does autonomous mean?

all factors that regulate specification are originated inside of the cell

• cell-intrinsic mechanism

what does conditional mean?

external from the cell influence the fate

what does syncytial mean?

Combination of both, only a few organisms

what is differentiation?

Cell that has originated from the single fertilized egg has stopped dividing mitotically (terminal cell division or terminal mitosis) and develops specialized structural elements and distinct functional properties.

explain what is happening in this diagram?

Diagram (A)

Clump of unspecified cells (grey) and clumps within (red and yellow)

• Take a red and put it into a dish

• Same thing with a single yellow cell

• Red: without other factors the cell becomes a muscle cell

• Yellow: without other factors the cell becomes a neuron

• They are both specified

 

Diagram (B)

Large patch of yellow cells and a singular red cell

• Red cell does not differentiate into a muscle cell and become a red neuron not determined

 

Diagram (C)

Single red cell is put into yellow cells and develops into muscle it is determined

explain this diagram:

Diagram (A)

• Development of muscle cells

• Undergoing cell division

• Pigment is always localized at the base of the cell and forms the tail of the tunicate

  • Can track the fate of the cell that are localized at the lower end of the cell

 

Diagram (B)

• 8 cell stage

• B4.1 always develop into muscle

 

Diagram (C)

• Separate pair of cells form each other

• Develop into the muscle

• Autonomous specification: can separate it form other pairs but it will always develop into what it is fated to be

• Remove b4.1 it will not develop a tail

Autonomous specification

what is the significance of Whittaker’s experiment?

Whittaker:

• Macho mRNA segregates asymmetrically in the cytoplasm to drive tail muscle development (B4.1)

• In situ hybridization put probes with paired gene and it will track said genes

• Black spot localizes to B4.1 therefore B4.1 expresses macho mRNA

  • Asymmetric cell division

• mRNA that makes muscle protein end up in a single pairing of the embryo

 

Control division

• Embryo divides normal and contain macho mRNA

Macho depleted

• Knock down macho: reduce the expression

  • Small development

Macho added to other blastomeres

• Over express macho:

  • Cell size is bigger

The localization or a crucial mRNA control the down stream and the development of the embryo

conditional specification:

Transplant in blastula

• Grey = dorsal

• Beige - ventral

(A) Take a chunk out of the back and transplant it to the stomach side of the blastula

• No diff. btwn he two tag poles

• They have adopted the same fate of the surrounding cells

(B) Glass needle to excise the back and the front of the embryo

• Still normal developed tag pole

what is the significance of this experiment?

• The fates of the cells in this area were not fixed and able to compensate

• Cellular communication btwn neighbouring cells

conditional specification:

Driesch's:

• Separate the four cell from each other and they develop on its own

• They still fully develop except smaller

what can we conclude from this?

Each of these cells is not restricted to becoming only part of the embryo

• the potential for a blastomere to adopt any cell fate is greater than its expected fate during normal development

• cell-cell interactions are critical for normal development

syncytial specification:

Larvae: at this stage of development there are red nuclei

• Single egg with many different. nuclei on their own

If nuclei can divide how can they adopt different fates that will define different parts of the embryo?

what are autonomous and conditional specification factors?

• If nuclei can divide how can they adopt different fates that will define different parts of the embryo

  • Morphogen gradients

  • Anterior end we secrete a factor Bicoid with is anterior and drops in concentration as we move towards the posterior end

  • Caudal does the opposite

• The nuclei that are situated in the anterior will develop into the head, the brain, the mouth

• In the middle, digestive tract, the gut

• Position is important

• Morphogen gradient determine what the cells are fated to be

Autonomous: transcription factors differentially expressed after cellularization

Conditional: positive relative to neighbouring nuclei

what is single-cell RNA sequencing?

it is a mechanism used to map the fates of individual cells during development

Single cell RNA sequencing:

• Take embryos at diff development stages

• Chop them up to that the tissue is dissociated into ind. Cells

• Pass cell through microfluidic chamber where each cell is separated form each other to bee engulfed by oil droplets that have reagents to sequence all of the genes that are expressed in that cell

• Lipid droplets can release mRNA and attach nucleotides identifier called bar codes to the mRNA and as the sequences at amplified we can vein the bases that are incorporated into the strand that is synthesized form each mRNA and read off the base pairs to determine the sequences that are expressed in the cell

