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. 2011;6(5):e20197.
doi: 10.1371/journal.pone.0020197. Epub 2011 May 26.

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

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

Myrto Bami et al. PLoS One. 2011.

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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.
Figure 2
Figure 2. Hoxb1 regulation of selected genes validated by RT-PCR.
(A) Hoxb1 mediated fold regulation of CRABPI, CRABPII and Lhx5 and Lhx9 expression in the short (s) and long (l) induction schemes. As a comparison, the regulation of two know Hoxb1 targets, Hoxb1 itself and Hoxb2 is shown. (B) Real – time PCR confirmation of differences in the expression of CRABPI and II and Lhx9 and 5 in Hoxb1 and Hoxb1+ cells.
Figure 3
Figure 3. Expression of CRABPI and CRABPII in the hindbrain of wt and Hoxb1−/− mouse embryos at 10.5 dpc.
(A – D) Ventricular views of flat mounted wt (A, C) and Hoxb1 −/− (B, D) mouse hindbrains stained with a CRABPI riboprobe (A, B) and a CRABPII riboprobe (C, D) at 10.5 dpc. r4-specific expression is denoted by arrows (A, C), arrowheads (A) and brackets (C) in wt hindbrains. r4-specific expression is lost in Hoxb1 −/− hindbrains and denoted by asterisks (B, D). Scale bar corresponds to 450 µm.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. 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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