ducts

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ducts
acini
Lineage relationships?
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?
precursor A
descendant lineage
?
precursor B
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R26R reporter locus (Phil Soriano)
INACTIVE
ACTIVE
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precursor A
descendant lineage
precursor B
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Lineage analysis of Cell Differentiation Pathways
All pancreatic endocrine cells go through a
Ngn3-expressing progenitor
in situ hybridization to detect endogenous ngn3
expression in the developing pancreas
Gu G, Dubauskaite J, Melton DA. (2002) Direct
evidence for the pancreatic lineage NGN3 cells
are islet progenitors and are distinct from duct
progenitors. Development 129, 2447-2457.
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hPAP expressed in all endocrine cell types
(insulin, somatostatin, glucagon, and
pancreatic polypeptide producing cells) But
Ngn3 is NOT expressed in the mature islet
cells Endocrine cells expressing hPAP are
lineally related to earlier progenitors that
expressed ngn3 transiently - ngn3 triggers
comittment to endocrine fate
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Ptf1a deletion complete loss of
exocrine cells
acinar-specific immunostaining patterns
selective expression in acinar cell lines
Function in early stages of pancreas formation?
Early and late functions for Ptf1a budding
pancreas from primitive embryonic foregut
generating acinar cells
Kawaguchi Y, Cooper B, Gannon M, Ray M, MacDonald
RJ, Wright CV. (2002) The role of the
transcript- ional regulator Ptf1a in converting
intestinal to pancreatic progenitors. Nat.
Genet. 32, 128-134.
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By gene targeting, exchange Ptf1a protein-coding
region with nls-CRE also generates Ptf1a null
allele
Functionally test the cell-specificity of
Ptf1a-expression by R26R
Yoshiya Kawaguchi (Nat. Gen. Sept 2002)
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R26R reporter locus (Phil Soriano)
INACTIVE
ACTIVE
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control
Ptf1a/CRE R26R
uniform high-level exocrine recombination
P1 pups (low level duodenal blue background)
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postnatal day 1
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Ptf1aCRER26R labels early dorsal
ventral pancreatic buds
hypothesis within foregut region, Ptf1a is a
critical transcriptional trigger that commits
cells to the pancreas fate
e10.5 mouse embryo
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duodenal blue background
PR
NORMAL
Ptf1a CRE/ R26R
Ptf1a CRE/CRE R26R
MUTANT
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PANCREAS
INS
Endocrine fates seen in pancreatic rudiment
GLU
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villus
crypt
Cells that would have committed to pancreas dont
simply die, or fail to proliferate, but turn
back into gut epithelium
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Plasticity of stem/precursor cells
embryogenesis and mature organ
Gene combinations that switch (appropriate)
cells between organ fates, without
compromising behavior in other ways
Applications normal tissue, and perhaps
ES/HSC/NSC differentiation
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Corecombination of R26R (or GFP variants) -
follow cell fates in real time?
temporally restricted cell type-specific Cre
activity in precursor populations - normal
tissue, and after gene inactivation/activation
example Gu et al. (Melton lab) Development 2002
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Inducible Cre Temporal control on
tissue-specific inactivation - pulse-chase
lineage labeling
loxP
loxP
pdx1
INACTIVE
CRE
CRE
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R26R reporter locus (Phil Soriano)
INACTIVE
Or GFP reporters - live imaging
ACTIVE
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in situ hybridization Ngn3-driven expression of
Cre-ER mimics the endogenous ngn3 expression
pattern
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Developing a genetically rigorous set of lineage
relationships relies on large number of
well-characterized, well-behaved genes (for
knock-ins) or well-characterized, well-behaved
promoters (for transgenic-based experiments)
Solid and detailed understanding of how to define
specific cell types along the differentiation
pathway
All helpful towards Defining progenitor cell
types in the organ of interest Perhaps
allowing tagging with specific markers (e.g. cell
surface protein for which there are
antibodies, or internal GFP) for FACS-based
purification and study of cells ex vivo
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