Early Vert Development (4)

Question 1

In summary, what did we find with the animal/vegetal sandwich experiments?

Answer 1

• endoderm is able to induce mesoderm

Question 2

In summary, what did we find with the rescue of UV ventralized embryos?

Answer 2

• dorsal endoderm (Nieuwkoop center) can induce dorsal mesoderm (Spemann’s organizer)
• Wnt signaling was driving force

Question 3

Given all the information, what can we conclude about the UV effect?

Answer 3

• microtubule formation is blocked by UV
• this causes GBP (and disheveled and wnt11 mRNA) to not make it to the right location
• end result is a ventralized embryo

Question 4

What is the Nieukoop center doing wrt organizer formation?

Answer 4

• GBP, disheveled –> TF siamois –> TF Goosecoid

Question 5

What is Goosecoid?

Answer 5

• transcription factor that specifies the Sp organizer

Question 6

What do we see with a GBP rescue experiment?

Answer 6

• injecting GBP into a UV treated ventralized embryo results in only half the average dorsal/anterior index
• this indicates that GBP is not suficient for a complete rescue of a ventralized embryo and something else is needed

Question 7

How is beta-catenin normally in each axis?

Answer 7

• ventral: beta-catenin degraded

- dorsal: beta-catenin stabilized

Question 8

On the dorsal side, what happens to the siamois gene?

Answer 8

• b-catenin proteins bind upstream to siamois gene
• this activates transcription of siamois protein
• siamois protein activates the goosecoid gene and goosecoid protein is transcribed

Question 9

On the ventral side, what happens to the siamois gene?

Answer 9

• Tcf3 proteins bind upstream to the siamois gene

- this represses the siamois gene and no siamois protein is transcribed

Question 10

What contributes to goosecoid expression?

Answer 10

• siamois protein (but does not produce strong enough goosecoid expression alone)
• Smad2/4 from TGF-B signal

Question 11

What is the TGF-B pathway that contributes to Goosecoid protein transcription?

Answer 11

• Vg1 –> goosecoid
• Vg1 –>Xnr1,2,3 (xenopus nodal-related) –> goosecoid
• (VegT) –> derriere activin nodal –> Xnr1,2,3 –> goosecoid

Question 12

What does goosecoid specify/activate?

Answer 12

• noggin, chordin and follistatin

- these specify/activate XBF-2 and Zic-r that define neural fate

Question 13

What concentrations lead to ventral endoderm?

Answer 13

• high concentrations of VegT and Vg1 (all endoderm specified by this)
• low concentrations of B-catenin
• nodal related low

Question 14

What concentrations lead to dorsal endoderm?

Answer 14

• overlap of VegT and Vg1 with b-catenin

- nodal related high

Question 15

What is seen throughout the fully formed adult body?

Answer 15

• high degree of diversification along axis (ex. brain, or bones in limb)

Question 16

What is the Xnr gradient?

Answer 16

• lower concentration on the ventral side and higher concentration on the dorsal side
• within the endoderm and affects the mesoderm

Question 17

Why is having a gradient advantageous?

Answer 17

• allows for scale
• different relations of response to signal (different response to low, medium or high)
• different cell responses could lead to different fates

Question 18

What are morphogens?

Answer 18

• factors that organize a field of surrounding cells into patterns
• very important mechanism in development

Question 19

What are the two criteria to define a morphogen?

Answer 19

1. form a gradient of concentration (usually emanating from a localized source)
2. act on cells in a concentration-dependent manner (i.e. different concentrations will lead to a different response from the same cell)

Question 20

What upregulates Xnr?

Answer 20

• GBP and disheveled upregulate Xnr

- this is why it has a higher concentration in the dorsal region

Question 21

What is the factor in mesoderm? What is it’s gradient and why?

Answer 21

• BMP4 in mesoderm
• higher concentration in ventral region and lower in dorsal
• because GBP and disheveled produce Xiro which inhibits BMP4
• morphogen gradient in mesoderm

Question 22

What information do morphogen gradients give? How does this apply to BMP4?