• We can then create plots from the data that group cell together based on similarities in gene expression

 

Spatial map:

• Clusters are cell that have similarity in gene expression

 

Developmental tree:

• Earliest cell fates and their divergence

how is differential gene expression accomplished during development?

transcription: epigenetics, transcription factors

pre-mRNA processing: alternative splicing

Translation: ribosomal selectivity, cytoplasmic localization of mRNA, miRNA and RNA interference

explain the anatomy of a gene

Gene b-globin - component in your hemoglobin that serves as a o2 carrier

• 5' to 3'

• We have regulatory elements that transcription factors and rna polymerase will bind to in order to transcribe the gene

• Transcription initiation cite: gene will start being transcribe

• Initiation codon: ATG

Yellow: exons code protein

Introns: junk DNA

• Exons splice together to forms coding sequence for protein

• Introns that get excised out

 

Transcription initiation cite: controls the precocity of translation, regulate binding of ribosomes

• 5' cap and 3' poly a tail

• 5'cap: protective cap from enzyme = nucleases

• 3' poly a tail: nuclear export signal so that mRNA can come out and enter the cytoplasm

mRNA has caps and exons that have been spliced, introns are gone

• Translate this

 

Start at ATG start site and ending at the terminator codon TAA

 

mRNA are transcribed form DNA form exons that are alternatively splice together, they contain untranslated regions at 5' end to the 3' end that are normally  used as protection, peptide that translation undergoes modification

 

An enhancer sequence: help to recruit rna polymerase promoted and loop around of form a structure so rna polymerase can be transported

 

Repressor sequences: doesn’t allow transcription to occur, do not facility the expression of down stream genes

how do enhancers and silencers modulate gene transcription?

enhancers bind transcription factors to induce tissue-specific gene expression

An embryo that is experiencing a gene in two diff regions:

Target genes A;

Purple brain enhancer sequence

• There are transcription factors that are expressed exclusively in the brain that can bind to the enhancer sequence recruit rna polymerase and allow the down stream genes to be expressed and achieve transcriptional control

  • Only gene that are specific to brain cells are being expressed by virtue of the presence of brain specific transcription factors

Green limb enhancer sequence

• Diff set of transcription factors that activate the same genes in a very different area of the body 

 

Only genes that are specific to brain cell are being expressed by brain specify transcription factors

Same thing for the limbs

 

This is an example of how the differential expression of transcription factors in diff regions of the body can activate the expression of the same gene in term they have diff functions in those diff regions of the body

how can we identify enhancers and silencers?

GFP reporter: zebrafish that has glowing eyes

• What type of gene was GFP fused to?

  • Eye gene, protein that is expressed in the cellular cell type of the retinal (tissue of the back of the eye which contain photoreceptors)

  • Photoreceptors: cones in colour and rods black and white

  • Cone carry visual  pigment proteins called opsin

  • Fused GFP to an opsin protein which is why we can see the GFP in the photoreceptor of the retina

  • In this case the GFP in indicating the presence of an enhancer

    • In the enhancer there are transcription factor that upregulate to expression of opsin genes

  • To indicate presence of an enhancer that specific for the photoreceptors of the eye in a zebra fish

• Lac z: beta galactosidase,

  • Substrate is lactose which is glucose and galactose

  • Turn blue when cleaved

  • Wherever there is more blue = more beta galactosidase = more expression of the gene that encodes beta galactosidase

 

NRSE: colour change in embryo

• When remove NSRE = more blue staining

• Role of NSRE is a repressor, restrict the expression on the L1 to the CNS

how do epigenetic modifications modulate access to genes?

what stage of development does this occur at?

Epigenetic modifications affect the structure of dna but not the sequence of dna

 

Epigenetic regulation by histone modification:

Adjust winding or the tightness by modifying the tail of the histones

• Methylate the tails = tighter

  • Gene expression goes down

• Acetylate tails of histones = loose

  • More gene expression

 

MeCP2: binds to methyl groups that are attached to CG base pairs in DNA

• removes acetyl groups from histone tails and add methyl groups 

• transcriptional repressor

• More we remove acetyl and more we add methyl groups the less gene expression we will have

how do transcription factors regulate gene transcription?

