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The role of Lhx2 in the hematopoietic stem cell function, liver development and disease
Umeå University, Faculty of Medicine, Molecular Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During embryonic development, generation of functional organs is dependent on proper interactions between different cell types. Elucidation of the mechanisms operating during organ formation might provide insights into the origin of many pathological disorders in the adult. Gene inactivation studies in mice have provided invaluable tool to study the function of genes critical for morphogenesis of distinct organs. A LIM-homeodomain transcription factor Lhx2 has previously been reported to play a role in fetal liver development and hematopoiesis, as its inactivation leads to lethal anemia due to underdeveloped liver. This thesis focuses on the function of Lhx2 in the development of these two organ systems.

Reciprocal signaling between ventral foregut endoderm and mesenchyme of the septum transversum regulates the liver formation, expansion and differentiation. A fully formed liver is composed of endoderm-derived hepatocytes and cholangiocytes and a variety of mesenchyme-derived cell types, such as endothelial cells and hepatic stellate cells. In early stages of liver development Lhx2 is expressed in the liver-associated septum transversum mesenchyme, a part of which becomes integrated into the liver organ and develops into hepatic stellate cells. Functional Lhx2 expression in the hepatic mesenchyme is necessary for normal liver outgrowth and differentiation. Loss of Lhx2 from developing hepatic stellate cells leads to their activation and excessive deposition of collagen fibres, resulting in hepatic fibrosis and severely distorted liver architecture. Transfection of Lhx2 to human stellate cell line downregulates genes associated with stellate cell activation and fibrogenesis. Thus, Lhx2 is the first gene identified to negatively regulate events leading to hepatic fibrosis. Elucidation of the molecular mechanisms involved in this process might therefore be instrumental for the development of novel therapies useful in treatment of this disorder.

Fetal liver is also a major site of hematopoiesis in the embryo and provides physiological conditions necessary for the efficient expansion of hematopoietic stem cells (HSCs). The hematopoietic defect observed in Lhx2-deficient embryos is cell-nonautonomous, indicating that Lhx2 might control secreted factors involved in the self-renewal of HSCs. This putative second role of Lhx2 has been investigated by analyzing the mechanism whereby Lhx2 expression generates in vitro self-renewing HSC-like cell lines. Interestingly, in agreement with the cell nonautonomous phenotype of the lethal anemia in Lhx2-/- embryos, the mechanism of self-renewal is dependent on Lhx2 expression and occurs via secreted factor(s). Identification of these factor(s) might potentially allow ex vivo expansion of HSCs for therapeutic purposes.

The Lhx2-immortalized HSC-like cell lines share many basic features with HSCs and self-renew in vitro in presence of Steel factor (SF). SF/c-Kit signaling mediates a wide variety of biological activities in cells at many different levels in the hematopoietic hierarchy. We used the HSC-like cell lines as an in vitro model system to compare signal transduction pathways from c-Kit receptor in stem cells versus differentiated hematopoietic cells. HSCs require PI-3K dependent activation of Raf1-Mek-Erk cascade for their survival and self-renewal in response to SF, whereas activation of Erk is PI-3K independent in committed myeloid and mast cells. Thus, the mode of SF/c-Kit signaling is dependent on the differentiation status of the cells.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi , 2004. , 108 p.
Keyword [en]
Cell and molecular biology, liver fibrosis, liver development, Lhx2, hematopoietic stem cells
Keyword [sv]
Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
URN: urn:nbn:se:umu:diva-373ISBN: 91-7305-748-7OAI: diva2:143297
Public defence
2004-12-10, Major Groove, 6L, Norrlands Universitetssjukhus, Umeå, 13:00 (English)
Available from: 2004-11-19 Created: 2004-11-19 Last updated: 2010-08-02Bibliographically approved
List of papers
1. Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2.
Open this publication in new window or tab >>Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2.
2004 (English)In: Gene Expression Patterns, ISSN 1567-133X, E-ISSN 1872-7298, Vol. 4, no 5, 521-528 p.Article in journal (Refereed) Published
Abstract [en]

