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March 15th, 2010 - Lab Meeting
midline cell fate
Outline
1. Hh and its role in midline cell fate
a. MG
b. Neurons
2. Wg and its potential role (or lack thereof) in midline cell fate
hh activates PMG specific gene expression patterns
WT
UAS-hh
hh[AC]
hh activate midline En in Tribolium
hh represses AMG specific gene expression patterns
WT
UAS-hh
hh[AC]
What might be happening?
Hh Hh
Hh
Hh Hh
en expression
1. hh from row G, H and the lateral CNS activate en expression in rows G?, H, A, B
2. From rows G-B, the MP4-6, MNB, and PMG arise
3. En in PMG represses AMG gene expression and promotes PMG gene expression
What might be happening?
Runt
Hh Hh
Hh
Hh Hh
Runt
en expression
1. Runt represses en expression and keeps it out of rows C-E
Does Hh direct gene expression in row F, or even more anterior? What about
neurons?
hh effects MP3 formation/fate
Tup
Elav
GFP
WT
hh[AC]
ple
GFP
WT
hh[AC]
ptc GFP
What kind of cells are made in hh mutants?
Cas Elav GFP
Runt GFP
En GFP
The simplest explanation is that there are 4 neurons, 2 MP1 and 2 MP4/5.
(Cas is also on in lots of MG, so Cas may be effected in a more complex way)
Conclusion: the effect of hh extends past the En+ cells
Outline
1. Hh and its role in midline cell fate
a. MG
b. Neurons
2. Wg and its potential role (or lack thereof) in midline cell fate
Reasons to think that wg is involved in midline cell fate acquisition
1. Its wg, it is involved in lots of cell patterning and cell fate
a. pubmed search for ‘wingless signaling’ yields 980 papers and 164 reviews
b. ‘wnt signaling’ yields 7,848 papers and 1,493 reviews
2. During segmentation, wg is in a regulatory loop with en and hh to producing a
signaling center
en
wg
hh
ptc
Reasons to think that wg is involved in midline cell fate acquisition
3. wg function regulates anterior cell fates in the epidermis and lateral CNS
a. in wg mutants, naked cuticle (anterior epidermal cell fate) is lost
b. in wg mutants, NB4-2 (anterior cell) cell fate is lost
Reasons to think that wg is involved in midline cell fate acquisition
4. wg is expressed in the midline cells early in their development
stage 8
stage 9
wg
En
stage 10
stage 11
stage 12
Reasons to think that wg is involved in midline cell fate acquisition
5. Ectopic wg and wg mutants show alterations in l(1)sc expression
stage 10
Bossing and Brand, 2006
Reasons to think that wg is NOT involved in midline cell fate acquisition
1. misexpression of wg in the midline has no effect on Runt/En/morphology
Bossing and Brand, 2006:
The earliest time we can express Wingless in all midline cells is
stage 10. At this stage, ectopic Wingless no longer interferes
with midline cell differentiation.
stage 11
Runt
En
GFP
stage 15
Reasons to think that wg is NOT involved in midline cell fate acquisition
2. misexpression of Wg downstream effectors in the midline has no effect on
Runt/En/morphology
UAS-arm[s10]
stage 11
stage 15
Runt En GFP
UAS-tcfΔN
stage 11
stage 13
Reasons to think that wg is NOT involved in midline cell fate acquisition
3. wg mutants still show Runt+ Sim+ cells and En+ posterior cells
wt
stage 13
Sim Runt
En GFP
wg[1-12]
stage 13
Reasons to think that wg is NOT involved in midline cell fate acquisition
4. Ectopic wg and wg mutants show alterations in l(1)sc expression….but we dont
know what is being
stage 10
assayed.
where is the midline?
can wg turn off l’sc in
non-midline cells, it
looks like it. Is this then
related to
segmentation?
Bossing and Brand, 2006
Reasons to think that wg is NOT involved in midline cell fate acquisition
5. In wg mutants ‘early’ en is absent, but ‘late’ en is not effected
Loss of ‘early’ En is
likely due to a loss of
the regulatory loop
wgenhhwg
the presence of en
seems due to the fact
that wg is not
regulating midline gene
expression
Bossing and Brand, 2006
So, what do I think
wg is probably NOT involved in midline cell cell fate/ differentiation
So, what do I think
Epidermal patterning and segmentation:
1. There is an early role for wg in establishing en and hh expression in the posterior
of the segment
2. wg regulates anterior cell fate by repressing the expression of genes that in the
wg mutant are activated ectopically in the anterior.
3. l(1)sc is expressed in the anterior of the segment due to segmentation genes
(which ones I don’t know).
l(1)sc
wg
En/hh
So, what do I think
Midline cell patterning and cell fate:
1. Misexpression of wg or downstream components does not alter midline cell fate
whereas hh and ci can. So, wg regulation of l’sc is probably not occurring at the
same time as hh regulation (probably earlier, during segmentation).
2. The presence of late en and absence of early en is good proof that wg plays no
role in hh regulation of en (and probably l’sc).
3. This likely means that wg repression of gene expression in the anterior of the
segment is not present. This might allow hh signaling to effect gene expression in
anterior cells (ie MP1, MP3), which it does in MP3.
4. Something makes the midline cells different from lateral CNS and epidermis, a
good candidate is single-minded. How does it do this?
How can one nail down the role of wg?
1. reporters of wg activity
a. Top-Flash (not tried in flies, just s2 cells, works in mice)
b. nkd-GFP
2. verify the expression of en and l’sc in wg mutants. If they are present in the
expected cells than wg plays no role.
What might be happening?
Hh
Hh
Hh
Hh
Hh Hh
Runt
en expression
3. Runt in Row C, D, E opposes Hh activity and keeps en expression off.
Evidence comes from Josephs work on Runt and En cross-repression
What might be happening?
Hh
Hh
Hh
Hh
Hh Hh
Runt
MP1
en expression
4. MP1 arises from Rows C, D, E (Runt+ cells).
5. odd expression in Rows A and B is unrelated to MP1 , odd expression is
intiated de novo in MP1
What might be happening?
Hh
Hh
Hh
Hh
Hh Hh
Runt
MP4
MP1
MP3
MP5
MP6
en expression
6. MP3 arises from row F
7. MP4-MNB and PMG arise from rows G-B
8. AMG arise from rows C-F
MNB