3D model of MITF (helix-loop-helix)

• Transcription factor that has three diff domains

  • Binds t specific enhancer sequence: DNA binding domain

  • Allow to bind to other protein, coregulator to help it function

  • C - terminal: that will bind to rna polymerase to help it enhance its transcriptional activity or to histone modifiers to modulate transcription

Change binding domain: will not function bc it cannot bind DNA

how are transcription factors categorized

Transcription factor are put into certain families based on the similarities in their DNA binding domains

what are Yamanaka factors and how can we use them?

Yamanaka transcription factors

• Transcription factors, protein that enable gene expression in order to promote differentiation from a pluripotent cell to a specified cell

• Transcription factors can be used to drive the process backwards

• Transform skin cell from a healthy or diseased pt and express in them four diff transcription factors using viruses

  • Can transform a skin cell back into a pluripotent stem cell and from their express your favourite transcription factors to turn those stem cell into anything you want

• Allows us to retain genotype from a pt that has a disease and study the phenotype of that disease in diff cell types

• Allows use to engineer disease relevant mutations

Ex. Can take cell from a healthy person, dedifferentiate them (erase their fate) and turn them into stem cells and induce neurons form them

1. Make cell type from disease and study phenotype

2. Healthy engineer disease and study the phenotype

how does alternative splicing differentia gene expression?

what stage of development does it occur during?

Dscam in the fruit fly: 115 exons

• Within each exons diff variants can be spliced together

• Function as an adhesion protein

(B) GFP expressing neurons and the processes coming out, parallel to each other

Knock out: remove Dscam:

• Allow neurite to cluster together

• Cant keep neurite separate from each other

 

The same gene encode multiple spliced isoforms that then vary the extent of neurite outgrowth and clumping in a given tract of the drosophila

how do ribosomal selectivity generate differential gene expression?

what stage of development does it occur during?

Rpl1 in mice: synthesize transcription factor gene = hox

• Ribosomes bind to hox to form the rib cage

• When protein in mutant it inhibits the affinity of ribosomes and gets additional malformed vertebrate

Rpl38: protein in ribosomal large subunit; expressed in somite that generate vertebrae

Rpl38 deficiency: cannot translate subset of Hox genes to specify vertebrae → deformed skeleton

how does cytoplasmic localization of mRNA generate differential gene expression in drosophila eggs?

mRNA:

• Nanos mRNA

• Drive the formation of features that are at the posterior end

 

Heat shock protein:

• Present everywhere but is being degraded through out the cytosol

• Remain intact at the posterior pole

 

Kinesin and diamine 

• Walk along microtubules

Microtubules:( -) nucleus and (+) farther away

• Kinesin walks form (+) end to (-) end, transporting cargo from the nucleus away

• Diamine is a  minus end directed motor, transporting cargo to the nucleus

• Always be transport from the nucleus away

• Demines go towards the nucleus

 

Cargo = Bicoid and Oscar mRNA

• Bicoid is attached to diamine, going to nucleus

• Oscar attached to kinesin, going away from the nucleus

• This way we achieve polarity in th way diff mRNA are being segregated inside the cell

• A lot of Oscar accumulating at the posterior end and a lot of Bicoid accumulating near the nucleus at the anterior end

how do microRNAs specifically regulate mRNA transcription and translation?

Lin-14: transcription factor required during first larval phase; no needed afterward

binding of small RNA’s (microRNAs) to repetitive sequence in 3’UTR of lin-14 mRNA triggers degradation of transcripts

miRNA structure: “hairpin loop” structures trigger protective “RNA interference” mechanism to inhibit transcription and translation of gene

hoe does RNA interference inhibit gene expression?