Liver development is based on reciprocal interactions between ventral foregut endoderm and adjacent mesenchymal tissues. Targeted disruption of the LIM-homeobox gene Lhx2 has revealed that it is important for the expansion of the liver during embryonic development, whereas it appears not to be involved in the induction of hepatic fate. It is not known whether Lhx2 is expressed in the endodermal or mesenchymal portion of the liver, or if the cells normally expressing Lhx2 are absent or present in the liver of Lhx2(-/-) embryos. To address this we have analyzed gene expression from the Lhx2 locus during hepatic development in wild type and Lhx2(-/-) mice. Lhx2 is expressed in cells of the septum transversum mesenchyme adjacent to the liver bud from embryonic day 9. The hepatic cords subsequently migrate into and intermingle with the Lhx2+ cells of the septum transversum mesenchyme. Lhx2 expression is thereafter maintained in a subpopulation of mesenchymal cells in the liver until adult life. In adult liver the Lhx2+ mesenchymal cells co-express desmin, a marker associated with stellate cells. At embryonic day 10.5, cells expressing the mutant Lhx2 allel are present in Lhx2(-/-) livers, and expression of Hlx, hepatocyte growth factor, Hex and Prox1, genes known to be important in liver development, is independent of functional Lhx2 expression. Thus, Lhx2 is specifically expressed in the liver-associated septum transversum mesenchyme that subsequently becomes an integral part of the liver and the formation of these mesenchymal cells does not require functional Lhx2.

Bone morphogenetic proteins; Desmin; Embryonic development; Epithelial–mesenchymal interactions; Fetal liver; Foregut diverticulum; Hex; Hepatocyte growth factor; Hlx; Ito cell; Liver bud; Lhx2; Lhx2 mutant phenotype; LIM-homeobox gene; Prox1; Septum transversum mesenchyme; Stellate cells; Transcription factor; Ventral foregut endoderm; Vimentin
National Category
Cell and Molecular Biology
urn:nbn:se:umu:diva-4064 (URN)10.1016/j.modgep.2004.03.001 (DOI)15261829 (PubMedID)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2010-08-02Bibliographically approved
2. Lhx2-/- mice develop liver fibrosis
Open this publication in new window or tab >>Lhx2-/- mice develop liver fibrosis
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2004 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 47, 16549-16554 p.Article in journal (Refereed) Published
Abstract [en]

Liver fibrosis is a wound-healing response to chronic injury of any type and is characterized by a progressive increase in deposition of extracellular matrix (ECM) proteins, the major source of which are activated hepatic stellate cells (HSCs). Because the LIM homeobox gene Lhx2 is expressed in HSCs and liver development in Lhx2 / mice is disrupted, we analyzed liver development in Lhx2 / embryos in detail. Lhx2 / embryos contain numerous activated HSCs and display a progressively increased deposition of the ECM proteins associated with liver fibrosis, suggesting that Lhx2 inhibits HSC activation. Transfection of Lhx2 cDNA into a human HSC line down-regulates expression of genes characteristic of activated HSCs. Moreover, the Lhx2 / liver display a disrupted cellular organization and an altered gene expression pattern of the intrahepatic endodermal cells, and the increased deposition of ECM proteins precedes these abnormalities. Collectively these results show that Lhx2 negatively regulates HSC activation, and its inactivation in developing HSCs appears therefore to mimic the signals that are triggered by the wound-healing response to chronic liver injury. This study establishes a spontaneous and reproducible animal model for hepatic fibrosis and reveals that Lhx2 expression in HSCs is important for proper cellular organization and differentiation of the liver.

hepatic stellate cells, cirrhosis, liver regeneration
urn:nbn:se:umu:diva-4275 (URN)10.1073/pnas.0404678101 (DOI)15536133 (PubMedID)
Available from: 2004-11-19 Created: 2004-11-19 Last updated: 2010-09-08Bibliographically approved
3. Multipotent hematopoietic progenitor cells immortalized by Lhx2 self-renew by a cell nonautonomous mechanism
Open this publication in new window or tab >>Multipotent hematopoietic progenitor cells immortalized by Lhx2 self-renew by a cell nonautonomous mechanism
2001 (English)In: Experimental Hematology, ISSN 0301-472X, E-ISSN 1873-2399, Vol. 29, no 8, 1019-1028 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Direct molecular and cellular studies of hematopoietic stem cells (HSCs) are hampered by the low levels of HSCs in hematopoietic tissues. To address these issues, we generated immortalized multipotent hematopoietic precursor cell (HPC) lines by expressing the LIM-homeobox gene Lhx2 (previously LH2) in hematopoietic progenitors derived from embryonic stem cells differentiated in vitro.

