STUDYING GENE EXPRESSION IN THE EMBRYO

Studying geneexpression

Most methods can be adapted to either tissue sections or whole mounts

Probe for protein ‘Western’ immunohistochemistry

Probe for mRNA ‘Northern’ hybridisation ‘in situ’ or in microarray

‘Reporter gene’ Lac Z: -galactosidaseGFP: green fluorescent protein

HYBRIDISATION - MAKING THE PROBE

(a) The normal process of transcription

mRNA made by RNA polymerase 2Base sequence equals coding strand except T > UHybridises with anti-sense RNA or cDNA

Promoter and enhancer regions Coding strand

Template strand

3’

5’5’ 3’

HYBRIDISATION - MAKING THE PROBE

(b) Production of anti-sense RNA

Anti-sense RNA made by viral polymerase acting on cloned DNABase sequence equals template strand except T > UHybridises with mRNA

Viral Promoter

3’

5’ 5’3’

Coding strand

Template strand

HYBRIDISATION – MAKING THE PROBE

(c) Production of cDNA

3’5’AAAA

cDNA made by reverse transcriptase of mRNABase sequence equals template strandHybridises with mRNA

mRNA

T T T T5’3’ cDNA

• mRNA molecules have poly A tail• reverse transcriptase can elongate an oligo dT primer

HYBRIDISATION – VISUALISING THE LOCATION

NATURE OF PROBE METHOD OF DETECTION

Radiolabelled Autoradiography

Fluorescently labelled Fluorescence microscopy

Digoxigenin labelled Anti-digoxigenin antibody coupled to(Digoxigenin coupled to UTP) alkaline phosphatase

• Can be done on fixed tissue sections or whole mounts• Except radioactivity which is not suitable for whole mounts• Whole mounts require permeabilisation to allow reagent access• Digoxigenin is a plant steroid which is antigenic allowing raising of antibodies

IN-SITU HYBRIDISATION – SUMMARY

Fixed tissue sectionor permeabilised whole mount

Probe for expressedmRNA using antisenseRNA or cDNA

Visualise locationby autoradiography,fluorescence or enzyme-linked antibody

5’ 3’

5’ 3’

1 2

3 4

Fix probe oligonucleotides representing portions of coding strands of known genes.

T T T T5’3’ cDNAs

AAAATotal cellular messenger

3’5’AAAA

Reverse transcriptase

cDNAs = template strands of DNAwith fluorescent label (F)

Degrade RNA

T5’3’

T T T

F

T

TT

TF

Hybridise

1 2

3 4

Fluorescent detection shows genes 2and 3 are expressed in this tissue

MICROARRAY

Using two probes allows comparison between tissues

RED Tissue 1

GREEN Tissue 2

YELLOW Both

BLACK Neither

IMMUNOHISTOCHEMISTRY

Fixed tissue sectionor permeabilised whole mount

Probe for expressedprotein using primaryantibody

Visualise locationusing second antibody,coupled to enzyme,fluorophore or gold

USE OF A REPORTER GENE

Engineer construct composed of regulatorysequence of interest and green fluorescent protein (GFP)gene

Inject into zygoteStudy expression ofGFP at different stagesof development

-galactosidase LacZ (E.coli) can also be used. An artificial substrate when cleaved by LacZ gives a coloured insoluble product

SUMMARY OF VISUALISATION METHODS

‘Northern’ = hybridisation to mRNA ‘Western’ = Binding of antibodies to proteins

mRNA

F1 2 F

Fluorescence

mRNA

R

Ag+ AgAu

Autoradiography/Immune gold

mRNA

ES

PD

SE

PEnzyme coupled to antibody

FOLLOWING GENE EXPRESSION – Pax6 AS AN EXAMPLE

What is pax6?Pax6 encodes a transcription factor required for normal eye, nervous and pancreatic development. It binds to enhancer elements of Pax6- regulated genes such as lens crystallin and those genes specifying and cells in the pancreas

Mutants in Pax6 cause severe abnormalities(Gilbert Fig 6.2A shows section through developing mouse brain in the region of the optic cup; from Fujiwara et al., 1994)

Normal Pax6 mutant

IN-SITU HYBRIDISATION – TISSUE SECTIONS

Pax6 mRNA detected by hybridisation with radioactive antisense cRNA (mouse)(Gilbert Fig. 4.17, from Grindley et al. 1995)