Drosha: makes individual pre-miRNA hairpins

RNAi:

• Double stranded rna that will enter an enzyme complex

• That will cut it up, unwind it and release one of the two strands

• Allow the single rna to base pair w/ the target

• The target will be cleaved and can no longer be expressed

• Depend heavily on how to design the double-stranded rna

  • If you don’t check the sequence and it binds to 10 million targets it will knock out 10 million genes

  • If you don’t design it properly and it doesn’t bind, it provides no knock out

RISC complex: separates dsDNA strand and aligns with 3’UTR of target mRNA

cleaves mRNA or blocks translation

recognition of target sequence depends on the stregnth of miRNA complementarity

how does in situ hybridization work and when would we use it?

procedure: Drosophila

Gene of interest expression in a patter: present in little stripes

• Single-stranded mRNA probe that binds to the gene of interest

• Wherever the probe binds the antibody will be able to localize the probe and turn the substrate from colourless to blue

1. Binding of the probe that is specific to the genes

2. Biding of antibodies to detect the epidotes that are connected to the probe

3. Colour change of the substrates to detect where the gene is expressed

application: to determine when are where a gene is expressed in an embryo

characterize gene expression

how does ChIP-Seq work and when would we use it?

Procedure:

• String of chromatin w/ a DNA sequence and transcription factor of interest

• Chop up DNA and observe the DNA protein complexes

• Antibodies that bind to transcription factor of interest

• Can precipitate that antibody down

• Analyse DNA that is pulled down by that pairing

• Remove transcription factor and study DNA

• Where in the genome where that transcription factor bind

Isolate chromatin

cross-link proteins (nucleosome or transcription factors) to DNA

bind proteins with specific antibodies

precipitate antibodies out of the solution with magnetic beads

separate protein from DNA and sequence DNA

Map sequences to the genome

Application: to determine where the transcription factors bind along a DNA sequence

• what gene a transcription factor regulate and want to know what DNA it binds to

  characterize gene expression

what is RNA-seq and when would we use it?

Procedure:

• Genes that are expressed in the eye of a chick embryo at three diff developmental stages

• Cut out the eye

• Prepare mRNA, which is a proxy or an indicator of which genes are being expressed 

• PCR rxn

• Read base pairs

• Match to database

• Tell which genes are being expressed

Application: determine how transcriptome differ in the same tissue at different stages

comparing gene expression at different stages of development

deep sequencing: gene expression in large tissue

characterize gene expression

what are the advantages and disadvantaged of rna sequencing at single cell vs bulk sequencing?

Advantage:

Single-cell: high resolution (can tell the full complement of genes of one cell at a time), pull components in one gene at a time

Deep sequencing: allows to analysis of the dev of an entire organ at one time,

what is CRISPR/ Cas9 and when would we use it?

procedure:

Guide rna

• Guide the cas9 toward the gene of interest

 

Cas9 is a nucleus which is able to cut DNA up

• w/ the guide rna it can be direct to the gene of interest  where it makes a double strand break and cuts through DNA of gene of interest

 

Trigger 2 diff repair mechanisms in the cell:

Non homologous end joining

• The cell will try to patch up the gap by inserting fresh nucleotides or depleting ones that are in the break

• Back fires on the cell, induce a mutation called frame shift mutation

  • The entire organ reading frame of a protein gets shifted and translation terminates too early

• Translation terminates too early

Provide the cell w/ a repair template

• Insert the gene of interest into the genome

• Will try to repair with the gene of interest that we have inserted

application: test gene function, repair mutations

1. What to understand the function of a gene, knock it out and see what happens to the cells

2. Fix a mutation, if the central gene is deleted you can put it right back in

how can we use CRIS/ Cas9 to repair mutations?

Use to knock our a cell but also introduce a new gene of interest into the genome of a cell

 

Why would we want to introduce a new gene into the cell?

• Study a gene of interest better

• Used to repair something faulty

• Can introduce wild-type gene into a diseased pt and resolve the outcome of the disease

what is GAL4 and when would we use it?

procedure:

Used in fruit flies

1. One construct encoded transcription factor = gal 4

   1. We place it down stream of an enhancer sequence that correspond to the tissue we want to express

2. Second construct that express a gene of interest; Pax 6

   1. Eye development

   2. Driven by the binding of the trans factor, Gal 4

 

Gal4 under brain pax 6 in the brain

 

Pax 6 can only be expressed by gal4

 

Gal 4 will only exist wherever you express it

 

Gal4 in jaw and pax6 for eyes

• Two normal eyes and two eye in the jaw bc of gal 4

application: activate or repress regulatory genes in specific tissues

what is cre-lox and when would we use it?

procedure:

Used in mammalian system

Two-component system - 2 targeting constructs

1. Enhancer-expressed downstream protein - cre recombinase in a tissue of interest

   1. Ex. If I want to express cre-recombinase in the liver I put it in the control of a livre specific enhancer 

   2. Enzyme that can catalase the grouping of two sequences and the corresponding excision of the DNA

   3. Cre recombinase pairs two loxP together and loops the exons around and exon 2 gets excised

2. Cre recombinase can knock out a gene

application: conditionally eliminate gene expression in specific cell types

what are the two ways cell-to-cell communication can ocur?

juxtacrine and paracrine

what is juxtacrine signalling?

