He Pa to Genesis One 060207

7/31/2019 He Pa to Genesis One 060207

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Hepatogenesis I

Liver development

HB 308

George Yeoh

Room 2.59 MCS Building

[email protected]


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Topics

Early liver developmentTissue interaction - role of morphogens and

cytokines

Liver enriched transcription factors

Transgenic mouse models

Research interests


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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 liverbud develops ventrally.

The liver metabolises nutrients absorbed from thegut. Therefore first organ to receive intake.

It removes toxic compounds which are absorbedby modifying them so they are soluble.


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Proximity of developing liver

(gut endoderm) and cardiac mesoderm

Liver

Septum transversum

Heart


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Cardiac mesoderm is necessary

for liver formation

In mid 1960s Le Douarin developed a

model using cultured fragments of tissuefrom chick embryos.

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 fragmentswould block hepatogenesis.


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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 theappearance 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 (9-9.5d) stage.

Endoderm cells harbour large numbers of aFGF receptorsand aFGF can substitute for cardiac mesoderm in a culturemodel.


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Albumin mRNA expression visualised

by in situ hybridisation in whole embryo

Why in situ hybridisation?

Why not immunohistochemistry?


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Analysis of Alb and AFP mRNA transcripts by RT-PCR in segments of embryonic gut endoderm

A&B) Only

ventralendoderm at

7-8 somite

stage (lane 5)

transcribes

Alb mRNA.

C) All of

endodermtranscribes

AFP mRNA


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Zarets 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.


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Mesenchyme interaction is

necessary for liver development

The septum transversum (ST) is mesenchymewhich comes from mesoderm.

It ultimately gives rise to the epicardium and thediaphragm.

Several research groups have proposed a role forthe ST in liver development.

Early experiments provided contradictory datawhich depended on whether ventral endodermcultures were contaminated with mesenchymalcells.


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Experiments suggesting a role for the ST and

Bmp4 in liver development

Cultures of ventral endoderm when care was takento exclude ST cells fail to develop into liver.

ST expresses high levels of Bmp4, and theimportance of Bmp4 is shown in knock intransgenic mice.

Bmp4 -/- mice fail to develop a liver bud.

Co-cultures of ventral endoderm and cardiacmesoderm exposed to noggin (an antagonist ofBmp4) do not express albumin.


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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)

FGFs

BMPs

BMPs


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Transcription factors

Strategy taken by liver developmentresearchers was to analyse liver specificgenes e.g. albumin, transferrin,

transthyretin, tyrosine aminotransferase etc.to identify motifs and transcription factorswhich affect them.

Four families of transcription factors -HNF1, HNF3, HNF4 and C/EBP werecharacterised. How?


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Anatomy of the TTR promoter

Note: In tandem arrangement and multiple sites


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Gene targeting experiments identify

critical transcription factors

Early experiments were unsuccessful.

Mice showed gross defects in gastrulation and gutdevelopment was disrupted.

These effects were so early as to preclude analysis of

their role in hepatogenesis, for if there is no gut, thenone 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, theembryos died at very early stages of development.


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HNF4 Knockouts

The phenotype for this KO is embryonic lethal.Gastrulation is impaired - no endoderm,

mesoderm or ectoderm. Reasoned that visceralendoderm requires HNF4

Duncan & co-workers made chimeric embryoswhere the visceral endoderm is HNF4 +/+ but theembryo itself is HNF4 -/-.

These underwent normal gastrulation but they didnot form liver.


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HNF4 chimeric mice

These develop a liver primordium which containshepatoblasts.

The hepatoblasts express some but not all livergenes.

Those that are expressed are expressed at lowerlevels than in HNF4 +/+ embryos.

Affect later stages in development? Experimentsusing conditional KOs are in progress to addressthis question.


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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 andother transcription factors by using embryoid bodies.

These are essentially clusters of embryonic cells andare equivalent to cells of the visceral endoderm whichproliferate but fail to develop further.

Although a liver did not develop, these embryoidbodies allowed Duncan to study the inter-regulation ofHNFs.


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HNF3 KO mice data

In this model HNF3beta positively regulatesHNF4 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.


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HNF signalling networkHNF3alpha HNF3beta

HNF4alpha HNF1alpha

++++ +++

+

+++

Network ensures that expression of one TF augments the

expression of others to maintain liver differentiated state.


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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 withsimilar 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.


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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 whichcontains only hemopoietic cells.


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Current view of important factors

in early liver development

Competency Specification Liver bud Growth

Differentiation

HNF4

Hex

HNF3

GATA


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Regulation of late stages of liver developmentIn 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 andstore 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 manyenzymes and these have to be induced in concert.


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Liver development progresses by theacquisition and loss of proteins and enzymes

Adult

Neonatal

Late suckling

Late fetal

Early fetal

E10 E15 E21Birth

3W

Alb & TN

AFP

M2-PK, Aldolase A

L-PK, Aldolase B

TAT, PEPCK, G-6-Pase

PAH

Greengard O., 1970

GK


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Expression of tyrosine aminotransferase (TAT) a

gluconeogenic enzyme is heterogeneous

Beta galactosidase (TAT) KI neonatal mouse

liver stained with x-gal


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Cultured fetal hepatocytes display heterogeneouspattern of tyrosine aminotransferase expression

Heterogeniety in vivo is observed in vitro,

therefore not due to microenvironment

All hepatocytes are ALB + Some hepatocytes are TAT+


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Liver develops as a heterogeneous

collection of hepatocytes

A

B

C

A

B A

CB A

Hom

ogeneousM

odel

HeterogeneousModel

A= stem cell?


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Liver cells at different developmental stagesexpress different sets of genes

FL14 FL19 ARLPNRL

ALB + + + +AFP + + - -

MPK + + - -

TAT - + + +

LPK - + + +

PAH - - + +


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Hormones co-ordinately activate transcription of

genes which code for enzymes required for liverfunction in the newborn

Genes have enhancers which confer i) liverspecificity and ii) hormone e.g. glucocorticoid,

adrenalin and glucagon responsiveness


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Summary

Liver develops from ventral endoderm

Initially, hepatoblasts with limited liver function aregenerated (this requires GATA and HNF3).

This process depends on interaction between endoderm

and cardiac mesoderm as this produces inducing factorssuch as Hex.

Later, hepatoblasts differentiate into hepatocytes. Thisrequires 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 initiatetranscription of many liver specific genes.

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