NF

HB100 m

Normal

FB

HB

OS 250 m

HB –HindbrainFB – Fore BrainNF – Neural foldOS – Optic stalk

8.5d

9.25d

250 m 250 m

250 m

LP

CN

RE

OV

OS

L

LP – Lens plateOV – Optic vesicleOS – Optic stalkCN – CorneaL – LensRE – Retinal epithelium

9.5d 10.5d

15.5d

IN-SITU HYBRIDISATION – WHOLE MOUNT

Mouse embryo, 10.5d

Pax6 mRNA detected by hybridisation with digoxigenin labelled antisense RNA followed by alkaline phosphatase-coupled antibody against digoxigenin(Gilbert Fig. 4.16, A from Li et al 1994, B from Gray et al 2004)

Chick embryo (35h)

REPORTING pax6 REGULATORY SEQUENCES USING lacZ

Fusion of the lens and cornea regulatory sequence of pax6 with the lacZ gene (mouse, 10.5d) (Gilbert Fig 5.7, from Williams et al 1998)

A B C D Pax6

Upstream enhancers of the mouse Pax6 gene

A PancreasB Lens and corneaC Neural tubeD Retina

DETECTING Pax6 PROTEIN EXPRESSION

Mouse (9.25d)

Primary antibody:Rabbit polyclonal antiserum raised against Pax6

Secondary antibody:Goat anti-rabbit antiserum with antibodies attached to horse radish peroxidase which can produce a coloured product(from Mastick et al 1997)

200 m

Forebrain

STUDYING THE FUNCTION OF GENES IN THE EMBRYO

Studying genefunction

Antisense RNA Blocks initiation of translation

Gene knock-out Can be constitutive or conditional

RNA interference dsRNA homologues of mRNA promote degradation of messenger

Notice that just showing where and when a gene is expressed does not give a definitive answer to its function

GENE KNOCK-OUT

Instead of adding genes to embryonic stem cells (as with reporter genes), you can also replace a gene with a non-functional component.

Cloned gene cut at restriction sites and gene replaced by, for example, an antibiotic resistance gene to aid selection of modified clone

Insertion into embryonic stem cells and selection of heterozygotes

Injection of modified stem cells into blastula inner cell mass

Chimaeric embryos and offspring produced, some of which have modification in germ line

Breed chimaera with wild type to produce heterozygotes

Breed heterozygotes to produce homozygotes for the knock-out

A Normal

B BMP7 knock-out(homozygote) BMP7 knock-out

(homozygote)Normal

Mouse embryos at day 17 of 21 day gestation

Conclusion:BMP7 is involved in eye development and in kidney development(Gilbert Fig. 4.20)

KNOCK-OUT OF THE BMP7 GENE

CONDITIONAL GENE KNOCK-OUT

Constitutive knock-out can be problematic in studying later effects of a knock-out if its early effects are lethal

Instead of replacing normal gene with non-functional component, replace with normal gene flanked by special recognition sequences for an inducible excision enzyme called CRE

Generate homozygote embryos

Induce excision enzyme at chosen stage of development to generate conditional knock-out

Inducer

Excision Enzyme

gene

Targetgene

Anti-sense RNA blocks initiation of translation of the sense messenger

Viral Promoter

3’

5’ 5’3’

Coding strand

Template strand

PROTEIN KNOCK-OUTPREVENTION OF TRANSLATION OF MESSENGER

PROTEIN KNOCK-OUT - DESTRUCTION OF MESSENGER

Antisense RNA fragment promotes cleavage of the cellular messenger

dsRNA for protein of interest is injected into a cell and is cleaved into small fragments

Antisense component of fragment associates with complementary sense sequence of mRNA using a protein called RISC

RISC

4 day mouse blastulas

Red fluorescent antibody binds to E-cadherin

4 day mouse blastulas(zygotes were injected with dsRNA for E-cadherin). This time red fluorescent antibody shows almost no reaction

PROTEIN KNOCK-OUT USE OF RNAi

Very little antibody reaction in BBlastomeres in B have failed to undergo compaction (bumpy appearance)

(Gilbert Fig. 4.23)

A

B