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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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Expression of Lhx5 in the chick hindbrain after                                Hoxb1 gain of function experiments.(A – F) Expression of Lhx9 in whole mount (A, B),                            flat mounted hindbrains (ventricular view) (C, D) and r1 transverse                            sections (E, F) of chick embryos electroporated at stage HH 10–11                            and analyzed 48 h PE by Lhx9 in situ hybridization                            alone (A, B) or in combination with Hoxb1 immunofluorescence (C –                            F). Lxh9 is expressed in the mantle layer of dorsal r1 in a thick stripe                            that subsequently thins out along the rhombic lip of the rest of the                            hindbrain (arrowheads A, C, E, F). This expression is lost at sites of                            Hoxb1 ectopic expression (asterisks B, D, E, F). Scale bar corresponds                            to 300 µm in C, D and to 150 µm in E, F.
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pone-0020197-g005: Expression of Lhx5 in the chick hindbrain after Hoxb1 gain of function experiments.(A – F) Expression of Lhx9 in whole mount (A, B), flat mounted hindbrains (ventricular view) (C, D) and r1 transverse sections (E, F) of chick embryos electroporated at stage HH 10–11 and analyzed 48 h PE by Lhx9 in situ hybridization alone (A, B) or in combination with Hoxb1 immunofluorescence (C – F). Lxh9 is expressed in the mantle layer of dorsal r1 in a thick stripe that subsequently thins out along the rhombic lip of the rest of the hindbrain (arrowheads A, C, E, F). This expression is lost at sites of Hoxb1 ectopic expression (asterisks B, D, E, F). Scale bar corresponds to 300 µm in C, D and to 150 µm in E, F.

Mentions: Lhx9 is broadly expressed in the mouse developing CNS in the forebrain, midbrain, hindbrain and spinal cord. In the mouse, its levels of expression, as detected by RNA in situ hybridization, in the hindbrain were relatively low with no specific r4 pattern [43]. Using a chick Lhx9 in situ probe [39] we found that cLhx9 is expressed in dorsal r1 and in a thin dorsal stripe in the developing chick hindbrain (arrowheads, Fig. 1A, C, D). Thus we choose to do our analysis in chick embryos by ectopically expressing Hoxb1 in the developing hindbrain. Chick embryos were electroporated with Hoxb1 expression vector at HH 10–11 and RNA in situ hybridization was performed 48h PE to detect cLhx9 expression. The expression of cLhx9 in the non-electroporated side was strong along the whole length of the hindbrain but, in the electroporated side, cLhx9 was down regulated in response to ectopic Hoxb1 expression. This was evident in whole mount embryos and flat mounted hindbrains (asterisks in Fig. 5B, C, D) and these findings were confirmed by cryosections (Fig. 5E, F).


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)

Expression of Lhx5 in the chick hindbrain after                                Hoxb1 gain of function experiments.(A – F) Expression of Lhx9 in whole mount (A, B),                            flat mounted hindbrains (ventricular view) (C, D) and r1 transverse                            sections (E, F) of chick embryos electroporated at stage HH 10–11                            and analyzed 48 h PE by Lhx9 in situ hybridization                            alone (A, B) or in combination with Hoxb1 immunofluorescence (C –                            F). Lxh9 is expressed in the mantle layer of dorsal r1 in a thick stripe                            that subsequently thins out along the rhombic lip of the rest of the                            hindbrain (arrowheads A, C, E, F). This expression is lost at sites of                            Hoxb1 ectopic expression (asterisks B, D, E, F). Scale bar corresponds                            to 300 µm in C, D and to 150 µm in E, F.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020197-g005: Expression of Lhx5 in the chick hindbrain after Hoxb1 gain of function experiments.(A – F) Expression of Lhx9 in whole mount (A, B), flat mounted hindbrains (ventricular view) (C, D) and r1 transverse sections (E, F) of chick embryos electroporated at stage HH 10–11 and analyzed 48 h PE by Lhx9 in situ hybridization alone (A, B) or in combination with Hoxb1 immunofluorescence (C – F). Lxh9 is expressed in the mantle layer of dorsal r1 in a thick stripe that subsequently thins out along the rhombic lip of the rest of the hindbrain (arrowheads A, C, E, F). This expression is lost at sites of Hoxb1 ectopic expression (asterisks B, D, E, F). Scale bar corresponds to 300 µm in C, D and to 150 µm in E, F.
Mentions: Lhx9 is broadly expressed in the mouse developing CNS in the forebrain, midbrain, hindbrain and spinal cord. In the mouse, its levels of expression, as detected by RNA in situ hybridization, in the hindbrain were relatively low with no specific r4 pattern [43]. Using a chick Lhx9 in situ probe [39] we found that cLhx9 is expressed in dorsal r1 and in a thin dorsal stripe in the developing chick hindbrain (arrowheads, Fig. 1A, C, D). Thus we choose to do our analysis in chick embryos by ectopically expressing Hoxb1 in the developing hindbrain. Chick embryos were electroporated with Hoxb1 expression vector at HH 10–11 and RNA in situ hybridization was performed 48h PE to detect cLhx9 expression. The expression of cLhx9 in the non-electroporated side was strong along the whole length of the hindbrain but, in the electroporated side, cLhx9 was down regulated in response to ectopic Hoxb1 expression. This was evident in whole mount embryos and flat mounted hindbrains (asterisks in Fig. 5B, C, D) and these findings were confirmed by cryosections (Fig. 5E, F).

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