Together, our results indicate that zebrafish RARs function as context-dependent transcriptional activators. Ectopic expression of WT zebrafish RARs does not affect development nor sensitize the embryo to RA treatment. Hyperactive RARs can activate RA-responsive targets, although different VP16 fusions exhibit differing abilities to function depending on the context analyzed. To our surprise, we did not find that zebrafish versions of dominant negative RARs were able to act as transcriptional repressors in the early embryo, even though a human dnRAR can produce phenotypes consistent with loss of RA signaling. Together, our studies suggest that, during early A-P patterning of the zebrafish embryo, RARs function primarily as transcriptional activators rather than via a binary repressor-activator mechanism. Thus, it seems that the binary model for RAR function does not apply to all in vivo scenarios.
Our analysis of zebrafish RA-responsive transgenes demonstrates that RARE sites alone cannot recapitulate the expression patterns of all RA-responsive target genes, such as some anterior hox
genes or dhrs3a
(Marletaz et al., 2006
; Waxman et al., 2008
). The post-gastrulation initiation of RARE reporter transgene expression in zebrafish, observed in our studies and by Perz-Edwards and colleagues (2001)
, contrasts with the expression of two similar mouse transgenes, which begins in pregastrula embryos and more closely recapitulates the expression of known target genes (Balkan et al., 1992
; Perz-Edwards et al., 2001
; Rossant et al., 1991
). This contrast suggests the intriguing notion that there are species-specific differences in RARE responsiveness. However, we cannot rule out the possibility that these differences simply reflect position-dependent influences upon differentially integrated transgenes.
Restricting the comparison to different zebrafish RA-responsive transgenes, it seems likely that differences in expression result from locus-specific influences. For example, the more restricted expression of our reporters, in contrast to the more expansive expression of previously reported transgenes (Perz-Edwards et al., 2001
), may reflect the ability of our longer transgenes to better insulate themselves against outside influences. In support of strong context-dependent influences on minimal RARE sites, a recent study has examined the zebrafish cyp26a1
promoter, which is RA-responsive due to two RARE sites, but whose expression is also known to be controlled by other signaling pathways (Hu et al., 2008
). Transgenes driven by the cyp26a1
promoter have spatial and temporal expression patterns closely resembling the early expression of the endogenous gene, which differ significantly from the expression patterns of the synthetic RARE reporter transgenes presented here and previously (Perz-Edwards et al., 2001
). This further suggests that other transcription factors, acting either within or outside the transgene sequences, heavily influence the temporal and spatial responsiveness of genes with RARE sites within the zebrafish embryo.
The context-dependent nature of influences on RARE sites in the zebrafish embryo fits with the notion that the function of zebrafish RARs may be highly context-dependent. 3 of the 4 zebrafish RARs (RARaa, RARab and RARgb) are expressed ubiquitously prior to gastrulation, while later RARgb is expressed ubiquitously at low levels (Waxman and Yelon, 2007
). Thus, the locations where RA signaling occurs are likely to be dictated primarily by the availability of RA and other collaborating factors, rather than by the RARs themselves. We found multiple distinctions between the abilities of the RARvp fusion proteins in three different functional assays: RARE reporter transgene expression, endogenous target gene expression, and CNS patterning phenotypes. In general, RARab-vps were able to more strongly induce expression from the transgenic reporter compared to the RARga-vps, although there were also subtle differences in activation ability among the RARab-vps and RARga-vps. In contrast to the differences found with the RARE reporter transgenes, the RARab-vps and RARga-vps similarly induced the ectopic expression of endogenous target genes. The endogenous promoters may represent a more permissive context, which buffers differences between specific RARs within the embryo. Because the expression of endogenous target genes can be repressed by an overload of RAR-vp expression or the lack of an F domain, coordinated input from other factors must also be necessary for proper regulation of target gene expression.