Juxtracrine signaling - direct contact

• Mediated by diff. membrane bound receptors that are expressed by the doner cell and the recipient cell

• Sometime it is two of the same receptor that interact w/ each other = homophilic binding

• Sometimes it is two diff receptors = heterophilic binding

• Single receptor in the cell that interacts w/ other protein components that are secreted just outside

  • That area is called the ECM, extracellular matrix

    • Provides both structure and means of adhesion for diff cell types

    • Has a number of signaling properties

• Cadherin biding, homophilic binding interact (yellow)

• Notch pathway, heterophilic binding interaction

  •  two membrane bound receptors: notch and delta

  • Induce a protease cascade to induce gene transcription down the line

what is paracrine signalling?

Paracrine signalling, cell can be some distance away

• Cell that secrete a signal and a cell that secretes a signal

• Usually some form of receptor activation by means of dimerization

  • Receptor come in two halves (monomers) but then come together and then activate a phosphorylation cascade that then activate and repress gene expression

• Sometime if the signaling cascade simply function to rearrange proteins that have already been  translated and are floating around the cell, they act to recruit more of the protein towards the plasma membrane in order to induce some sort of developmental affect, this is a rapid response bc we do not need to go through gene expression form scratch

• Any change that involves an increase or a decrease in gene expression is going to take much longer

Morphogen secreted factors that express diff membrane receptors

• Secreted factors coming from a single source, a number of cell arranged at diff distances from the source, that express possible diff membrane receptors that react differently to higher or lower threshold of a morphogen

how are cell types able to self organize?

No random mass of cell

The cell types are able to self organize, they find each other in space

• The epidermal cells (green) form the periphery of the new cell mass

• Neural tissue (blue) is concentrated on the inside of the mass

• Helps spontaneously

• Consequence of diff in surface tension of the two cell type

• More tightly bound the cells are to each other, more they are likely to be positioned at the core of the mass

• Less surface tension = more likely to be found at the periphery of the mass

 

what mediates the difference in cell adhesion in the surface tensions during organization?

• Cadherins

  • Adhesions molecules that bind calcium

  • Calcium must be present in order to for cadherin to work

  • Cadherins work by binding together one expressed in recipient and one in doner

  • Intracellular domains help assemble the cytoskeleton

  • Induce signal that induces gene expression down the line

  • More cadherins are expressed the more surface tension we have

what are the components of the ECM

ECM components:

proteoglycan complex

• Stabilize ligan receptors interactions

• Ligan and receptors bind to certain affinities it is possible that a weak affinity interaction can be strengthened by the presence of heparin and sulfate groups

Integrins:

• Imbedded in plasma membrane connect thee external extracellular matrix components to the  cytoskeleton

• They have signaling roles

• Can function in assembly of actin polymerization complexes that will allow the cytoskeleton to assemble and grow out in certain directions

• Control intercellular level on calcium

EMT: three cells that look identical, epithelial

• Fixed

• Two forms of adhesion junctions

  • Integrin and adherens

  • It is possible for one of these cells to receive a paracrine signal, some other cell that secretes a factor that will then down regulate the expression of the adherens proteins in the adhesion proteins and fewer integrin being expressed that is dissolves the connection btwn the skin cell and the floor plate

  • Dissolve the connection

    • Cutting the cell lose

    • And able to squeeze through the gap and take on diff. structure

Development of CNS: neural cress cell

• Cell that cut free and differentiate into diff parts of the CNS other than neurons

what is induction?

one cell population influences the development and behabior of neighbouring cells

what is competence?

the ability of cells to respond to an inductive signal

what is instructive interaction?

signal is necessary to initiate new gene expression in the responding cell to specify its fate

what is permissive interaction?

responding cell has already been specified and needs only an environment that allows expression of these traits

Xenopus:

