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Principles of Developmental Biology > Lecture 2/3- Basic principles and techniques > Flashcards

Lecture 2/3- Basic principles and techniques Flashcards

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1
Q

What are the 3 germ layers and what cells/tissues derive from them?

A
  1. Ectoderm: skin cells, neurons, pigment cells
  2. Mesoderm: cardiac/skeletal/smooth muscle, tubule kidney cells, RBC
  3. Endoderm: lung, thyroid and pancreatic cells
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2
Q

Name some processes which underlie embryonic development

A
  1. Pattern formation
  2. Morphogenesis
  3. Cell differentiation
  4. Growth
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3
Q

Describe pattern formation in a developing embryo

A
  • 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
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4
Q

Describe morphogenesis in a developing embryos

A
  • 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
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5
Q

Describe the process of cell differentiation in a developing embryo

A
  • 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
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6
Q

Describe the process of growth in a developing embryo

A
  • Increase in mass or size
  • Continuous process throughout development and life
  • Occurs through cell proliferation, enlargement and accretion of extracellular matrix tissues
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7
Q

How are developmental process studied?

A

Embryology: observational biology and experimental manipulation

Developmental biology: study of genes and proteins

Animal models and use of genetics

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8
Q

Give some examples of animal models used on developmental biology studies

A

Drosophila, fish, frog, rat, mice, bird

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9
Q

What experimental techniques could be used to see where and when a gene is expressed in the developing embryo?

A
  • In situ hybridisation
  • Northern blot
  • RT-PCR
  • Micro-array (inc single cell RNA sequencing)
  • Reporter lines (transgenic)
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10
Q

Outline the method of in situ hybridisation

A
  1. mRNAs will have a particular sequence in which an antisense probe can be made that is complimentary to the sequence
  2. Label probe with DIG
  3. Fix embryo and incubate with the antisense probe
  4. The transcribing mRNA will bind to the probe in the cells which are expressing the specific transcript
  5. By attatching an anti-DIG-AlkPho to the probe, the specific cells can be visualised as a blue stain
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11
Q

Outline the use and method of reporter lines

A
  1. Make a reporter line which reports the expression of a particular gene
  2. Dependant on the idea that a gene will be expressed because of the regulatory elements that control its expression
  3. Remove the regulatory sequence from the gene of interest and clone downstream a reporter gene (e.g GFP)
  4. Introduce this construct into an animal to become inherited (transgenic animal)
  5. Protein translated and transcribed from this can be visualised as will fluorescence green
  6. Allows you to image protein expression without killing the embryo and look a particular developing tissue in real time
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12
Q

Outline the method of microarray assay

A
  1. Enables the study of when a gene is expressed
  2. Involves the bulk analyses of which mRNA transcripts are expressed in particular sample
  3. Isolate RNA, generate cDNA and apply fluorescent tags to each cDNA
  4. Finally hybridises in bulk to an array of the genome
  5. Where they bind let’s you say a particular mRNA was present
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13
Q

Outline the method of RNAseq analysis

A
  1. Dissect tissue and isolate and separate the individual cells
  2. Place cells in an oil droplet with a barcode which allows you to sequence the transcriptome of every single individual cell
  3. Compare the gene expression profiles of each single cell
  4. 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
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14
Q

Which techniques could provide data regarding spatial expression of the gene?

A
  • In situ hybridisation

* Reporter lines

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15
Q

Which techniques could provide data regarding the temporal expression pattern of a gene?

A

Most techniques of analysed at distinct time points

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16
Q

What experimental techniques could confirm if the protein was expressed at the same time and place as the gene?

A
  • Western blot analyses of fusion protein constructs

* Immunohistochemistry

17
Q

Outline the method of immunofluorscence

A
  • 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
18
Q

Outline the method for fusion protein constructs

A
  • 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
19
Q

What experimental techniques can be used to identify of a gene/protein is essential for development and its function?

A
  • Gain of function (transgenesis)

* Loss of function (forward/reverse genetics/KO)

20
Q

Define loss and gain of function

A

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

21
Q

Outline the method of forward genetics?

A
  • 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
22
Q

Outline the method of reverse genetics

A
  • 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
23
Q

What experimental techniques could confirm how a gene is regulated?

A
  • Tissue ablation
  • Tissue graft
  • Tissue transplantation
  • Bead/cell implantation
24
Q

Outline the grafting experiment method

A
  • 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
25
Q

Outline the limb bud experiment method

A
  • Transplantation of the ZPA to an ectopic location results in the mirror image duplication of digits
  • Further experiments mimic the ZPA by soaking beads in Shh
  • Or removing/inhibiting Shh/ZPA fails to get any digits
26
Q

What can be discovered through fate maps and lineage analyses?

A

What tissues/organs are derived from a cell that expresses a gene

27
Q

What experimental techniques are used for fate maps and lineage analyses

A
  • Cell/tissue transplantation
  • Cell/tissue labelling with dye
  • Cell/tissue genetically labelled (electroporation, GFP transgenic lines, brainbow mice and zebrabow)

Principles of Developmental Biology (16 decks)

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