Most of the hyperactive zebrafish RARs can eliminate the MHB, similar to the reported effects of increased RA signaling in Xenopus
(Blumberg et al., 1997
; Koide et al., 2001
). However, recent studies in zebrafish have not suggested a conserved role for RARs to act as repressors in the same context, although it cannot be ruled out that this could be due to an inability to adequately deplete the zebrafish RARs (Linville et al., 2008
). Interestingly, we have found that the individual zebrafish RAR-vps have differing potency in affecting the anterior CNS, although these differences did not correlate with their ability to activate the RARE reporter transgene. These differences in ability to affect the CNS seem to be additional indicators of context-specific modifiers at the transcriptional level in the embryo. Together, the distinctions between the assays revealed by the hyperactive RARs indicate that transgenic reporter activation can at times be misleading relative to the effect on endogenous targets. Thus, multiple convergent assays should be performed to achieve a complete assessment of RAR function in a given in vivo
In contrast to the abilities of the hyperactive RARs, zebrafish dnRARs are not capable of acting as transcriptional repressors in the early zebrafish embryo, suggesting that zebrafish RARs may have minimal endogenous requirement as transcriptional repressors. To our surprise, even replacing the RARab A- or F-domains with an Enr domain does not allow the chimeric proteins to function as transcriptional repressors in the zebrafish embryo. We have found that fusing the Enr domain to a zebrafish retinoid X receptor (a related nuclear hormone receptor and heterodimeric partner of the RARs) can specifically affect its function and strongly abrogate normal development when injected into zebrafish embryos (JSW and DY, unpublished observations), indicating that the Enr domain can alter transcriptional function of members of the nuclear hormone family of receptors in zebrafish. Furthermore, both the zebrafish dnRAR and RAR-enr proteins are able to function as transcriptional repressors in heterologous cell culture signaling assays. It is possible that the ineffectiveness of the ectopically expressed dnRAR and RAR-enr proteins in zebrafish embryos is due to technical limitations, such as a lack of significant overexpression above endogenous protein levels or variability between expression levels of different proteins. However, we think these scenarios are unlikely, based on the observed robust expression of tagged versions of these proteins (Fig. S2
What we have found more perplexing is that a human dnRARa can cause phenotypes resembling loss of RA signaling in the zebrafish embryo, suggesting it is able to function as a transcriptional repressor (Roy and Sagerström, 2004
; Waxman et al., 2008
) in this context. We presume that there are structural differences between human and zebrafish RARs that are responsible for their context-dependent functional differences, but it is not yet clear where these differences reside. The human and zebrafish dnRARs used are 78% identical and 85% similar, with most of the differences in the variable A domain and hinge region/D domain. Initial assessments of human-zebrafish chimeric dominant negative proteins, made by fusing the human RARa A-C domains to the D-ΔF domains of the zebrafish RARab dominant negative protein and vice versa, were not informative, since both chimeras were able to act as weak dominant negative proteins relative to the human dnRARa (Fig. S8
). Therefore, it appears that aspects of both halves of the human protein are required to confer ability to act as a repressor within the context of the early zebrafish embryo. Future studies employing additional chimeras will be necessary to discern which residues are important for the context-specific differences. Altogether, the inability of the zebrafish dnRARs to function in the zebrafish embryo, in contrast to the ability of the human dnRARa, emphasizes the inherent differences of these homologous proteins and the limitations of a binary model for RAR function in some developmental contexts.
Together, our data suggest there is significant context dependence for the responsiveness of RA signaling at the transcriptional level. Putting these results into a broader perspective, such context dependence is not surprising in light of mechanisms for transcriptional regulation of developmental genes in other settings. Elegant studies of gene regulatory networks in organisms such as Drosophila, sea urchins, and frogs have demonstrated that precise tissue-specific regulation of gene expression often involves multiple cis-regulatory modules harboring multiple transcriptional elements that correspond to transcription factors with context-dependent functions (Levine, 2010
; Levine and Davidson, 2005
; Wilczynski and Furlong, 2010
). Future studies will be aimed at elucidating the precise function of RARs in a variety of contexts and the nature of their interactions with partners in cis-regulatory modules containing RAREs.
The acquisition of RA signaling and its control of hox
gene expression has been associated with development of the chordate body plan (Marletaz et al., 2006
). Although RA signaling has been shown to regulate hox1
expression in amphioxus (Schubert et al., 2005
), it has been shown recently that the thalacian urochordates have lost all of the major components important for RA signaling (RAR, Cyp26 and Raldh) and do not require it for A-P patterning (Canestro and Postlethwait, 2007
). While ascidian urochordates have retained the RA signaling machinery, it appears that they may not require RA signaling for A-P patterning either (Canestro and Postlethwait, 2007
). It is clear that RA signaling is required for A-P patterning in zebrafish (Begemann et al., 2001
; Grandel et al., 2002
; Maves and Kimmel, 2005
); however, our results suggest that the regulation of RA target genes may have a strong dependence on additional transcriptional regulators in zebrafish embryos. Based on studies of RA-responsive genes and the more expansive expression of RARE reporter transgenes in mice (Balkan et al., 1992
; Oosterveen et al., 2003
; Rossant et al., 1991
; Sharpe et al., 1998
), we also hypothesize that RA signaling in tetrapods may function more independently at the transcriptional level.
Assimilating the observations from basal chordates, zebrafish, and tetrapods, we propose two possible models for the evolution of RAR function. In the first model, the ancestral role of RA signaling and RARs during early A-P patterning could have been subordinate to or redundant with other necessary transcription factors. If the role of RA signaling were minimal, acting only in concert with other transcription factors in the common chordate ancestor, it would be easier for these other factors to compensate for loss of RA signaling in the urochordates and maintain proper embryonic A-P patterning. In contrast, tetrapod RARs could have become better able to interact with regulators of transcriptional machinery and therefore less dependent on interactions with other cis-regulators, allowing RA signaling to take on a more singular role in regulation of A-P patterning. Thus, RARs may have evolved from being transcription factors with minimal singular competence during A-P patterning of the chordate body plan to having a more pronounced individual role in tetrapods. Alternatively, in the second model, the manner in which the zebrafish RARs function could be a derived characteristic of teleosts, or even zebrafish specifically. In this scenario, there would be little or no functional difference between RARs from amphioxus and mammals, while teleost RARs have lost the ability to act as repressors in some contexts. Other explanations would therefore be needed to explain why and how proper A-P patterning is maintained in the absence of RA signaling in urochordates. Future studies comparing RARs and the regulation of RA signaling target genes in chordates will allow for an enhanced understanding of the evolution of RA signaling and its role in A-P patterning of the chordate body plan.