Two signaling pathways: Shh and Wnt Flashcards Preview

BMS 2 Week 3 > Two signaling pathways: Shh and Wnt > Flashcards

Flashcards in Two signaling pathways: Shh and Wnt Deck (32):
1

Hedgehog family 

  1. Indian Hh: Necessary for development of Bone and cartilage 
  2. Sonic Hh: Necessary for development of CNS
  3. Desert Hh: Necessary for development of peripheral nerves 

2

Pathway without presence of Hh. 

  1. Ptc and Smo are in transmembrane compartments and you have Gli proteins inside.
  2. Ptc is constitutively inhibiting the Smo protein in cell
  3. When that happens, full length Gli proteins are transported from tip of cilium to basal body to be phosphorylated by PKA
  4. This causes full length phosphorylated Gli proteins are partially proteolyzed by proteasome into a shorter protein
  5. That protein is a transcriptional repressor (Glirep)
  6. This Gli repressor goes into nucleus and binds with cofactors that inhibit target genes. 

3

Ci 

Gli homolog in flies 

4

Cell with presence of Hh

  1. When Hh is present and binds to Ptc, the Ptc transmembrane is internalized in vesicle.
  2. Now Smo is disinhibited and moves from bottom to tip of cilium
  3. Smo prevent Gli proteins from being phosphorylated by PKA
  4. Full length Gli proteins travel from tip to basal body and are not proteolysed in proteasome
  5. Full length Gli protein remains in cell (Gliact)
  6. Gliact goes into nucleus and binds with cofactors that will  activates target genes. 
  7. Gliact activates Ptc and Gli1 genes (positive feedback)
    1. Creation of more Ptc receptor allows more binding of Hh

*Gliact and Glirep bind to the same target genes. The difference in their processing determines if they are bound to cofactors that activate or repress those genes. 

5

Where does Shh signal transduction occurs?

Shh signaling involves primary cilium ONLY in vertebrates

  • These are membrane extensions that occur
  • There is one per cell
  • These protrusions are arranged around a basal body and microtubules (MT) extend to push plasma membrane out into cilium.
  • These cilia are immobile but they are important signaling centers almost like “antennae”
  • Many of components we just talked about are shuttled into and out of cilium in both cells that don’t have signaling and in cells in which signaling is occurring.

 

6

Ciliary defects

i. Vision (rods and cones contain modified cilium)

ii.Hearing (hair cells of inner ear contain cilium)

iii.Smell (odorant receptors are GPCRs that localize to cilia)

iv.Left-right organ location (situs inversus) (embryonic nodal cell cilia)

v.Kidney function

vi.Hedgehog signaling

7

Transport within Cilia 

  1. Microtubules there that maintain structural integrity of the primary cilium
  2. Gli protein are shuttled from bottom to top of primary cilium through activity of IFT (intraflagellar transport proteins) which link to dynein and kinesin motors allowing the Gli to be transported back and forth up and down primary cilium.
  3. Hh signaling and primary cilium function can be compromised by mutations in IFT proteins. 

8

Shh localization in body

Shh’s expression in embryos was found to localize to two areas that were believed to have important signaling function.

  • In the limb: Zona polarizing activity (ZPA) is a tissue that has polarizing functions when you transplant it. This is where Shh is expressed.

    • If ZPA is taken out of one bird and placed into another, duplication of limbs occurs

  • ii.In the CNS: The notochord and floor plate secrete Shh resulting in formation of floor plate and motor neurons. 

    • You can transplant a notochord, floor plate or other Shh-secreting cells such that you get a duplicated floor plate and motor neurons

9

Shh KO Mice phenotype 

  1. Shh KO mice have severe defects in the development of CNS and limb buds
    1. Mice have single midline eye cyclopia
    2. Mice have single brain vesicle instead of two brain vesicles that give rise to cortex holoprosencephaly

10

How does Shh control digit pattern by graded inhibition?

  1. In tissues during development and in adults, Shh diffuses away from source and decreasing concentrations until they taper to 0.
  2. We would predict that the status of Gli proteins within cells along gradient would be relative to the levels of Shh outside of proteins
    1. At highest levels of Shh more Gliact present
    2. At intermediate levels of Shh equal amt of Gliact and Glirep
    3. At lower levels of Shh more Glirep present 

11

Wt Shh and Gli3

  1. Normal digits formed 

12

Shh -/-

Gli3 +/+

Limb

  1. Digits are suppressed, maybe a tiny limb bud will form. 

13

Shh -/-

Gli -/-

Limb

  1. Extra digit formation
    1. Shows that you don't need GliAct made from binding of Shh to PCT in order to get digit formation. 

