Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of Lhx5 in mouse and chick hindbrain                            after Hoxb1 loss and gain of function experiments,                            respectively.(A–C) Expression of Lhx5 in ventricular views of                            flat mounted hindbrains (A, B) and r4 transverse sections (C, D) using                                Lhx5 in situ hybridization alone (A, B) or in                            combination with Hoxb1 immunofluorescence (C, D) of wt (A, C) and                                Hoxb1−/− (B, D) 10.5 dpc                            embryos. Lhx5 is expressed in two characteristic                            stripes in the mantle layer of r4 (A, C denoted by brackets) that expand                            substantially in the absence of Hoxb1 (brackets in B, D). (E–H)                            Expression of Lhx5 in flat hindbrains (E, F) and r2                            transverse sections (G, H) of chick embryos electroporated at stage HH                            10–11 and analyzed 48 h PE by in situ hybridization for chick Lhx5                            and immunofluorescence for Hoxb1 (E–H). Expression of                                Lhx5 in the non-electroporated side is restricted                            at two dorsomedial r2 and r3 stripes (arrowheads E–H) and this                            expression is abolished upon Hoxb1 electroporation (asterisks                            E–H). Scale bar corresponds to 325 µm in A, B, to 100                            µm in C, D, G, H and to 125 µm in E, F.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3102681&req=5

pone-0020197-g004: Expression of Lhx5 in mouse and chick hindbrain after Hoxb1 loss and gain of function experiments, respectively.(A–C) Expression of Lhx5 in ventricular views of flat mounted hindbrains (A, B) and r4 transverse sections (C, D) using Lhx5 in situ hybridization alone (A, B) or in combination with Hoxb1 immunofluorescence (C, D) of wt (A, C) and Hoxb1−/− (B, D) 10.5 dpc embryos. Lhx5 is expressed in two characteristic stripes in the mantle layer of r4 (A, C denoted by brackets) that expand substantially in the absence of Hoxb1 (brackets in B, D). (E–H) Expression of Lhx5 in flat hindbrains (E, F) and r2 transverse sections (G, H) of chick embryos electroporated at stage HH 10–11 and analyzed 48 h PE by in situ hybridization for chick Lhx5 and immunofluorescence for Hoxb1 (E–H). Expression of Lhx5 in the non-electroporated side is restricted at two dorsomedial r2 and r3 stripes (arrowheads E–H) and this expression is abolished upon Hoxb1 electroporation (asterisks E–H). Scale bar corresponds to 325 µm in A, B, to 100 µm in C, D, G, H and to 125 µm in E, F.

Mentions: We then examined whether Hoxb1 can repress Lhx5 and Lhx9 expression in vivo. To study the expression of Lhx5 in the mouse hindbrain and specifically in r4 we performed whole mount in situ hybridization using a specific Lhx5 probe [38]. At 10.5 dpc in the hindbrain, Lhx5 is expressed in two dorsoventral stripes along r1–r6 in a rhombomere specific pattern. In wt r4 there is a paucity of Lhx5 expression in the ventral domain corresponding to the site of motor neuron progenitors whereas expression in the dorsal stripe is weaker compared to that of r2 and r3 and similar to that of r5 and r6 (brackets, Fig. 4A). In Hoxb1−/− r4 Lhx5 expression increases in both the dorsal and ventral domains and becomes similar with the expression pattern of r2 and r3 (brackets, Fig. 4B). Thus r4 expression of Hoxb1 and Lhx5 appeared to be mutually exclusive. This was confirmed, by Lhx5 and Hoxb1 immunofluorescence on wt r4 transverse sections (Fig. 4C). In Hoxb1−/− r4 expression of Lhx5 expanded in both ventral and dorsal expression domains. This was consistent with the in situ hybridization results and suggested that Hoxb1 may repress expression of Lhx5. To address this, we ectopically expressed Hoxb1 in the hindbrain of HH stage 10–11 chick embryos using in ovo electroporation. The embryos were analyzed 48 h post electroporation (PE) (HH stage 20) by whole mount in situ hybridization with the chick Lhx5 in situ hybridization probe [40] and Hoxb1 immunofluorescence. The cLxh5 at HH is expressed in two dorsomedial stripes in r2 and r3 (arrowheads Fig. 4E). Expression of cLhx5 was specifically down regulated in the areas where Hoxb1 was ectopically expressed (asterisks, Fig. 4E, F) and this was confirmed by r2 transverse sections showing that dorsal expression of Lhx5 was lost in the electroporated side of the embryo (Fig. 4G, H).


