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Hepatogenesis I
Liver development
HB 308
George Yeoh
Room 2.59 MCS Building
[email protected]
Topics
[Early liver development
[Tissue interaction - role of morphogens and
cytokines
[Liver enriched transcription factors
[Transgenic mouse models
[Research interests
Early liver morphogenesis
[Liver is derived from endoderm.
[It forms from a diverticulum (bud) which
branches out from the primitive gut.
[The pancreas develops dorsally, while the liver
bud develops ventrally.
[The liver metabolises nutrients absorbed from the
gut. Therefore first organ to receive intake.
[It removes toxic compounds which are absorbed
by modifying them so they are soluble.
Proximity of developing liver
(gut endoderm) and cardiac mesoderm
Liver
Heart
Septum transversum
Cardiac mesoderm is necessary
for liver formation
[In mid 1960’s Le Douarin developed a
model using cultured fragments of tissue
from chick embryos.
z
z
z
Piece of primitive gut (endoderm) cannot
develop into liver by itself.
Requires interaction with cardiac mesoderm to
produce glycogen storing hepatocytes.
A physical barrier between the two fragments
would block hepatogenesis.
Zaret & coworkers using a mouse model and
current molecular and cell biology techniques
established the following:
[ Endoderm is capable of synthesising alpha-fetoprotein.
[ Albumin expression in endoderm coincides with the
appearance of the first hepatoblasts, therefore Alb mRNA
is used as marker for first hepatocytes.
[ In the mouse, liver forms from ventral endoderm at
between the 4-6 somite (8-8.5d) and the 7-8 somite (99.5d) stage.
[ Endoderm cells harbour large numbers of aFGF receptors
and aFGF can substitute for cardiac mesoderm in a culture
model.
Albumin mRNA expression visualised
by in situ hybridisation in whole embryo
Why in situ hybridisation?
Why not immunohistochemistry?
Analysis of Alb and AFP mRNA transcripts by RTPCR in segments of embryonic gut endoderm
A&B) Only
ventral
endoderm at
7-8 somite
stage (lane 5)
transcribes
Alb mRNA.
C) All of
endoderm
transcribes
AFP mRNA
Zaret’s findings (cont/d)
[ There is a “window of opportunity”, for by day 11.5, the
endoderm (gut) can no longer be induced to produce
hepatoblasts. It has irreversibly committed to gut.
[ Dorsal endoderm in culture will readily differentiate into
hepatoblasts (albumin mRNA positive), even in the
absence of cardiac mesoderm.
[ Explant co-culture experiments suggest that dorsal
mesoderm exerts an inhibitory influence on dorsal
endoderm which prevents it differentiating into liver and
ensures it becomes gut. Hence there are positive and
negative “differentiation” factors.
Mesenchyme interaction is
necessary for liver development
[The septum transversum (ST) is mesenchyme
which comes from mesoderm.
[It ultimately gives rise to the epicardium and the
diaphragm.
[Several research groups have proposed a role for
the ST in liver development.
[Early experiments provided contradictory data
which depended on whether ventral endoderm
cultures were contaminated with mesenchymal
cells.
Experiments suggesting a role for the ST and
Bmp4 in liver development
[Cultures of ventral endoderm when care was taken
to exclude ST cells fail to develop into liver.
[ST expresses high levels of Bmp4, and the
importance of Bmp4 is shown in knock in
transgenic mice.
[Bmp4 -/- mice fail to develop a liver bud.
[Co-cultures of ventral endoderm and cardiac
mesoderm exposed to noggin (an antagonist of
Bmp4) do not express albumin.
Current knowledge of the role of FGF
and Bmp4 in liver development
FGF secreted by
cardiac mesoderm (red)
induces ventral
endoderm to produce
hepatoblasts.
BMP produced by the
septum transversum primitive epicardium
and diaphragm (yellow)
converts hepatoblasts to
hepatocytes.
E8 (7 somites)
E9 (14 somites)
FGF’s
BMP’s
BMP’s
Transcription factors
[Strategy taken by liver development
researchers was to analyse liver specific
genes e.g. albumin, transferrin,
transthyretin, tyrosine aminotransferase etc.
to identify motifs and transcription factors
which affect them.
[Four families of transcription factors HNF1, HNF3, HNF4 and C/EBP were
characterised. How?
Anatomy of the TTR promoter
Note: In tandem arrangement and multiple sites
Gene targeting experiments identify
critical transcription factors
[Early experiments were unsuccessful.
[Mice showed gross defects in gastrulation and gut
development was disrupted.
[These effects were so early as to preclude analysis of
their role in hepatogenesis, for if there is no gut, then
one can not analyse how liver develops from gut!
[Conclude that liver TFs are essential for gut
development.
[Also some TFs were required for extra embryonic
membranes to form. If these are defective, the
embryos died at very early stages of development.
HNF4 Knockouts
[The phenotype for this KO is embryonic lethal.
