1. STUDYING GENE EXPRESSION IN THE EMBRYO Studying gene expression 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:  -galactosidase GFP: green fluorescent protein

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

3. 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 mRNA Viral Promoter 3’ 5’ 3’ Coding strand Template strand

4. HYBRIDISATION – MAKING THE PROBE (c) Production of cDNA 3’ 5’ AAAA cDNA made by reverse transcriptase of mRNA Base sequence equals template strand Hybridises with mRNA mRNA TTTT 5’ 3’ cDNA mRNA molecules have poly A tail reverse transcriptase can elongate an oligo dT primer

5. HYBRIDISATION – VISUALISING THE LOCATION NATURE OF PROBEMETHOD OF DETECTION RadiolabelledAutoradiography Fluorescently labelledFluorescence microscopy Digoxigenin labelledAnti-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

6. IN-SITU HYBRIDISATION – SUMMARY Fixed tissue section or permeabilised whole mount Probe for expressed mRNA using antisense RNA or cDNA Visualise location by autoradiography, fluorescence or enzyme-linked antibody

7. 5’ 3’ 5’ 3’ 1 2 34 Fix probe oligonucleotides representing portions of coding strands of known genes. TTTT 5’ 3’ cDNAs AAAA Total cellular messenger 3’ 5’ AAAA Reverse transcriptase cDNAs = template strands of DNA with fluorescent label (F) Degrade RNA T 5’ 3’ TTT F T T T T F Hybridise 1 2 34 Fluorescent detection shows genes 2 and 3 are expressed in this tissue MICROARRAY Using two probes allows comparison between tissues RED Tissue 1 GREEN Tissue 2 YELLOW Both BLACK Neither

8. 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

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

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

11. 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

12. 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 HB 100  m Normal FB HB OS 250  m HB –Hindbrain FB – Fore Brain NF – Neural fold OS – Optic stalk 8.5d 9.25d 250  m LP CN RE OV OS L LP – Lens plate OV – Optic vesicle OS – Optic stalk CN – Cornea L – Lens RE – Retinal epithelium 9.5d 10.5d 15.5d

13. 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)

14. 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 Pancreas B Lens and cornea C Neural tube D Retina

15. 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

16. STUDYING THE FUNCTION OF GENES IN THE EMBRYO Studying gene function 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

17. 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

18. 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

19. 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 Target gene

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

21. 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

22. 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 RNA i Very little antibody reaction in B Blastomeres in B have failed to undergo compaction (bumpy appearance) (Gilbert Fig. 4.23) A B