70
C. Rochette-Egly
software allowed a gene network analysis of each gene and the identification of stage
specific gene expression signatures. Interestingly, Affymetrix and Agilent developed
arrays with long oligonucleotide sequences directly synthesized in situ, on the surface. Such microarrays (Affymetrix GeneChip
® ) have been used extensively during
the 15 last years to analyze the contribution of RA signaling to lineage specific differentiation (Mendoza-Parra et al. 2016, 2011; Simandi et al. 2010) and to select
early direct RA target genes (Su and Gudas 2008).
In a comparison of wild type (WT) and RARγ null F9 cells, both in the absence
and in the presence of RA, the group of Gudas (Su and Gudas 2008) highlighted the
function of RARγ in the regulation of several Hox genes and of genes encoding signaling pathways (sfrp2, Tie1), membrane proteins (Emp1) and enzymes involved in
RA metabolism (Cyp26a1) or glucose metabolism (Fbp-2) (Table 3.1). As RARγ null
F9 cells do not differentiate in response to RA (Al Tanoury et al. 2014; Taneja et al.
1997), these results highlighted the importance of the RARγ subtype in development.
The group of Gronemeyer also compared the RA-regulated genes during endodermal F9 (Mendoza-Parra et al. 2011) and neuronal P19 cells (Mendoza-Parra et al.
2016) differentiation at different time points after the addition of RARγ or RARα
agonists. Remarkably, in F9 cells, the RARγ-specific agonist, (and not the RARαspecific agonist), induced a pattern of differential gene expression that was similar to
that induced by RA, corroborating that RARγ is driving differentiation in these cells
(Taneja et al. 1997). In contrast, in P19 cells, the RARα-specific agonist (and not the
RARγ agonist) fully recapitulated the transcriptome profile of P19 cells, consistent
with the fact that neuronal differentiation of these cells is driven by RARα. In these
studies, a biphasic global gene induction was also observed, with a peak at 2 h and
a strong wave at 48–72 h. Interestingly, 60% of genes were commonly regulated in
both cell lines, albeit with different kinetics in some cases. These commonly RAregulated genes are classical RA-induced genes (e.g., Rarb, Cyp26a1, or Hoxa1) and
down-regulated pluripotency factors (Sox2, Nanog). As expected, the P19-specific
RA-induced genes were involved in neuronal fate commitment (Ascl1, Gata3, Gbx2
and Tal2), while the F9-specific genes (Gata6, Gli2, Sox7 and cdx1) were involved
in endoderm or mesoderm development (Table 3.1).
Recently, an alternative approach to the construction of arrays was created and
licensed by Illumina. This novel technology (Bead Array Microarrays) consists
of synthesizing oligonucleotides on small beads and in depositing these beads in
microwells on the surface of an array substrate. This technology has been used to
analyze the RA-regulated genes during the RA-induced neuronal differentiation of
hESCs (Illumina Sentrix
® Human HT-12 V3 BeadChips) (Fathi et al. 2011) and
mESCs (Illumina
® Mouse WG-6V1.0 BeadChips) (Akanuma et al. 2012) (Table
3.1). Thousands of genes were found to be RA-regulated with dynamic expression
patterns and stage-specific expression signatures. The group of Gudas (Kashyap
et al. 2013) also used BeadChips (Illumina
® MouseRef-8 v2.0) to compare WT and
RARγ-/- mESCs and corroborated that most of the RA-regulated genes (homeobox
genes involved in morphogenesis, axis formation, and tissue patterning and genes
involved in RA metabolism) are dependent upon functional RARγ signaling (Table
3.1).
C. Rochette-Egly
software allowed a gene network analysis of each gene and the identification of stage
specific gene expression signatures. Interestingly, Affymetrix and Agilent developed
arrays with long oligonucleotide sequences directly synthesized in situ, on the surface. Such microarrays (Affymetrix GeneChip
® ) have been used extensively during
the 15 last years to analyze the contribution of RA signaling to lineage specific differentiation (Mendoza-Parra et al. 2016, 2011; Simandi et al. 2010) and to select
early direct RA target genes (Su and Gudas 2008).
In a comparison of wild type (WT) and RARγ null F9 cells, both in the absence
and in the presence of RA, the group of Gudas (Su and Gudas 2008) highlighted the
function of RARγ in the regulation of several Hox genes and of genes encoding signaling pathways (sfrp2, Tie1), membrane proteins (Emp1) and enzymes involved in
RA metabolism (Cyp26a1) or glucose metabolism (Fbp-2) (Table 3.1). As RARγ null
F9 cells do not differentiate in response to RA (Al Tanoury et al. 2014; Taneja et al.
1997), these results highlighted the importance of the RARγ subtype in development.
The group of Gronemeyer also compared the RA-regulated genes during endodermal F9 (Mendoza-Parra et al. 2011) and neuronal P19 cells (Mendoza-Parra et al.
2016) differentiation at different time points after the addition of RARγ or RARα
agonists. Remarkably, in F9 cells, the RARγ-specific agonist, (and not the RARαspecific agonist), induced a pattern of differential gene expression that was similar to
that induced by RA, corroborating that RARγ is driving differentiation in these cells
(Taneja et al. 1997). In contrast, in P19 cells, the RARα-specific agonist (and not the
RARγ agonist) fully recapitulated the transcriptome profile of P19 cells, consistent
with the fact that neuronal differentiation of these cells is driven by RARα. In these
studies, a biphasic global gene induction was also observed, with a peak at 2 h and
a strong wave at 48–72 h. Interestingly, 60% of genes were commonly regulated in
both cell lines, albeit with different kinetics in some cases. These commonly RAregulated genes are classical RA-induced genes (e.g., Rarb, Cyp26a1, or Hoxa1) and
down-regulated pluripotency factors (Sox2, Nanog). As expected, the P19-specific
RA-induced genes were involved in neuronal fate commitment (Ascl1, Gata3, Gbx2
and Tal2), while the F9-specific genes (Gata6, Gli2, Sox7 and cdx1) were involved
in endoderm or mesoderm development (Table 3.1).
Recently, an alternative approach to the construction of arrays was created and
licensed by Illumina. This novel technology (Bead Array Microarrays) consists
of synthesizing oligonucleotides on small beads and in depositing these beads in
microwells on the surface of an array substrate. This technology has been used to
analyze the RA-regulated genes during the RA-induced neuronal differentiation of
hESCs (Illumina Sentrix
® Human HT-12 V3 BeadChips) (Fathi et al. 2011) and
mESCs (Illumina
® Mouse WG-6V1.0 BeadChips) (Akanuma et al. 2012) (Table
3.1). Thousands of genes were found to be RA-regulated with dynamic expression
patterns and stage-specific expression signatures. The group of Gudas (Kashyap
et al. 2013) also used BeadChips (Illumina
® MouseRef-8 v2.0) to compare WT and
RARγ-/- mESCs and corroborated that most of the RA-regulated genes (homeobox
genes involved in morphogenesis, axis formation, and tissue patterning and genes
involved in RA metabolism) are dependent upon functional RARγ signaling (Table
3.1).