Potential entry points for loss of function analysis

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Potential entry points for loss of function
analysis
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Potential entry points for loss of function
analysis
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Potential entry points for loss of function
analysis
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Dicer inactivation-in c elegans
Dcr1/- X Dcr1/-
Dcr-/-
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Dicer inactivation-in c elegans
Dcr1/- X Dcr1/-
Dcr-/-
WT
Dcr-/-
(Science, 293, 2269)
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Dicer inactivation-in c elegans
Dcr1/- X Dcr1/-
Dcr-/-
Dcr-/-
WT
(G D, 15, 2654)
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Dicer inactivation-in c elegans

• Dcr1 is required for germ line formation
• Dcr1 is not essential for later development
• Behaviors?
• Amplification? (maternal species)

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Dicer inactivation-in c elegans

• Dcr1 is required for germ line formation
• Dcr1 is not essential for later development
• Behaviors?
• Amplification? (maternal miRNAs)

Lin4 and Let7 phenotype
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Dicer inactivation-in zebra fish
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Dicer inactivation-in zebra fish
Dcr1 not required for early gastralation/specifica
tions events, but needed for later events. miRNA
production?
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Dicer inactivation-in zebra fish
(dead end germ line depletion)
(Mir430)
(Science, 308, 833)
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Dicer inactivation-in zebra fish
(Science, 308, 833)
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Dicer inactivation-in zebra fish
(Science, 308, 833)
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Dicer inactivation-in zebra fish
(Science, 308, 833)
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Dicer inactivation-in zebra fish
(Science, 308, 833)
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Dicer inactivation-in zebra fish
(Science, 308, 833)
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Dicer inactivation-in zebra fish
Dicer inactivation-in zebra fish

• miRNAs
• mostly dispensable for cell type
  specification, but critical for
  morphogenesis--intercellular communication.
• Probably not required for activation of cell
  fate specification genes

Zebrafish and c. elegans Differential effect on
germ line formation, or non-autonomous effect?
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Dicer inactivation-in zebra fish
Piwi RNA in germ line niche Dicer function for
mouse germ line formation
WT Dicer-/-
Spindle and DNA
(G D 61,644)
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Dicer inactivation-in zebra fish
Piwi RNA in germ line niche Dicer function for
mouse germ line formation
(G D 61,644)
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Dicer inactivation-in mouse
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Dicer inactivation-in mouse
T expression Oct 4 expression
(Nature Genetics, 35, 215)
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Dicer inactivation-in mouse
T expression Oct 4 expression
What cells are missing?
(Nature Genetics, 35, 215)
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Dicer inactivation-in mouse
Conditional alleles
Yeast, C elegans, Drosophila, Zebrafish, Mammals
,
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Dicer inactivation-in mouse
Conditional alleles
Yeast, C elegans, Drosophila, Zebrafish, Mammals
integrases Cre/LoxP, FRT/Flpe, PhiC31
PLoS ONE. 2 e162
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Dicer inactivation-in mouse
Conditional alleles
(G D 19, 489)
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Dicer inactivation-in mouse
What do you expect?
(G D 19, 489)
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Dicer inactivation-in mouse
What do you expect?
(G D 19, 489)
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Dicer inactivation-in mouse
miRNAs required for differentiation
(G D 19, 489)
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Dicer inactivation-in mouse

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Functional comparison between TFs and miRNA
miRNA vs transcription factors

Mode of action
post-transcription transcription
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Dicer inactivation-in mouse

Heterochromatin formation affected in Dicer-/- ES
cells
?
Methylation pattern
miRNA
?
Dnmts
(G D 19, 489)
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Dicer inactivation-in mouse

Heterochromatin formation affected in Dicer-/- ES
cells
?
Methylation pattern
miRNA
?
Dnmts
(G D 19, 489)
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Dicer inactivation-in mouse

Specific functions

• 110, 000 miRNA molecules/ES cell.
• More than half belongs to four clusters,
  related with cell cycle
• more than 300 different types

(PNAS 104, 18097)
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Dicer inactivation-in mouse

Zp3- Cre to produce oocyte-specific deletion
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Dicer inactivation-in mouse

MyoD-Cre for muscle
(DB 311, 359)
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Dicer inactivation-in mouse

(DB 311, 359)
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Dicer inactivation-in mouse

(DB 311, 359)
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Dicer inactivation-in mouse

(PNAS 103, 2209)
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Dicer inactivation-in mouse

(PNAS 103, 2209)
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Deletion of individual miRNAs
Only two examples so far in mouse Mir208 and
Mir155

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Deletion of individual miRNAs
Only two examples so far in mouse Mir208 and
Mir155

Accompanied by abnormal immune cell deposition
/ -/-
Science 316, 604 and 608)
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Deletion of individual miRNAs
Only two examples so far in mouse Mir208 and
Mir155

Science 316, 604 and 608)
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Deletion of individual miRNAs
Only two examples so far in mouse Mir208 and
Mir155

Survival
Pathogen challenge
Science 316, 604 and 608)
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Deletion of individual miRNAs
Only two examples so far in mouse Mir208 and
Mir155

IG production
Pathogen challenge
Science 316, 604 and 608)
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Deletion of individual miRNAs
Only two examples so far in mouse Mir208 and
Mir155

T-cell proliferation
Science 316, 604 and 608)
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Redundancy or the functional mode of miRNA?