Developmental biology 3 (Prof. Dale)

Question 1

What were the 2 theories accounting for tissue formation have proposed by Aristotle (384-322 BCE) ?

Answer 1

Preformation Vs epigenesis

Question 2

What does preformation theory say ?

Answer 2

Preformation: organs and tissues are preformed and correctly positioned in the fertilised egg. They simply enlarge during embryonic development.

Question 3

What does epigenesis theory say ?

Answer 3

Epigenesis: organs and tissues are formed gradually, complexity of the embryo increasing with time. Aristotle preferred this explanation.

Question 4

How did Aristotle provide us with one of the earliest descriptions of embryonic development ?

Answer 4

He cracked open chicken eggs on every day of their 3 week incubation and described the embryos with his naked eye (+subsequently provided 2 explanations for appearance of organs).

Question 5

What did Antonie van Leeuwenhoek (1632-1723) mean when he said “[…] all manner of great and small vessels, so various and so numerous that I do not doubt that they be nerves, arteries and veins… And when I saw them, I felt convinced that, in no full grown body, are there any vessels which may not be found likewise in semen.” in 1677 ?

Answer 5

Van Leeuwenhoek believed that organs were preformed

in the spermatozoa i.e. he thought he could see a fully formed human (homunculus) in the head of each sperm.

Question 6

What is the problem of preformation (having organs preformed in either the egg or the spermatozoa) ?

Answer 6

Children inherit characteristics from BOTH parents.

Question 7

How does the development of the chick embryo provide evidence against preformation (and in favour of epigenesis) ?
Was this evidence widely accepted by the early 18th C. ?

Answer 7

• examining developing chick embryos –> complexity increases with time –> embryo not preformed
• Aristotle could see this without the aid of microscopes
• remarkably, this was not evident to many late 17th/early 18th C. scientists using dyes to stain the embryos and microscopes to see them!

Question 8

What are the different levels of potency (in decreasing order) that a cell can have ?

Answer 8

Totipotent (all cell types) > pluripotent (cells from 3 germs layers but not extra embryonic tissues) > multipotent > oligopotent > unipotent

Question 9

What pathway must a totipotent cell follow to become a sk muscle cell ?

Answer 9

Fertilized egg –> ICM –> mesoderm –> somite –> myotome –> sk muscle

Question 10

What are the 3 different phases of cell commitment ?

What happens during these phases ?

Answer 10

(1) Specification = cells receive instructions on what they are to become but they do not become fully committed –> can be changed experimentally by moving them to a new environment
(2) Determination = cells become fully committed to their fate –> cannot be changed by moving them to a new environment
(3) Differentiation = cells acquire those characteristics that distinguish them from all other cell types –> often involves transcription of tissue-specific genes

Question 11

What are the 3 major factors that contribute to cell commitment ?

Answer 11

1. Localised Determinants
2. Embryonic Induction
3. Morphogen Gradients

Question 12

What did Wilhelm Roux (1850-1924) suggest in 1888 concerning localised determinants ?
What theory does this seem to echo ?

Answer 12

• cell fates may be specified by maternal localised determinants, laid down in the cytoplasm of the egg during oogenesis
• each major cell-type having its own determinant
• only cells that inherit the determinant adopt the specified fate
  This could look like preformation at the molecular level.

Question 13

Edwin Conklin (1963-1952) found in 1905 that eggs of the ascidian Styela partita contained a crescent of yellow cytoplasm that was subsequently found in larval muscle cells.
What did he suggest concerning this yellow cytoplasm ?

Answer 13

Conklin suggested that this yellow cytoplasm contained a muscle determinant.

Question 14

Hiroki Nishida and Kaichiro Sawada identified in 2001
macho-1, a maternal mRNA that is localised to blastomeres containing yellow cytoplasm.
What did they find concerning macho-1 ?
What can we therefore say about macho-1 ?

Answer 14

• deleting macho-1 = loss of muscle formation
• injecting macho-1 into a different blastomere = ectopic muscle formation
  Macho-1 = a transcription factor that regulates muscle-specific genes (e.g. m-actin, m-myosin, myf, tbx6, snail).

Question 15

How did Hans Driesch (1867-1941) show in 1893 that sea urchins did not develop using local determinants ?

Answer 15

Driesch dissociated cleavage stage sea urchin embryos –> individual blastomeres formed small but otherwise nearly normal larvae ==> Regulation means that sea urchin eggs cannot be a mosaic of localised determinants

Question 16

What did Driesch’s observe in his see urchin experiment ?

What did this imply about regulation ?

