Molecular tool box for studying gene function in neural development For Eye Development Course

Studying gene function in neural development Differential Gene Expression (The Paradigm of Developmental Biology) Differential gene expression from genetically identical nuclear DNA creates different cell types in different parts of the embryo. approx. 30 ’ 000 human genes, but … lens cells express crystallin protein differentiated neurons express N-tubulin protein oligodendrocytes express myelin-associated glycoprotein developing roof plate cells express BMP4 signal protein developing floor plate cells express Shh signal protein

Studying gene function in neural development Gene Expression (The Central Dogma of Molecular Biology) DNARNA PROTEIN transcription translation GENE GENE PRODUCTS GENE FUNCTION

I.Investigation of gene expression: gene A is expressed in the appropriate tissue and at the appropriate stage to mediate process B. II.Gain-of-function experiment: gene product of gene A is sufficient for mediating process B. III.Loss-of-function experiment: gene A or gene product of gene A is normally required for process B. IV.Analysing of experiment by investigating the resulting phenotype or marker gene expression. Studying gene function in neural development Experimental Design ( “ gene A functions in process B ” )

I.Methods for analysing gene expression (Protein, RNA, Reporter gene (DNA)) II.Methods for expressing gene products (ectopic expression, over-expression, expression rescue of Protein, RNA or DNA) III.Methods for inhibiting gene function (inhibiting Protein function, disrupting or inhibiting RNA, disrupting gene (DNA)) Studying gene function in neural development Methods tool box (IV. Analyse experiments with tissue-specific molecular markers.)

Antibodies: –gel-based methods ( “ biochemistry ” ): Western Blot, Proteomics –tissue-based methods ( “ in situ ” ): immuno-histochemistry (whole-mount, histology sections) Protein fusion constructs –artificial chimera protein: fuse endogenous protein coding sequence to (lacZ (gene) =  -gal (protein)) GFP etc. I. Methods for analysing gene expression Detecting protein expression/localisation

Western Blot Protein extraction Protein gelBlotFilm st. 0 st. 5 st. 10 st. 20st. 30 st. 50 Protein A linked to the enzyme Alkaline Phosphatase (AP) or HRP Protein A is expressed from stages 10 to 30 during Xenopus development (chemiluminescent)

Immunohistochemistry Protein A linked to the enzyme Alkaline Phosphatase (AP) or linked to fluorescent molecule coloured precipitate endogenous protein in fixed embryonic tissue

Gel-based methods ( “ Molecular Biology ” ) –Northern Blot (qualitative, detecting different gene products) –RNase Protection Assay (quantitative), antisense RNA probe forms dsRNA with target mRNA and protects it from RNaseH digest (RNaseH only digests ssRNA). –Microarrays ( Analysing the RNA expression of hundreds of genes ) –RTPCR (high sensitivity, qualitative, advanced methods are quantitative) Tissue-based methods ( “ in situ ” ) –RNA in situ hybridisation (whole-mount, histology sections) I. Methods for analysing gene expression Detecting RNA expression

Northern Blot RNA extraction RNA gelBlotFilm st. 0 st. 5 st. 10st. 20 st. 30 st. 50 Gene A is expressed maternally and zygotically from stages during Xenopus development zygotic RNA maternal RNA AAAAAAAAAAAA endogenous mRNA of gene A: radioactively labelled DNA probe Maternal and zygotic mRNA from gene A is different size

DNA gel Film st. 0 st. 5 st. 10st. 20 st. 30 st. 50 Gene A is expressed from stages 20 onward during Xenopus development AAAAAAAAAAAA reverse transcription TTTTTTTTT AAAAAAAAAAAA cDNA of gene A: RNA extraction PCR with radioactive nucleotides TTTTTTTTT endogenous mRNA of gene A: radioactive DNA PCR product: RT-PCR gene-specific PCR primers

RNA in situ hybridisation

cis-regulatory DNA elements drive expression of reporter protein. –Lac Z: very sensitive but only in fixed dead tissue –Green Fluorescent Protein (GFP): less sensitive but in live embryos mutated GFP: CFP (cyan), RFP (red), YFP (yellow) DsRed is a red fluorescent protein I. Methods for analysing gene expression Reporter gene DNA constructs ß-galactosidase enhancer prom. GFP enhancer prom.

II. Experimental expression of gene products Introducing extra gene product Introducing an extra gene (Transgenics) Fast Less reliable Slower, more difficult more reliable

Introducing extra protein? doesn ’ t usually work, not enough protein can be delivered, because there is no amplification step. Introducing extra RNA: usually works well, because one mRNA molecule produces many proteins. Good for early embryonic stages, since proteins are immediately produced. Introducing DNA gene constructs: Un-integrated DNA constructs cause mosaic expression, but can be useful if ubiquitous promoters are used to express proteins that function cell-non- autonomously (signals). II. Experimental expression of gene products Introducing extra gene product

Microinjection (RNA and DNA): works well with large cells, i.e. early Xenopus and Zebrafish embryos Electroporation (DNA): works fairly well with Chick neural tissue. Lipofection: Lipofection reagent facilitates passing of DNA through hydrophobic cell membranes II. Experimental expression of gene products Delivering extra gene product (into cells)

