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Stem cell function and organ development: analysis of Lhx2 function in hematopoietic stem cells and eye development
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). (Leif Carlsson)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Stamcellsfunktion och organutveckling : studier av blodstamceller och ögonutveckling (Swedish)
Abstract [en]

When a multicellular organism suffers damages to tissues/organs it heals itself by either substituting the lost cellular matrix by scar formation or by regenerating the lost tissue. Regeneration likely occurs by a recapitulation of the developmental process that formed the organ. Many processes regulating organ development are based on epithelial-mesenchymal interactions and a strict control of organ specific stem/progenitor cells. Elucidation of the molecular basis of these processes is therefore vital in order to develop novel therapies in regenerative medicine. The LIM homebox gene Lhx2 is interesting in this context since Lhx2 has been shown to be important for the formation of several organs by regulating epithelial-mesenchymal interactions and progenitor cell function. Targeted inactivation of Lhx2 leads to a lethal anemia due to malformed liver and severe neural abnormalities such as hypoplasia of the forebrain and anophtalmia. Thus, elucidation of the mechanisms of the function of Lhx2 in different organ systems would give important insights into the molecular mechanisms regulating epithelial-mesenchymal interactions and stem/progenitor cell function.

To elucidate the function of Lhx2 in the hematopoietic system Lhx2 was initially expressed in hematopoietic progenitor cells derived from ES cells differentiated in vitro using retroviral vectors. This approach led to the generation of hematopoietic stem cell (HSC)-like cell lines suggesting that Lhx2 could impact HSC function. However neither the specificity nor the efficiency of the Lhx2-induced phenotype could be determined using this approach. To be able to elucidate the function of Lhx2 in the hematopoietic system, an ES cell line with inducible Lhx2 expression was generated. Lhx2 expression induces self-renewal of a distinct hematopoietic progenitor cell from which HSC-like cell lines were established. Down-regulation of Lhx2 in these HSC-like cell lines leads to a rapid loss of stem cell character, providing a good model to study the molecular function of Lhx2 in hematopoietic stem/progenitor cells. A global gene expression analysis was performed comparing the Lhx2+ stem cell population to the Lhx2- differentiated progeny. This approach identified genes putatively linked to self-renewal/differentiation of HSCs. A considerable proportion of the genes showed an overlapping gene expression pattern with Lhx2 expression in tissue of non-hematopoietic origin suggesting that Lhx2 function in stem/progenitor cells partly overlap with Lhx2 function during organ development.

In order to define other Lhx2-dependent progenitor cell populations and to generate a tool to analyze the function of Lhx2 in organ development a new transgenic mouse model was generated. By using a specific part of the Lhx2 promoter to drive expression of Cre recombinase in vivo (Lhx2-Cre mice) we have been able to define the first eye committed progenitor cells in the forebrain. By using the Lhx2-Cre mice it will be possible to distinguish the function of genes during eye development from their function in the patterning of the forebrain e.g. the eye field transcription factors. Conditional inactivation of Lhx2 in these eye specific progenitor cells causes an immediate developmental arrest. The transgene is also active in Lhx2-/- embryonic forebrain, but re-expression of Lhx2 in Lhx2-/- progenitor cells only promote formation of retinal pigment epithelium cells. Analysis of genes expressed by the Lhx2+ stem cell population allowed us to define novel genes putatively linked to Lhx2 function in eye development. Thus, we have defined the progenitor cells in the forebrain committed to eye development and the expansion and patterning of these progenitors are dependent on Lhx2. Although commitment to eye development is Lhx2-independent, Lhx2 might be important for the acquisition of the oligopotent fate of these progenitor cells.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2010. , 112 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1365
Series
978-91-7459-058-6, ISSN 0346-6612 ; 1365
Keyword [en]
Lhx2, hematopoietic system, hematopoietic stem cell, progenitor, embryonic stem cell, embryoid body, eye development, forebrain, eye field transcription factor
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:umu:diva-35933ISBN: 978-91-7459-058-6 (print)OAI: oai:DiVA.org:umu-35933DiVA: diva2:350110
Public defence
2010-10-01, Major Groove, Byggnad 6L, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2010-09-14 Created: 2010-09-10 Last updated: 2010-09-14Bibliographically approved
List of papers
1. Lhx2 promotes self-renewal of a distinct multipotent hematopoietic progenitor cell in embryoid bodies
Open this publication in new window or tab >>Lhx2 promotes self-renewal of a distinct multipotent hematopoietic progenitor cell in embryoid bodies
2008 (English)In: PLoS one, ISSN 1932-6203, Vol. 3, no 4, e2025- p.Article in journal (Refereed) Published
Abstract [en]

The molecular mechanisms regulating the expansion of the hematopoietic system including hematopoietic stem cells (HSCs) in the fetal liver during embryonic development are largely unknown. The LIM-homeobox gene Lhx2 is a candidate regulator of fetal hematopoiesis since it is expressed in the fetal liver and Lhx2−/− mice die in utero due to severe anemia. Moreover, expression of Lhx2 in embryonic stem (ES) cell-derived embryoid bodies (EBs) can lead to the generation of HSC-like cell lines. To further define the role of this transcription factor in hematopoietic regulation, we generated ES cell lines that enabled tet-inducible expression of Lhx2. Using this approach we observed that Lhx2 expression synergises with specific signalling pathways, resulting in increased frequency of colony forming cells in developing EB cells. The increase in growth factor-responsive progenitor cells directly correlates to the efficiency in generating HSC-like cell lines, suggesting that Lhx2 expression induce self-renewal of a distinct multipotential hematopoietic progenitor cell in EBs. Signalling via the c-kit tyrosine kinase receptor and the gp130 signal transducer by IL-6 is necessary and sufficient for the Lhx2 induced self-renewal. While inducing self-renewal of multipotential progenitor cells, expression of Lhx2 inhibited proliferation of primitive erythroid precursor cells and interfered with early ES cell commitment, indicating striking lineage specificity of this effect.

National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-2625 (URN)10.1371/journal.pone.0002025 (DOI)18431502 (PubMedID)
Available from: 2007-10-10 Created: 2007-10-10 Last updated: 2010-09-14Bibliographically approved
2. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
Open this publication in new window or tab >>Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
Show others...
2006 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, 75- p.Article in journal (Refereed) Published
Abstract [en]

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.

Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.

Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.

Keyword
Animals, Cell Differentiation, Down-Regulation, Doxycycline/metabolism/pharmacology, Embryo/cytology/metabolism, Gene Expression Regulation, Hematopoietic Stem Cells/drug effects/*metabolism, Homeodomain Proteins/genetics/*metabolism, In Situ Hybridization, Mice, Models; Biological, Oligonucleotide Array Sequence Analysis, Tetracycline/metabolism/pharmacology, Transcription Factors/genetics/*metabolism
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-5917 (URN)10.1186/1471-2164-7-75 (DOI)16600034 (PubMedID)
Available from: 2007-12-03 Created: 2007-12-03 Last updated: 2017-12-14Bibliographically approved
3. Lhx2 is required for expansion of progenitor cells committed to eye development
Open this publication in new window or tab >>Lhx2 is required for expansion of progenitor cells committed to eye development
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-35992 (URN)
Available from: 2010-09-13 Created: 2010-09-13 Last updated: 2010-09-14Bibliographically approved

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