14

Shh +/+

Gli -/-

Limb

  1. Extra digit formation occurs

15

Shh -/-

Gli 3 +/-

Limb

  1. Digit formation is suppressed but not as much as in Shh -/- and Gli3 +/+ genotype. There are maybe 1 or 2 digits formed. 

16

Shh -/-

Gli3 +/+

17

How do Shh signaling and balance betwee Glirep3/Gliact work within CNS

Shh -/-

Glirep +/+

  1. Phenotypes of knockout of Shh but not Glirep3 results in holoprocencephaly (one vesicle) and cyclopia (one eye) due to no Gliact being present but presence of Glirep3. 

18

Shh -/-

Glirep3 +/-

CNS

Some exencephaly or overgrowth of dorsal part of brain due to not enough Glirep3 being produced to prevent growth in dorsal regions. 

19

Wt Shh and Gli3rep

Normal CNS development 

20

Shh +/+

Gli3rep -/-

A lot of exencephaly results due to not having Gli3rep to block overgrowth of doral regions. 

21

Shh -/-

Gli3rep -/-

Results in exencephaly due to no Gli3rep being present to regulate growth of brain in dorsal region. 

 

Results in cyclopia and holoprosencephaly due to not having Gliact present for proper formation of anterior structures. 

 

*Therefore, Gli3rep KO does not rescue formation of these structures because Gliact has to be present for formation of midline structures such as hypothalamus, spinal cord, interneurons, and motor neurons. 

22

Shh and Dentate Gyrus

Shh helps maintain granule cells in dentate gyrus of hippcampus. 

-KO of Smo in granule cells results in an KO of Shh pathway. Therefore, less granuel cells can be observed in dentate gyrus and hippocampus. 

23

Holoprosencephaly 

  1. Caused by
    1. Mutations that downregulate SHH
    2. Mutations that downregulate Gliact (Gli2)
    3. Mutations that upregulate Glirep (Gli3)
  2. Results in incomplete midline formation (due to Gliact being blocked which is responsible for midline formation)
  3. Range of severities
    1. Cyclopia (one eye)
    2. Single forebrain vesicle
    3. Absence pituitary, corpus callosum, and optic nerves
    4. Midline clefting of palate
    5. Single maxillary central incisor (mild)

Phenotype limited to brain/CNS due to developing structures having different sensitivities to reduced gene dosage. 

24

Cyclopamine

Plant alkaloid that binds and inhibits Smo

25

Smo

7 alpha helix G-protein 

26

Jervine

Inhibits Smo 

27

Shh and Cholesterol 

Mature Shh has cholesterol bound to C-termius

 

  1. Shh's long precursor undergoes a self-cleavage reaction that requires the addition of cholesterol.

  2. The processed form of the protein containing a covalent cholesterol adduct in the C-terminal is the active signaling species

  3. Loss of Shh occurs when anything blocks the addition of cholesterol to Shh

28

Smith-Lemli-Opitz syndrome

-DHCR7 encodes enzyme critical for cholesterol synthesis

-Mutation in DHCR7 leads to loss of cholesterol

-Loss of cholesterol results in same phenotype as loss of Shh aka Holoprosencephaly 

 

29

Mutations in Gli3

Autosomal Dominant syndromes

1. Greig

2. Pallister Hall

3. Postaxial polydactyly 

  • Gli3 +/- 

30

Upregulation of Shh 

Results in:

  1. Gliblastoma 
    1. Affects astrocytes
  2. Meduloblastoma
    1. Arises in cerebellum due to abnormality of granule neurons in rhombic lip 
    2. Any mutations in Shh that cause its upregulation can result in Mb
      1. I.e. loss of one Ptch 1 allele such that nothing is blocking Smo mimics upregulation of Shh

31

Wnt and B-Catenin pathway 

Pathway when Wnt is present

  1. Wnt binds to correceptors Frizzled and Arrow
  2. Prevents B-catenin phosphorylation
  3. PP2A phosphatase removes any phosphate groups attached to B-catenin
  4. B-catenin translocates to nucleus and binds to TCF diplacind negative co-factors Graucho

Pathway when Wnt is not present

  1. Wnt not present
  2. B-Catenin is phosphorylated by Axin/APC/Gsk3
  3. B-catnin is substrate for proteolysis

32

Two pathways for Wnt

  1. Cannonical
    1. requires regulating B-catenin entry into nucleus
    2. Mediates embryonic development
  2. Non-cannonical
    1. Does not involve B-catenin 
    2. Pathway involved in tissue polarity, cell migration