78
C. Rochette-Egly
approaches were performed after exogenous RA applications that may produce opposite, toxic, or teratogenic effects, depending on the concentration, stage or duration
of RA exposure (Hermsen et al. 2013; Pickering et al. 2017). Thus, to decipher
the gene pathways regulated by RA, methods interfering with endogenous retinoid
signaling, such as genetic loss of function, morpholinos, or RAR/RXR antagonists
(which minimize the influence of endogenous RA), should be promising. To date,
only a few studies have used such strategies.
In one study, Xenopus embryos were treated with panRAR antagonists at the
blastula stage until the late neurula stage (Arima et al. 2005). A large number of
putative RAR target genes were identified, including known RA-responsive genes,
some of them overlapping with the known expression patterns for RARs or RALDH2.
More recently, in zebrafish gastrula, using RNA-seq (SOLiD 5500 sequencer), a
list of RA-responsive genes has been built up after RAR inhibition with specific
morpholinos (Samarut et al. 2014).
In the context of mouse embryonic development, WT and Retinaldehyde Dehydrogenase 2 (Raldh22/2) null-mutant mouse embryos (the latter being unable to
synthesize RA from maternally derived retinol) were compared. Using Affymetrix
Mouse Genome 430 2.0 GeneChip
® arrays, Chen et al. analyzed the transcriptomic
changes that occur in E8.8 foregut explants during induction of primary lung buds
(Chen et al. 2007). This study provided evidence of a novel regulatory mechanism
in which endogenous RA maintains low levels of Tgfβ signaling in the prospective
lung field of the foregut to allow Fgf10 expression and induction of primary lung
buds. On the other hand, with Affymetrix Mouse Gene 1.0 ST GeneChip
® arrays,
Dollé et al. analyzed the transcriptomic changes in anterior (forebrain) and posterior
(trunk) tissues, at early stages preceding the appearance of overt phenotypic abnormalities (Paschaki et al. 2013). Bioinformatic analysis of the data confirmed that RA
controls the expression of canonical RA target genes with RAREs (RARb, Hoxa1,
Cdx1, Stra6, Dhrs3, Crabp2). Interestingly, it also highlighted novel RA-regulated
genes belonging to several major embryonic signaling pathways (Fgf, Bmp, Tgfb,
Wnt, Hedgehog). Though it is currently unknown whether there is a direct effect of
RA on the expression of these genes, these studies suggested there is a combinatorial
regulation of embryonic RA-dependent genes by several families of transcriptional
regulators (Cunningham and Duester 2015; Paschaki et al. 2013).
Though transcriptomic analyses have begun to yield valuable information in
embryonic tissues, the information on the molecular targets of RA signaling is still
fragmentary. Indeed, studies at the chromatin level (ChIP-seq) have thus far been
hindered by a lack of high-grade, specific RAR antibodies and by the fact that they
require large amounts of material. Deciphering the molecular events that regulate
in vivo RA signaling remains a huge challenge. To accomplish this goal will require
the development of more sensitive, genome-wide, high throughput, studies that can
be performed on specialized embryonic tissues.
C. Rochette-Egly
approaches were performed after exogenous RA applications that may produce opposite, toxic, or teratogenic effects, depending on the concentration, stage or duration
of RA exposure (Hermsen et al. 2013; Pickering et al. 2017). Thus, to decipher
the gene pathways regulated by RA, methods interfering with endogenous retinoid
signaling, such as genetic loss of function, morpholinos, or RAR/RXR antagonists
(which minimize the influence of endogenous RA), should be promising. To date,
only a few studies have used such strategies.
In one study, Xenopus embryos were treated with panRAR antagonists at the
blastula stage until the late neurula stage (Arima et al. 2005). A large number of
putative RAR target genes were identified, including known RA-responsive genes,
some of them overlapping with the known expression patterns for RARs or RALDH2.
More recently, in zebrafish gastrula, using RNA-seq (SOLiD 5500 sequencer), a
list of RA-responsive genes has been built up after RAR inhibition with specific
morpholinos (Samarut et al. 2014).
In the context of mouse embryonic development, WT and Retinaldehyde Dehydrogenase 2 (Raldh22/2) null-mutant mouse embryos (the latter being unable to
synthesize RA from maternally derived retinol) were compared. Using Affymetrix
Mouse Genome 430 2.0 GeneChip
® arrays, Chen et al. analyzed the transcriptomic
changes that occur in E8.8 foregut explants during induction of primary lung buds
(Chen et al. 2007). This study provided evidence of a novel regulatory mechanism
in which endogenous RA maintains low levels of Tgfβ signaling in the prospective
lung field of the foregut to allow Fgf10 expression and induction of primary lung
buds. On the other hand, with Affymetrix Mouse Gene 1.0 ST GeneChip
® arrays,
Dollé et al. analyzed the transcriptomic changes in anterior (forebrain) and posterior
(trunk) tissues, at early stages preceding the appearance of overt phenotypic abnormalities (Paschaki et al. 2013). Bioinformatic analysis of the data confirmed that RA
controls the expression of canonical RA target genes with RAREs (RARb, Hoxa1,
Cdx1, Stra6, Dhrs3, Crabp2). Interestingly, it also highlighted novel RA-regulated
genes belonging to several major embryonic signaling pathways (Fgf, Bmp, Tgfb,
Wnt, Hedgehog). Though it is currently unknown whether there is a direct effect of
RA on the expression of these genes, these studies suggested there is a combinatorial
regulation of embryonic RA-dependent genes by several families of transcriptional
regulators (Cunningham and Duester 2015; Paschaki et al. 2013).
Though transcriptomic analyses have begun to yield valuable information in
embryonic tissues, the information on the molecular targets of RA signaling is still
fragmentary. Indeed, studies at the chromatin level (ChIP-seq) have thus far been
hindered by a lack of high-grade, specific RAR antibodies and by the fact that they
require large amounts of material. Deciphering the molecular events that regulate
in vivo RA signaling remains a huge challenge. To accomplish this goal will require
the development of more sensitive, genome-wide, high throughput, studies that can
be performed on specialized embryonic tissues.