MATERIALS AND METHODS: To validate further the relevance of the HPC lines as a model for normal HSCs, we analyzed in detail the growth requirements of HPC lines in vitro.

RESULTS: Lhx2 immortalized the HPC lines by a putatively novel and cell nonautonomous mechanism. Self-renewal of the HPC lines is dependent on functional Lhx2 expression. Most early-acting hematopoiesis-related growth factors show synergistic effects on the HPC lines, whereas late-acting factors do not induce differentiation by themselves. Transforming growth factor-beta(1) is a potent inhibitor of proliferation of the HPC lines. HPC lines form cobblestone areas with high efficiency when seeded onto stromal cell lines, and the cobblestone area-forming cell can be maintained in these cultures for several months.

CONCLUSIONS: Our data show that, in many respects, HPC lines are similar to normal hematopoietic progenitor/stem cells on the cellular level, in contrast to most previously described multipotent hematopoietic cell lines. The cell nonautonomous mechanism for immortalization of the HPC lines suggests that Lhx2 regulates, directly or indirectly, soluble mediators involved in self-renewal of the HPC lines.

urn:nbn:se:umu:diva-4276 (URN)10.1016/S0301-472X(01)00666-X (DOI)11495708 (PubMedID)
Available from: 2004-11-19 Created: 2004-11-19 Last updated: 2010-09-08Bibliographically approved
4. Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines
Open this publication in new window or tab >>Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines
Show others...
2004 (English)In: Blood, ISSN 0006-4971, Vol. 104, no 1, 51-57 p.Article in journal (Refereed) Published
Abstract [en]

The Steel factor (SF) and its receptor c-Kit play a critical role for various cell types at different levels in the hematopoietic hierarchy. Whether similar or distinct signaling pathways are used upon c-Kit activation in different cell types within the hematopoietic hierarchy is not known. To study c-Kit signaling pathways in the hematopoietic system we have compared c-Kit downstream signaling events in SF-dependent hematopoietic stem cell (HSC)–like cell lines to those of mast cells. Both Erk and protein kinase B (PKB)/Akt are activated by ligand-induced activation of the c-Kit receptor in the HSC-like cell lines. Surprisingly, phosphoinositide-3 (PI-3) kinase inhibitors block not only PKB/Akt activation but also activation of Raf and Erk. SF-induced activation of Ras is not affected by inhibition of PI-3 kinase. In mast cells and other more committed hematopoietic precursors, the activation of Erk by SF is not PI-3 kinase dependent. Our results suggest that a molecular signaling switch occurs during differentiation in the hematopoietic system whereby immature hematopoietic progenitor/stem cells use a PI-3 kinase–sensitive pathway in the activation of both Erk and PKB/Akt, which is then switched upon differentiation to the more commonly described PI-3 kinase–independent mitogen-activated protein (MAP) kinase pathway.

1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors/*metabolism, Animals, Cell Line, Chromones/pharmacology, Enzyme Activation, Enzyme Inhibitors/pharmacology, Femur/cytology, Hematopoietic Stem Cells/cytology/*enzymology, MAP Kinase Signaling System/*physiology, Mice, Mice; Inbred C57BL, Mitogen-Activated Protein Kinases/metabolism, Morpholines/pharmacology, Phosphorylation, Protein-Serine-Threonine Kinases/metabolism, Proto-Oncogene Proteins/metabolism, Proto-Oncogene Proteins c-akt, Proto-Oncogene Proteins c-kit/*metabolism, Proto-Oncogene Proteins c-raf/metabolism, Stem Cell Factor/metabolism, ras Proteins/metabolism
urn:nbn:se:umu:diva-15405 (URN)10.1182/blood-2003-07-2554 (DOI)14996702 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2010-01-29Bibliographically approved

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