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Directed neural differentiation of mouse embryonic stem cells is a sensitive system for the identification of novel Hox gene effectors.

Bami M, Episkopou V, Gavalas A, Gouti M - PLoS ONE (2011)

Bottom Line: We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9.Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation.The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Laboratory, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

ABSTRACT
The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes.

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ES differentiation and Hoxb1 induction scheme, comparison of gene                            expression profiling results.(A) Graphic representation of ESTet-On/Hoxb1 cell                            differentiation towards neural stem cells (NSCs) for the identification                            of Hoxb1 target genes. The induction length is shown in red (days) and                            blue arrows indicate the time point of microarray gene expression                            analysis. (B) Venn diagram of genes differentially regulated in the long                            and short Hoxb1 induction schemes. (C) Pie charts of up                            and down regulated genes in the two induction schemes.
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pone-0020197-g001: ES differentiation and Hoxb1 induction scheme, comparison of gene expression profiling results.(A) Graphic representation of ESTet-On/Hoxb1 cell differentiation towards neural stem cells (NSCs) for the identification of Hoxb1 target genes. The induction length is shown in red (days) and blue arrows indicate the time point of microarray gene expression analysis. (B) Venn diagram of genes differentially regulated in the long and short Hoxb1 induction schemes. (C) Pie charts of up and down regulated genes in the two induction schemes.

Mentions: The generation and neural differentiation of the mouse ESTet-On/Hoxb1 cells were as described previously [31]. For the short Hoxb1 induction scheme doxycycline (dox) was added during the last day of the selection period and for one additional day during the expansion stage (Fig. 1A). Gene expression profiling was carried out for biological triplicates for both dox induced (Hoxb1+) and uninduced (Hoxb1−) cells as described earlier [31] and the Affymetrix Mouse Genome 430A array was used. Microarray data are deposited in the public access Array Express database (Experiment ID E-MIMR-441). The list of regulated genes for the short induction scheme was restricted to genes with 0.75> fold regulation >1.3 and genes that were also present in the long induction scheme.


Directed neural differentiation of mouse embryonic stem cells is a sensitive system for the identification of novel Hox gene effectors.

Bami M, Episkopou V, Gavalas A, Gouti M - PLoS ONE (2011)

ES differentiation and Hoxb1 induction scheme, comparison of gene                            expression profiling results.(A) Graphic representation of ESTet-On/Hoxb1 cell                            differentiation towards neural stem cells (NSCs) for the identification                            of Hoxb1 target genes. The induction length is shown in red (days) and                            blue arrows indicate the time point of microarray gene expression                            analysis. (B) Venn diagram of genes differentially regulated in the long                            and short Hoxb1 induction schemes. (C) Pie charts of up                            and down regulated genes in the two induction schemes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102681&req=5

pone-0020197-g001: ES differentiation and Hoxb1 induction scheme, comparison of gene expression profiling results.(A) Graphic representation of ESTet-On/Hoxb1 cell differentiation towards neural stem cells (NSCs) for the identification of Hoxb1 target genes. The induction length is shown in red (days) and blue arrows indicate the time point of microarray gene expression analysis. (B) Venn diagram of genes differentially regulated in the long and short Hoxb1 induction schemes. (C) Pie charts of up and down regulated genes in the two induction schemes.
Mentions: The generation and neural differentiation of the mouse ESTet-On/Hoxb1 cells were as described previously [31]. For the short Hoxb1 induction scheme doxycycline (dox) was added during the last day of the selection period and for one additional day during the expansion stage (Fig. 1A). Gene expression profiling was carried out for biological triplicates for both dox induced (Hoxb1+) and uninduced (Hoxb1−) cells as described earlier [31] and the Affymetrix Mouse Genome 430A array was used. Microarray data are deposited in the public access Array Express database (Experiment ID E-MIMR-441). The list of regulated genes for the short induction scheme was restricted to genes with 0.75> fold regulation >1.3 and genes that were also present in the long induction scheme.

Bottom Line: We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9.Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation.The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Laboratory, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

ABSTRACT
The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes.

Show MeSH
Related in: MedlinePlus