Lecture 2/3- Basic principles and techniques

• Ectoderm: skin cells, neurons, pigment cells • Mesoderm: cardiac/skeletal/smooth muscle, tubule kidney cells, RBC • Endoderm: lung, thyroid and pancreatic cells

• Pattern formation • Morphogenesis • Cell differentiation • Growth

• A process by which cells are organised in time and spaced to produce a well ordered structure within the embryo • Body plain and body axes are established • A/P, D/V and L/R ends are recognised • Development is spatially and temporally predictable

• Cell and tissue movement and changes in cell behaviour that give the developing organ its 3D shape • Underpinned by changes in cell adhesion, migration, death and shape

• Process by which cells become different from each other and acquire specialised properties • Governed by changes in cell expression which dictate the repertoire of proteins synthesised • Progressive restriction of pluripotency and the progressive gain of specialised properties over time • Stem cell - specification - determination - differentiation - maturation

• Increase in mass or size • Continuous process throughout development and life • Occurs through cell proliferation, enlargement and accretion of extracellular matrix tissues

Embryology: observational biology and experimental manipulation Developmental biology: study of genes and proteins Animal models and use of genetics

Drosophila, fish, frog, rat, mice, bird

• In situ hybridisation • Northern blot • RT-PCR • Micro-array (inc single cell RNA sequencing) • Reporter lines (transgenic)

• mRNAs will have a particular sequence in which an antisense probe can be made that is complimentary to the sequence • Label probe with DIG • Fix embryo and incubate with the antisense probe • The transcribing mRNA will bind to the probe in the cells which are expressing the specific transcript • By attatching an anti-DIG-AlkPho to the probe, the specific cells can be visualised as a blue stain

• Make a reporter line which reports the expression of a particular gene • Dependant on the idea that a gene will be expressed because of the regulatory elements that control its expression • Remove the regulatory sequence from the gene of interest and clone downstream a reporter gene (e.g GFP) • Introduce this construct into an animal to become inherited (transgenic animal) • Protein translated and transcribed from this can be visualised as will fluorescence green • Allows you to image protein expression without killing the embryo and look a particular developing tissue in real time

• Enables the study of when a gene is expressed • Involves the bulk analyses of which mRNA transcripts are expressed in particular sample • Isolate RNA, generate cDNA and apply fluorescent tags to each cDNA • Finally hybridises in bulk to an array of the genome • Where they bind let’s you say a particular mRNA was present

• Dissect tissue and isolate and separate the individual cells • Place cells in an oil droplet with a barcode which allows you to sequence the transcriptome of every single individual cell • Compare the gene expression profiles of each single cell • Allows you to calculate how many populations of cells are present and aids in the discovery of new transcripts and see what RNA is being expressed

• In situ hybridisation * Reporter lines

Most techniques of analysed at distinct time points

• Western blot analyses of fusion protein constructs * Immunohistochemistry

• Every protein has a certain shape (epitopes) which can be recognised by a specific primary antibody • Incubate a specific protein in a primary antibody • The antibody has a complementary shape to the epitope • A secondary antibody coupled with a fluorescent tag binds to the primary antibody • This allows the proteins location to be indirectly visualised in a particular cell, at a particular place, at a particular time

• Take gene and close GFP into sequence with coding sequence of the gene • When the gene is transcribed, hybrid mRNA is made which is in the transcript of the gene • This makes a hybrid protein which is introduced to a transgenic animal • The gene and proteins can therefore be visualised in a living organism

• Gain of function (transgenesis) * Loss of function (forward/reverse genetics/KO)

GoF: mutation in a gene that confers a gain in the activity of the protein product encoded by a mutated gene LoF: mutation in a gene that disrupts the expression or function of the protein encoded by the mutated gene

• Seeks to identify a gene whose mutation caused a particular phenotype • Expose an animal model to a chemical mutagen which will randomly introduce mutations into the genome • Mutagenised one generation and breed our generations, looking for a specific phenotype • Then positional clone out the mutated gene to identify the gene whose disruption lead to a particular phenotype

• Seeks to characterise the phenotype of particular mutated gene • Selectively deleting a gene through selectively recombine out of the genome a target gene • Homologous recombination- ES cell transfection - ES cell selection - ES cell injection into blastocysts- implantation into animal - selection chimeric animal- breed - genome will be normal except missing the particular gene

• Tissue ablation • Tissue graft • Tissue transplantation • Bead/cell implantation

• Spemann and Mangold grafted organiser cells into an ectopic position • Demonstrated organiser cells are the source of secreted factors that activate adjacent cells which change their fate and assume a neural fate