Transgenic mouse (elaborate procedure, mammalian embryo) Transgenic Xenopus (efficient procedure, vertebrate embryo) Transgenic Drosophila (P-element transformation) –Plasmid 1: –Plasmid 2: –TRANSPOSASE integrates plasmid 1 into the Drosophila genome II. Experimental expression of gene products Transgenics (introducing an artificial gene into the genome) white + genepromoter::PROTEINPP Transposase gene

Inhibiting protein function: good Disrupting RNA or inhibiting RNA function: better Disrupting the DNA of the gene: best III. Experimental inhibition of gene function Inhibiting gene products and genes

Pharmacology: small inhibitory molecules, delivery often easy, specificity often difficult to assess. Dominant-negative protein constructs: mutated proteins that perturb normal function of the endogenous gene product. Fairly advanced knowledge of protein function required. Specificity often difficult to assess, because they inhibit may related gene products from several different genes. Depletion of endogenous protein using antibodies? works well for extract biochemistry, but mostly unsuccessful or misleading if used in in vivo experimental systems. III. Experimental inhibition of gene function Inhibiting protein function (good)

double stranded RNA, dsRNA: Disrupts target RNA. –RNAi: (long dsRNA) Works well in Drosophila and C.elegans, but doesn ’ t work well in neural tissue in C.elegans. –siRNA: (21nt small dsRNA) Works in mammalian tissues. Morpholino antisense oligonucleotides (MO): Inhibits RNA function (interferes with translation initiation or RNA splicing); Xenopus (microinjection), Zebrafish (microinjection), Chicken (delivery difficult). III. Experimental inhibition of gene function Inhibiting RNA (better) (desrupting RNA) (inhibiting function) “ knock down ” “ silencing ”

Targeted knock out (reverse genetics): requires homologous recombination Mouse knock out technology Integrational mutagenesis: Drosophila P-element mediated integration of exogenous DNA into genome to disrupt endogenous genes. Cloning of affected gene easy because exogenous DNA can be used as a marker. Classic genetics (forward genetics): start with a mutant phenotype, map mutation (difficult), identify mutated gene III. Experimental inhibition of gene function Disrupting the gene (best) “ knock out ”

Molecular Methods Tool Box I. AnalysisII. OverexpressionIII. Inhibition Protein Western Blot Proteomics immuno- histochemisty protein chimera (adding proteins to in vitro reactions) pharmacological inhibitors, dominant-negative proteins, protein depletion using antibodies RNA Northern Blot RNase protection assays microarrays RT-PCR RNA in-situ microinjectionRNAi & siRNA Morpholinos DNA (gene) Reporter genes(microinjection) Electroporation, lipofection, transgenics Knock-out Integrational mutagenesis Classic genetics

Question Several powerful molecular tools (or methods) are available to the developmental neurobiologist for studying differential RNA expression in the developing central nervous system of vertebrate embryos. Describe and compare the methods of Northern Analysis (Northern Blot) and RNA in situ hybridisation and their application. Minimal Model Answer: Northern Analysis (alternative correct term Northern Blot) involves the isolation and preparation of RNA from tissue (RNA from tissue taken from different parts of the embryo or from different embryonic stages can be loaded into different lanes and compared with each other), separation of RNA molecules on a gel in an electric field according to differences in mobility (according to size or mass is considered correct), transfer from the gel to a membrane (blotting paper considered correct) and detection of a particular RNA fragment with the help of a labelled (radioactive or epitope-labelled considered correct) antisense probe that hybridises specifically to the particular RNA fragment, followed by detection and imaging (e.g. film) of the resulting bands in columns representing the different lanes of the original gel. RNA in situ hybridisation involves the fixing of embryos or of embryonic tissue under conditions that preserve RNA, incubation with an epitope-labelled (radioactive not taught but also correct if applied to tissue sections) antisense RNA probe that will specifically hybridise to a particular RNA in the tissue, incubation with an enzyme conjugated antibody that recognises and binds the epitope label on the antisense RNA probe, incubation with a colourless substrate that is converted into a coloured precipitate only where the enzyme-conjugated antibody bound to the antisense RNA probe reveals the presence of the particular RNA studied and imaging of the resulting embryo or tissue. More advanced answer: Advantages of Northern Analysis include good resolution for detecting different size gene products (different RNA products from the same gene created by alternative promoters and/or alternative splicing) and of stage-specific differences of expression (depending on the accuracy of the original tissue isolation). Disadvantages include little quantitative information on levels of gene expression and limited resolution for detecting spatial (not cell to cell) differences of expression (depending on accuracy of original tissue isolation). Advantages of RNA in situ hybridisation include good resolution for detecting spatial differences of expression (at least cell to cell, if not on the subcellular level), relatively good information on the temporal differences of expression (depending on the accuracy of the staging). Disadvantages include little quantitative information on different levels of gene expression and usually limited specific information on expression of alternative gene products (depending on the particular probe used). Question for “Tool box for studying gene function in neural development” lecture in PY4302 course (2004).