STUDYING GENE EXPRESSION IN THE EMBRYO Studying gene

STUDYING GENE EXPRESSION IN THE EMBRYO Studying gene expression Probe for m. RNA ‘Northern’ hybridisation ‘in situ’ or in microarray Probe for protein ‘Western’ immunohistochemistry ‘Reporter gene’ Lac Z: b-galactosidase GFP: green fluorescent protein Most methods can be adapted to either tissue sections or whole mounts

HYBRIDISATION - MAKING THE PROBE (a) The normal process of transcription m. RNA made by RNA polymerase 2 Base sequence equals coding strand except T > U Hybridises with anti-sense RNA or c. DNA Promoter and enhancer regions Coding strand 5’ 3’ Template strand

HYBRIDISATION - MAKING THE PROBE (b) Production of anti-sense RNA Anti-sense RNA made by viral polymerase acting on cloned DNA Base sequence equals template strand except T > U Hybridises with m. RNA Viral Promoter Coding strand 5’ 3’ 3’ Template strand 5’

HYBRIDISATION – MAKING THE PROBE (c) Production of c. DNA made by reverse transcriptase of m. RNA Base sequence equals template strand Hybridises with m. RNA 5’ m. RNA 3’ AAAA TTTT 3’ c. DNA 5’ • m. RNA molecules have poly A tail • reverse transcriptase can elongate an oligo d. T primer

HYBRIDISATION – VISUALISING THE LOCATION NATURE OF PROBE METHOD OF DETECTION Radiolabelled Autoradiography Fluorescently labelled Fluorescence microscopy Digoxigenin labelled (Digoxigenin coupled to UTP) Anti-digoxigenin antibody coupled to 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 section or permeabilised whole mount Probe for expressed m. RNA using antisense RNA or c. DNA Visualise location by autoradiography, fluorescence or enzyme-linked antibody

MICROARRAY Total cellular messenger 5’ AAAA 3’ Reverse transcriptase 5’ F 3’ AAAA TTTT 3’ 5’ c. DNAs Degrade RNA TTTT 5’ F c. DNAs = template strands of DNA with fluorescent label (F) 3’ 1 3 3’ 3 4 Using two probes allows comparison between tissues RED Tissue 1 4 2 T Fluorescent detection shows genes 2 and 3 are expressed in this tissue 2 Fix probe oligonucleotides representing portions of coding strands of known genes. 5’ Hybridise 1 T T T GREEN Tissue 2 YELLOW Both BLACK Neither

IMMUNOHISTOCHEMISTRY Fixed tissue section or permeabilised whole mount Probe for expressed protein using primary antibody Visualise location using second antibody, coupled to enzyme, fluorophore or gold

USE OF A REPORTER GENE Engineer construct composed of regulatory sequence of interest and green fluorescent protein (GFP)gene Inject into zygote Study expression of GFP at different stages of development b-galactosidase Lac. Z (E. coli) can also be used. An artificial substrate when cleaved by Lac. Z gives a coloured insoluble product

SUMMARY OF VISUALISATION METHODS ‘Northern’ = hybridisation to m. RNA l 1 l 2 F F Fluorescence m. RNA Ag+ ‘Western’ = Binding of antibodies to proteins Ag Au R Autoradiography/ Immune gold m. RNA E D m. RNA S P E Enzyme coupled to antibody S P

FOLLOWING GENE EXPRESSION – Pax 6 AS AN EXAMPLE What is pax 6? Pax 6 encodes a transcription factor required for normal eye, nervous and pancreatic development. It binds to enhancer elements of Pax 6 regulated genes such as lens crystallin and those genes specifying a and b cells in the pancreas Mutants in Pax 6 cause severe abnormalities (Gilbert Fig 6. 2 A shows section through developing mouse brain in the region of the optic cup; from Fujiwara et al. , 1994) Normal Pax 6 mutant

IN-SITU HYBRIDISATION – TISSUE SECTIONS 9. 5 d 8. 5 d 10. 5 d LP NF OV 250 mm HB 100 mm 250 mm 15. 5 d CN 9. 25 d FB HB L RE OS 250 mm LP – Lens plate OV – Optic vesicle OS – Optic stalk CN – Cornea L – Lens RE – Retinal epithelium Normal OS 250 mm HB –Hindbrain FB – Fore Brain NF – Neural fold OS – Optic stalk Pax 6 m. RNA detected by hybridisation with radioactive antisense c. RNA (mouse) (Gilbert Fig. 4. 17, from Grindley et al. 1995)

IN-SITU HYBRIDISATION – WHOLE MOUNT Mouse embryo, 10. 5 d Chick embryo (35 h) Pax 6 m. RNA 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)

REPORTING pax 6 REGULATORY SEQUENCES USING lac. Z A B C D Pax 6 Upstream enhancers of the mouse Pax 6 gene A Pancreas B Lens and cornea C Neural tube D Retina Fusion of the lens and cornea regulatory sequence of pax 6 with the lac. Z gene (mouse, 10. 5 d) (Gilbert Fig 5. 7, from Williams et al 1998)

DETECTING Pax 6 PROTEIN EXPRESSION Forebrain 200 mm Mouse (9. 25 d) Primary antibody: Rabbit polyclonal antiserum raised against Pax 6 Secondary antibody: Goat anti-rabbit antiserum with antibodies attached to horse radish peroxidase which can produce a coloured product (from Mastick et al 1997)

STUDYING THE FUNCTION OF GENES IN THE EMBRYO Notice that just showing where and when a gene is expressed does not give a definitive answer to its function Studying gene function Gene knock-out Can be constitutive or conditional Antisense RNA Blocks initiation of translation RNA interference ds. RNA homologues of m. RNA promote degradation of messenger

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

KNOCK-OUT OF THE BMP 7 GENE A Normal B BMP 7 knock-out (homozygote) Normal BMP 7 knock-out (homozygote) Mouse embryos at day 17 of 21 day gestation Conclusion: BMP 7 is involved in eye development and in kidney development (Gilbert Fig. 4. 20)

CONDITIONAL GENE KNOCK-OUT Constitutive knock-out can be problematic in studying later effects of a knock-out if its early effects are lethal Inducer 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 Target gene Excision Enzyme gene

PROTEIN KNOCK-OUT PREVENTION OF TRANSLATION OF MESSENGER Anti-sense RNA blocks initiation of translation of the sense messenger Viral Promoter Coding strand 5’ 3’ 3’ Template strand 5’

PROTEIN KNOCK-OUT - DESTRUCTION OF MESSENGER ds. RNA for protein of interest is injected into a cell and is cleaved into small fragments RISC Antisense component of fragment associates with complementary sense sequence of m. RNA using a protein called RISC Antisense RNA fragment promotes cleavage of the cellular messenger

A 4 day mouse blastulas Red fluorescent antibody binds to E-cadherin PROTEIN KNOCK-OUT USE OF RNAi Very little antibody reaction in B Blastomeres in B have failed to undergo compaction (bumpy appearance) (Gilbert Fig. 4. 23) B 4 day mouse blastulas (zygotes were injected with ds. RNA for E-cadherin). This time red fluorescent antibody shows almost no reaction