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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.