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.
Table 1.
Hoxb1-regulated biological processes.
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.
Table 2.
Hoxb1 regulated genes.
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.
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.
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.