[Gastrulation is impaired - no endoderm,
mesoderm or ectoderm. Reasoned that visceral
endoderm requires HNF4
[Duncan & co-workers made chimeric embryos
where the visceral endoderm is HNF4 +/+ but the
embryo itself is HNF4 -/-.
[These underwent normal gastrulation but they did
not form liver.
HNF4 chimeric mice
[These develop a liver primordium which contains
hepatoblasts.
[The hepatoblasts express some but not all liver
genes.
[Those that are expressed are expressed at lower
levels than in HNF4 +/+ embryos.
[Affect later stages in development? Experiments
using conditional KOs are in progress to address
this question.
HNF3 Knockouts
[The HNF3beta KO dies even earlier than the HNF4
KO.
[ Duncan & co-workers used a different approach to
study the interrelationship between HNF3beta and
other transcription factors by using embryoid bodies.
[These are essentially clusters of embryonic cells and
are equivalent to cells of the visceral endoderm which
proliferate but fail to develop further.
[Although a liver did not develop, these embryoid
bodies allowed Duncan to study the inter-regulation of
HNFs.
HNF3 KO mice data
[In this model HNF3beta positively regulates
HNF4 and HNF1.
[However, a combination of HNF3alpha and
HNF3beta is less active than HNF3beta alone.
[ This is because HNF3alpha is a less effective
activator than HNF3beta, so that in this context, it
is an “inhibitor”.
HNF signalling network
HNF3alpha
HNF3beta
+++
+
+++
+
HNF1alpha
HNF4alpha
+++
Network ensures that expression of one TF augments the
expression of others to maintain liver differentiated state.
C/EBPalpha Knockouts
[ Targeted disruption of C/EBPalpha only affects postnatal
liver.
[ Liver development right up until birth is normal. However
the animals die soon after birth.
[ They are not able to induce the enzymes which are
required for glucose synthesis. In addition, the hepatocytes
fail to accumulate glycogen during the period preceding
birth.
[ C/EBP alpha mRNA levels increase substantially with
similar profile and kinetics as many liver enzymes which
are induced after birth. It is therefore a very good
candidate as a regulator of perinatal liver development.
Hex - a transcription factor which affects the
earliest stages of liver differentiation
[Hex is a homeobox transcription factor.
[Highly expressed in ventral endoderm.
[Hex -/- mice do not develop a hepatic
primodium (bud).
[No alpha-fetoprotein or albumin expressing
cells are seen by in situ hybridisation.
[However, there is a “liver capsule” which
contains only hemopoietic cells.
Current view of important factors
in early liver development
Competency
Specification
Liver bud
Growth
Hex
HNF3
GATA
Differentiation
HNF4
Regulation of late stages of liver development
[In the perinatal period, the fetus/newborn and
therefore the liver has to cope with a rapidly
changing environment.
[Before birth, there is a need to synthesise and
store glycogen so specific enzymes have to be
made.
[After birth, there is a need to make glucose as
there is no longer a maternal source.
[The gluconeogenic pathway involves many
enzymes and these have to be induced in concert.
Liver development progresses by the
acquisition and loss of proteins and enzymes
Alb & TN
AFP
M2-PK, Aldolase A
L-PK, Aldolase B
TAT, PEPCK, G-6-Pase
PAH
GK
E10
E15
Early fetal
Late fetal
Neonatal
Late suckling
E21
Birth
3W
Adult
Greengard O., 1970
Expression of tyrosine aminotransferase (TAT) a
gluconeogenic enzyme is heterogeneous
Beta galactosidase (TAT) KI neonatal mouse
liver stained with x-gal
Cultured fetal hepatocytes display heterogeneous
pattern of tyrosine aminotransferase expression
All hepatocytes are ALB + Some hepatocytes are TAT+
Heterogeniety in vivo is observed in vitro,
therefore not due to microenvironment
Liver develops as a heterogeneous
collection of hepatocytes
A
B
C
B
B
C
Heterogeneous Model
Homogeneous Model
A
A
A
A= stem cell?
Liver cells at different developmental stages
express different sets of genes
FL14
FL19
PNRL
ARL
ALB
+
+
+
+
AFP
+
+
-
-
MPK
+
+
-
-
TAT
-
+
+
+
LPK
-
+
+
+
PAH
-
-
+
+
Hormones co-ordinately activate transcription of
genes which code for enzymes required for liver
function in the newborn
[Genes have enhancers which confer i) liver
specificity and ii) hormone e.g. glucocorticoid,
adrenalin and glucagon responsiveness
Summary
[ Liver develops from ventral endoderm
[ Initially, hepatoblasts with limited liver function are
generated (this requires GATA and HNF3).
[ This process depends on interaction between endoderm
and cardiac mesoderm as this produces inducing factors
such as Hex.
[ Later, hepatoblasts differentiate into hepatocytes. This
requires further interaction with the septum transversum
(which provides Bmp4).
[ In the perinatal stages more functions are acquired by
hepatocytes and this is driven by hormones which initiate
transcription of many liver specific genes.