Answer 16

Each cell of the 4-cell sea urchin (and mammalian) embryo has greater potential than they normally exhibit –> they can form more tissues when isolated from their neighbours than when left in contact with them
Hyp: cells communicate with each other to restrict their potency

Question 17

What is embryonic induction ?
What does it require ?
What are examplar signalling proteins ?

Answer 17

Embryonic induction = the process whereby a cells fate is changed by signals from an adjacent group of cells
It requires two types of cell = signalling cell + responding cell
Signalling proteins: Activin, BMP, EGF, FGF, Hedgehog, IGF, Nodal, PDGF, TGFß + Wnt

Question 18

What did Pieter Nieuwkoop (1917-1996) observe when he isolated the animal and vegetal fragments from axolotl blastulae ?
Which fragment makes the futur mesoderm ?
What did he conclude from this ?

Answer 18

• animal and vegetal fragments –> differentiate into epidermis + endoderm respectively
• mesoderm –> also formed when animal and vegetal fragments were combined
• mesoderm –> only formed by the animal fragment
  Concl: mesoderm formation –> induced by the vegetal fragment

Question 19

How did Nieuwkoop suggest that mesoderm induction occured ?

Answer 19

• mesoderm –> induced in the equatorial region of amphibian blastulae by a signal from the vegetal hemisphere
• this signal –> induces expression of mesoderm specific genes such as brachyury

Question 20

How can the Xenopus blastula stage animal caps be used as an assay to identify mesoderm inducing factors (MIFs) ?
What results will this yield ?
Why do caps w/ MIFs elongate ?

Answer 20

By incubating them in media containing candidate molecules.
Animal caps –> only form epidermis when incubated alone (specification) Vs form mesodermal tissues (e.g. notochord and muscle) when a MIF is added
Caps w/ MIFs elongate because the induced notochord undergoes convergent-extension.

Question 21

What are the 2 main families of MIFs ?
Which proteins belong to these different families ?
Are any of these proteins limited to the vegetal hemishphere of Xenopus blastulae ?

Answer 21

• Transforming Growth Factor ß (TGFß) family: Activin, BMP 2/4, Growth & Differentiation Factor 3, Nodal, Vg1 (GDF1)
• Fibroblast Growth Factor (FGF) family: FGF1/2/4/8
• Only GDF3, Nodal, and Vg1 are localised to the vegetal hemisphere of Xenopus blastulae

Question 22

Which is the key signalling molecule for mesoderm induction ?

Answer 22

Inhibition studies in zebrafish, frogs + mice suggest that Nodal is the key signalling molecule.

Question 23

How does mesoderm induction occur ?

Answer 23

• Nodal = primary mesoderm inducing signal released by the vegetal hemisphere –> induces brachyury (bra) expression in the mesoderm
• brachyury –> encodes a T-box transcription factor that regulates expression of fgf4
• FGF signalling (ERK2*) –> maintains expression of brachyury
• +ve feedback circuits like this –> commonly used by embryonic genetic networks

Question 24

What are morphogens ?
How are they synthesized/destroyed ?
How do cells use them for communication/induction ?

Answer 24

• molecules that induce multiple cell fates in a concentration dependent manner
• synthesised by source cells, morphogens diffuse across a layer of unspecified cells to form a gradient (w/ highest concentration closest to the source)
• a “sink”may destroy the morphogen
• cells determine their fate by reading the concentration of the morphogen

Question 25

What model did Lewis Wolpert propose to explain how morphogens worked in 1969 ?

Answer 25

The “french flag” model (cells above a threshold concentration T1 become blues cells, those between T1 and T2 white cells, and those below T2 red cells).

Question 26

How is the spinal chord organized embryologically ?

Answer 26

• distinctive dorsal-ventral pattern –> can be revealed by the distribution of distinct transcription factors
• ventral midline lines just above notochord + contains a specialised structure = floor plate
• above floor plate, in distinctive order –> a number of different ventral interneurons (PV0-PV3) + motoneurons

Question 27

Which structure regulates spinal chord patterning ?

Answer 27

The notochord.

Question 28

How do we know that cell fate within the ventral half of the neural tube is specified by the notochord ?

Answer 28

Because the notochord induces ectopic floor plate + motor neurons when grafted to the side of the neural tube.
Motor neurons can also be induced when a floor plate is grafted into the side of the neural tube.

Question 29

What does Shh from the notochord induce ?

Answer 29

The floor plate (+Shh expression in the floor plate).

Question 30

What does Shh from the floor plate generate ?

How do neural tube cells respond to different [Shh]s ?

Answer 30

• A gradient across the ventral half of the neural tube
• concentration = high ventrally + low dorsally
• neural tube cells –> respond to different concentrations of Shh by activating expression of different genetic programs, specifying at least five different cell-types.