Look at how the lens of the eye is fused by two diff tissue types

• Periphery (light blue), ectodermal tissue

• Inside (darker blue), optic vesical

• Normal epidermic tissue has changed its shape (caved in)

  1. Optic vesicle is inducing the ectodermal tissue to become the lens of the eye 

• Ectodermal tissue overline the tissue of the optic vesical, no lens is being produced

  1. In region one it is situated in the head

  2. In region two the same collection of cell is present in the trunk

  3. The position of these two cell layers matters and there are perhaps more inducing factors in the head which induce the differentiation of the lens in the head

• Cut out the optic vesicle

  1. Ectoderm stays normal

  2. We've removed the optic vesicle

  3. The optic vesicle is responsible for sending the majority of the inducing signals that turn the ectoderm into the lens

• Taken a piece of tissue that is completely irrelevant to lens formation

  1. The ectoderm has not changed, normal

Negative control: random tissue will not induce a lens

• Induction of the lens depend on optic tissue in the right part of the embryo, only in the head and not in the trunk

Is this an example of instructive or permissive interaction?

instructive: Induction of the lens depend on optic tissue in the right part of the embryo, only in the head and not in the trunk

Rat Heart:

• Open a rat and excised the heart

• Took all the cell away by using a detergent call SDS

• The heart becomes more transparent, left with just the ECM

• Inject cardio myocytes, type of stem cell that are capable of differentiating into all the different cells of the heart 

• The cardio myocytes know how to reconstruct the whole heart

Is this an example of instructive or permissive interaction

This is a permissive interaction because the cardiomyocytes know they are going to become heart cells but they need to be in those conditions in order to do so

what are morphogens?

morphogens are paracrine signalling molecules that determine cell fate by regulating gene expression differently at different concentrations

morphogen: a diffusible molecule that can determine cell fate by its concentration

Xenopus:

• Express a secreted protein called activin in a gradient

(A) a cluster of unspecified cells at the center there are beads serving as a negative control

(B) coat them in a low [activin] and observe gene expression of the cells that are close to the beads and those that are far away

• The cells that re closest to the bead express a gene called xbra

(C) increase the coated of activin on the beads

• And assess genes expression to the beads that are closest and further away

• Cell that are closest to the bead are expressing a new gene called Goosecoid

 

Cross section:

• Goosecoid = dependent on high [activin]

• As we lose the activin gradient we loose Goosecoid and are left with xbra and then nothing at all

Why would we use this technique?

it is a way of setting up an artificial morphogen gradient and determining how cells are influenced, how their gene patterns are influenced by their proximity to those beads

What is FGF?

Receptors that are rich in the amino acid tyrosine

Receptors are phosphorylated

• Why tyrosine targeted?

  • Functional group is OH

  • This is how the phosphate group is added

  • Only certain amino acid that can be phosphorylated

• When  the ligan come it forces the receptors to dimerize with each other w/ some kind of phosphorylation mechanism

• The phosphorylated internal residues  in the cytoplasm can than activate down stream proteins like ras and raf

• Eventually they will the potential transcription factors to activate gene transcription inside the nucleus

what happen if there is a mutations along the FGF process?

FGF doesn't solely act through the RTK signalling pathway:

• FGF activates the JAK-STAT signaling pathway

• Phosphorylation-dependent cascades can in turn activate different genes

 

FgfR3 mutation that makes it constitutively active

• The pathway is always on

• This happens in the context of cartilage growth

• A mutation that can be inherited where FGF is constantly on, phosphorylate a downstream effector called STAT

  •  Activates an inhibitor of cell division in the cells that form cartilage and bones

  • Can't grow and when FGF pathways is constantly on

    • Limb size of embryo will be smaller and stubbier

    • Lethal mutation

How is FGF constitutively on

A mutation in the gene for FgfR3 causes the premature constitutive activation of the STAT pathway and the production of phosphorylated Stat1 protein. This transcription factor activates genes that cause the premature termination of chondrocyte cell division in the growth plates. The result is thanatophoric dysplasia, a condition of failed bone growth that results in the death of the newborn infant because the thoracic cage cannot expand to allow breathing.

what is hedgehog processing and secretion?