Answer 22

• “positional information”
• high BMP4: ventral mesoderm
• intermediate BMP4: intermediate mesoderm
• low BMP4: dorsal mesoderm

Question 23

What are key experiments that need to be performed in order to demonstrate that a molecule functions as a morphogen?

Answer 23

• can test the affect of different concentrations

- add more or knock down and then add back in different concentrations

Question 24

What experiment can be used to test whether BMP4 functions as a morphogen in the mesoderm?

Answer 24

• test different levels of BMP4
• assay: score for tissues
• control: no BMP4 or simply a normal embryo

Question 25

What exactly was done to examine BMP4?

Answer 25

- injected different amounts of BMP4 RNA to all cells - removed dorsal lip and cultured in isolation - examined what type of mesoderm was being formed

Question 26

What tissues result for different amounts of BMP4?

Answer 26

``` (dorsal/low) - notochord - muscle - pronephros - blood (ventral/high) ```

Question 27

Do morphogen gradients pattern the ectoderm during neural induction?

Answer 27

- yes/probably | - many different domains created by morphogens

Question 28

What is the "einsteck" experiment?

Answer 28

- dorsal lip transplant | - remove dorsal lip from donor and put into host

Question 29

What are the results of the "einsteck" experiment?

Answer 29

- depending what stage the dorsal lip is at (young or advanced), it can induce different axial regions - young dorsal lip: extra head - advanced dorsal lip: extra tail

Question 30

What results from a transplantation experiment using mesoderm at the neural plate stage?

Answer 30

- take different sections of mesoderm and transplant to early gastrula - induce different axial regions depending on which part of mesoderm taken - indicates that mesoderm is not uniform in the types of tissues produced

Question 31

What hypothesis does the mesoderm transplantation experiment lead to?

Answer 31

- hypothesis: there are molecules that act along the A-P axis in a graded manner - signals from underlying mesoderm (archenteron roof) instruct the neural plate in a graded manner

Question 32

What did researchers find when searching for differentially-expressed mesodermal genes along the A-P axis?

Answer 32

- cerberus phenotype (named after three headed dog)

Question 33

What does injection of cerberus into the vegetal blastomeres result in?

Answer 33

- ectopic head structure but no secondary axis

Question 34

Where is cerberus expressed?

Answer 34

- anterior most endomesoderm | - first tissue that involutes in the organizer

Question 35

What factors does cerberus act on?

Answer 35

- inhibits nodal, BMP4 and wnt

Question 36

What results from a cerberus knockdown?

Answer 36

- head region is not lost following a cererus knockdown | - indicates there is functional redundancy

Question 37

What other factors were found to be expressed in dorsal endomesoderm?

Answer 37

- looking for functional redundancy with cerberus | - found Dickopf (thick headed) and Frzb

Question 38

What resulted from Dickkopf knock-down?

Answer 38

- diminished head formation

Question 39

What resulted from Frzb gain-of-function experiments?

Answer 39

- increases head size

Question 40

What was Frzb found to do molecularly?

Answer 40

- antagonizes wnt pathway by binding to wnt and blocking receptor

Question 41

What do cerberus, Frzb and Dickkopf have in common? What hypothesis results?

Answer 41

- all expressed in head (anterior) - all inhibit wnt - wnt signaling is required for posterior (trunk) formation and is inhibited during anterior (head) formation

Question 42

What might happen if wnt is overexpressed in endomesoderm?

Answer 42

- may override wnt inhibition - may form more tail structure - result: over-expression of wnt8 in dorsal mesoderm leads to loss of head structures

Question 43

Hypothesis: Does wnt function as a morphogen on neural tissue? What experiment was done to test this?

Answer 43

- separate neural tissue from other influences to test wnt on it - isolate ectoderm: on own becomes skin - dissociate ectoderm and re-aggregate: becomes neural - assay: use RT-PCR to identify genes in specific areas

Question 44

What genes were found to be expressed in the previous experiment?

Answer 44

``` (Anterior) - forebrain: Bf1 - forebrain+midbrain: Otx2 - hindbrain: Krox20 (posterior) ```