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 mouse and chick hindbrain                            after Hoxb1 loss and gain of function experiments,                            respectively.(A–C) Expression of Lhx5 in ventricular views of                            flat mounted hindbrains (A, B) and r4 transverse sections (C, D) using                                Lhx5 in situ hybridization alone (A, B) or in                            combination with Hoxb1 immunofluorescence (C, D) of wt (A, C) and                                Hoxb1−/− (B, D) 10.5 dpc                            embryos. Lhx5 is expressed in two characteristic                            stripes in the mantle layer of r4 (A, C denoted by brackets) that expand                            substantially in the absence of Hoxb1 (brackets in B, D). (E–H)                            Expression of Lhx5 in flat hindbrains (E, F) and r2                            transverse sections (G, H) of chick embryos electroporated at stage HH                            10–11 and analyzed 48 h PE by in situ hybridization for chick Lhx5                            and immunofluorescence for Hoxb1 (E–H). Expression of                                Lhx5 in the non-electroporated side is restricted                            at two dorsomedial r2 and r3 stripes (arrowheads E–H) and this                            expression is abolished upon Hoxb1 electroporation (asterisks                            E–H). Scale bar corresponds to 325 µm in A, B, to 100                            µm in C, D, G, H and to 125 µm in E, F.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020197-g004: Expression of Lhx5 in mouse and chick hindbrain after Hoxb1 loss and gain of function experiments, respectively.(A–C) Expression of Lhx5 in ventricular views of flat mounted hindbrains (A, B) and r4 transverse sections (C, D) using Lhx5 in situ hybridization alone (A, B) or in combination with Hoxb1 immunofluorescence (C, D) of wt (A, C) and Hoxb1−/− (B, D) 10.5 dpc embryos. Lhx5 is expressed in two characteristic stripes in the mantle layer of r4 (A, C denoted by brackets) that expand substantially in the absence of Hoxb1 (brackets in B, D). (E–H) Expression of Lhx5 in flat hindbrains (E, F) and r2 transverse sections (G, H) of chick embryos electroporated at stage HH 10–11 and analyzed 48 h PE by in situ hybridization for chick Lhx5 and immunofluorescence for Hoxb1 (E–H). Expression of Lhx5 in the non-electroporated side is restricted at two dorsomedial r2 and r3 stripes (arrowheads E–H) and this expression is abolished upon Hoxb1 electroporation (asterisks E–H). Scale bar corresponds to 325 µm in A, B, to 100 µm in C, D, G, H and to 125 µm in E, F.
Mentions: We then examined whether Hoxb1 can repress Lhx5 and Lhx9 expression in vivo. To study the expression of Lhx5 in the mouse hindbrain and specifically in r4 we performed whole mount in situ hybridization using a specific Lhx5 probe [38]. At 10.5 dpc in the hindbrain, Lhx5 is expressed in two dorsoventral stripes along r1–r6 in a rhombomere specific pattern. In wt r4 there is a paucity of Lhx5 expression in the ventral domain corresponding to the site of motor neuron progenitors whereas expression in the dorsal stripe is weaker compared to that of r2 and r3 and similar to that of r5 and r6 (brackets, Fig. 4A). In Hoxb1−/− r4 Lhx5 expression increases in both the dorsal and ventral domains and becomes similar with the expression pattern of r2 and r3 (brackets, Fig. 4B). Thus r4 expression of Hoxb1 and Lhx5 appeared to be mutually exclusive. This was confirmed, by Lhx5 and Hoxb1 immunofluorescence on wt r4 transverse sections (Fig. 4C). In Hoxb1−/− r4 expression of Lhx5 expanded in both ventral and dorsal expression domains. This was consistent with the in situ hybridization results and suggested that Hoxb1 may repress expression of Lhx5. To address this, we ectopically expressed Hoxb1 in the hindbrain of HH stage 10–11 chick embryos using in ovo electroporation. The embryos were analyzed 48 h post electroporation (PE) (HH stage 20) by whole mount in situ hybridization with the chick Lhx5 in situ hybridization probe [40] and Hoxb1 immunofluorescence. The cLxh5 at HH is expressed in two dorsomedial stripes in r2 and r3 (arrowheads Fig. 4E). Expression of cLhx5 was specifically down regulated in the areas where Hoxb1 was ectopically expressed (asterisks, Fig. 4E, F) and this was confirmed by r2 transverse sections showing that dorsal expression of Lhx5 was lost in the electroporated side of the embryo (Fig. 4G, H).

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