Hedgehog processing and secretion: also control limb regulation, not only FGF. Demonstrates functional redundancy

 

Ligand hedgehog:

• Translated in the cell

• That it goes through several post translational modification

  1. It is cleaved, normally an n and c terminus, but it gets cleaved and it is rly only the n terminus that receives maximum signaling activity (the c terminus is biological quite compared to the n)

  2. N terminus has active signaling is also modified

     • Modified by a palmitic and cholesterol residue

     • It is the interaction of the cholesterol residue w/ a membrane receptor called dispatched that allows HH to be secreted from the cell

     • HH requires both residues in order to for the secondary structures

       • They exist a secreted monomer but thanks to  the moodiest they can aggregate

       • What is the affect of the aggregation on the biological activity of HH?

         • When the HH monomers are more stable their biological activity will last longer and over longer distances

what is the hedgehog signal transduction pathway?

Hedgehog signal transduction:

(A) no hedgehog

• Has a classical receptor call patched

  • Negatively regulates smoothened by putting it in a vesicle and preventing it from doing anything else, degraded and inactive

  • Downstream of that we have a transcription factor called CI which stays complexed and tethered to microtubules where it cant do much transcription factor that is active in the nucleus but once it is tether it is inactive

  • It can be cleaved by a protease called slim

    • CI can enter the nucleus where it act as a repressor

(B) hedgehog is present

• Hedgehog and patched are internalised by the cell and smoothened can escape and be targeted and phosphorylated

  • In it phosphorylated form is relives the flammation of the complex that is normally (Kirby complex) captures and inhibits transcription factor CI

  • CI is cut lose from the complex and can enter the nucleus and activate gene expression

What happens in mother-ingested teratogen, shutting

down the hedge hog pathway?

• Supressed differentiation, only partial development on the NS

• Cyclopia - one eye

what is wnt

Wnt can self regulate itself

• Negative feedback loops are able to regulate the amount of a ligand that is good for some processes but in excess can be bound

• Can feed back on its self

  • When it is secreted it can bind to two receptors called LRP5 and frizzled  which initiates the transcription of a factor called notum

    • Notum is a protease meaning that it can cleave proteins and has an affinity for lipids  (phospholipid moieties that happen to be on wnt)

    • Notum can take the phospholipid from wnt and make it incapable of binding to LRP5 and frizzled

    • Wnt requires the phospholipid moieties in order to divide and activate

    • This is a way of silencing wnt w/o suppressing the expression of wnt itself

      • Remove a critical feature from it and it cant function ( too much wnt with generate notum to silence wnt (negative feedback loop)

what are the two main pathways that wnt uses to function?

Canonical:

• Converges on a transcription factor called ß-catenin (pink)

• When no wnt is presence we have no downstream activation of is receptors LRP5 and frizzled

  • ß-catenin is sequestered in a complex which targets it for degradation

  • Get shuttled over to the proteasome  that cuts up ß-catenin and stop it form having any transcriptional activity

• When wnt is present

  • Favours the recruitment of the other complex proteins away from ß-catenin

  • Enters the cytosol and activates gene expression

Non-canonical:

• Involves wnt and same receptors but down stream effector are different

• Rho-GTPases that affect the development of the cytoskeleton and the release of calcium in an intercellular source like the endoplasm reticulum

  • Ca functions as a second messenger which can in turn regulate many down stream targets

what are TGF-ß factors?

Smad - phosphorylatable  proteins that also have transcriptional activity

• Many factors that are part of the TGF-ß family

• Have the dimerization of two diff receptor domains that cross for phosphorylation of either tyrosine or cytosine residues

• The phosphorylation of a Smad protein

  • Can also phosphorylate other isoforms of Smad down the line and can either activate or repress gene transcription

what is the notch signalling pathway?

When they interact w/ each other it promotes the cleavage of the cytosol domain of notch by the protease inside the cell and that fragment of notch has transcriptional activity which can displace a repressor and can activate a transcriptional complex to express that target gene

asymmetric division vs symmetric division vs adult stem cells

asymmetric division: 1 self-renewing stem cell and 1 daughter cell committed to differentiation

symmetric division: 2 self-renewing stem cells or 2 daughter cells committed to differentiation

adult stem cells: have limited potency and are committed to differentiation into specific cell types

• Brain stem cells

• limited potency

• can only be another neural cell