Principles and basic techniques of dev bio(L1-3)

Question 1

What is the point of developmental biology?

Answer 1

Its aim is to discover how a single cell becomes a multicellular organism with cells that have a specific organisation, characteristic and function. Research done in developmental biology can be applied to new research in ageing, stem cells, degenerative and regenerative medicine, cancer and genetics.

Question 2

How is the population developing?

Answer 2

New technology is leading to an ageing population (UK population is ageing faster than its growth. Between 2015-2020, the general population will grow by 3%. Over 65s will grow by 12% and the over 85s will grow by 18%. 55% of the UK welfare budget is spent on pensioners

Question 3

What are the 2 main opposing theories of development?

Answer 3

1. Epigenetics (Aristotle - 345 BC) - organisms develop progressively through generations of new forms and structures.
2. Preformation (Nikolaas Hartsoeker - 1695) organisms develop from miniature versions of themselves (homunculus)
   Obviously, Aristotle was right, his theory was finally accepted upon discovery of the cell by Cooke in 1665.

Question 4

How did Cooke discover the cell?

Answer 4

He placed sections of tissues under the microscope (which he had invented) and saw that there were compartments. He assumed cells must arise from existing cells - and that it was, therefore, the basic unit of life.

Question 5

What is cell theory?

Answer 5

All organisms are composed of one or more cells. the cell is the most basic unit of structure, function and organisation in all organisms. All cells arise from pre-existing cells.

Question 6

What is the germ plasma determinant theory? How was it proven?

Answer 6

Proposed by Weismann in the 1880s. Germ cells contain different determinants (all germ cells have the same set). Somatic cells only contain certain determinants which is why they are differentiated.
Roux did an experiment in 1888 where he killed half of a 2 cell embryo and found only half of the embryo developed. This proved Weismann’s theory. However, Dreisch repeated this is a 4 ell embryo but instead of killing them he split them up, they all developed into full embryos. This shows that the cells are autonomous unless communicated with - the dead cell couldn’t be communicated with. This is induction theory. Therefore, Weismann was not completely right - somatic cells also have determinants but choose which to express via cell-cell interactions. Therefore, all cells have the same DNA but express different genes which is what causes them to differentiate differently.

Question 7

What are the different types of cell-cell communication? Give examples of each

Answer 7

1. paracrine - cells secrete ligands which care recognised by a receptor on another cell e.g. Shh, Wnt, TGFb, BMP, FGF signalling.
2. Autocrine - the ligand is secreted and then binds to a receptor on the same cell - Shh, Wnt, TGFb, BMP, FGF
   Juxtacrine - A cell with a membrane-bound ligand binds to a membrane-bound receptor on neighbouring cells e.g. Notch
3. Endocrine - secreted on a large scale and transmitted to cells far away - not really used in dev bio.

Question 8

Explain the different types of signals (general)

Answer 8

Signals can be instructive (initiates a new program) or permissive (provides a favourable environment for a specific programme). Signals can act as morphogens (instruct specific cell fates according to their concentration).

Question 9

What are the different levels at which a cell can control its gene expression?

Answer 9

• The production of mRNA (chromatin and miRNAs)
  -The processing/stability of mRNA (splicing etc)
• The production of the protein
• The activity of the protein. (production of a protein doesn’t always lead to function)
  Enhancers can mediate the expression of a gene. They enable different sets of TFs expressed at the same gene in different tissues at different times. E.g Shh in the CNS and the limb bud.

Question 10

What are the different germ layers and what do they derive?

Answer 10

Ectoderm - external tissues - skin, neurones of brain, pigment cells
Endoderm - internal organs - lungs, thyroid, pancreas.
Mesoderm - middle tissues - cardiac, skeletal, smooth muscle, blood, kidneys

Question 11

What are the general processes that underlie embryonic development?

Answer 11

Pattern formation, morphogenesis, cell differentiation, growth.

Question 12

Explain the funnel and hour glass models

Answer 12

The early embryonic development of vertebrates share many similar features - the funnel model is used to show this (all vertebrates start of the same and gradually become different as development goes on) - this was suggested by Ernst Haeckel in 1868. Later, Von Baar suggested that early embryogenesis and gastrulation were quite different between vertebrates, but the intermediate development is quite similar (the hourglass model)

Question 13

What different methods could you use to find the location of the expression of certain genes, along with their function and importance?

Answer 13

In situ hybridisation, northern blots, RT-PCR, Microarrays, reporter lines (transgenic lines). Use an in-situ hybridisation or reporter line to find the spatial expression of a gene, and use northern blots of microarrays to find temporal expression (compare tissues at different developmental stages)

Question 14

How could you find out if the protein is expressed with the same timing as the gene?

Answer 14

Use a western blot of immunohistochemistry ( to look at the protein rather than the mRNA).

Question 15

What are fusion protein constructs and how are they made?

Answer 15

(different to reporter lines) - made by inserting the GFP mRNA into the reading frame so a protein with a GFO domain on it is produced. These can also be used for finding the distributions of proteins - so you join 2 genes together so the protein is actually a hybrid of 2.

Question 16

How do you conduct forward genetic experiments?

Answer 16

Forward genetics involve mutating the animal (the male) using chemical mutagenic agents e.g. ENU, to create organisms with mutated genes. You then breed them out and look for phenotypes you’re interested in. You can then investigate which gene has been mutated to cause this. You cross the mutants with a wt, then cross their offspring. You will then get heterozygous animals with you can cross breed to find any recessive phenotypes.

Question 17

How do you make conditional knockouts?

Answer 17

Make conditional knockouts by flanking the gene with LoxP sites. The gene will remain intact until the egg is crossed with a gamete expressing Cre restriction endonuclease. It will cut the gene out and cause a knockout. this is useful for if the gene you’re looking at causes early deaths when knocked out, but you need the animal to survive for longer, you can cause the expression of Cre to be delayed until a certain developmental stage.

Question 18

What technique can be used to demonstrate a signal’s inductive function? Give an example

Answer 18

Tissue manipulation. E.g. the Spemann and Mangold experiment in 1924. When a dorsal blastopore lip was grafted onto the other side - lead to a secondary axis forming. A similar thing was done by Saunders in 1968 and Riddle in 1993 in a chick limb bud to look at the inductive properties of the ZPA.

Question 19

Explain how observational biology can be useful in analysing cell lineage

Answer 19

Observational biology involves making fate maps to decipher cell lineage. You use a mixture of techniques like tissue transplantation and dye/genetic labelling to label specific cells and look at their development to find their progeny and lineage.
E.g. Brainbow mice.
- Use homologous recombination to create transgenic reporter lines, however, you introduce multiple fluorescent markers, all flagged by slightly different LoxP sites. You introduce Cre which causes excision in a different place in different types of cells. This means different fluorescent proteins are expressed in different cells. So you can label all the different cells in culture at once and track what they become to make a fate map. This was done with the mouse motor cortex (hence brainbow)