Rest plays a central role in regulation of gene expression required for cell proliferation and differentiation in a variety of contexts. Recent analysis of Rest function has led to proposals of a number of novel and seemingly contradictory roles for Rest. How the myriad of potential interactions mediated by Rest is translated into biologically relevant outcomes is poorly understood. We provide in vivo genetic evidence for an essential function for zebrafish Rest in regulation of the Hh pathway.
Rest has been best characterized as a transcriptional repressor, and we hypothesized that Rest directly or indirectly represses one or more Hh signaling components. Several lines of evidence implicate the Gli transcription factors, particularly Gli2a, as the key Rest target. First, Rest knockdown enhanced Hh target gene expression in multiple tissues implying that Rest regulates a fundamental aspect of the Hh pathway. Second, the effects of both positive and negative alterations in Hh signaling were enhanced with Rest loss of function. This is consistent with excess Gli activator function in contexts of strong Hh signaling, and excess Gli repressor activity when Hh signaling is reduced. Thus, excess Gli activity could account for the exaggerated response to both high and low levels of Hh signaling. In addition, our epistatic experiments reveal that Rest interacts with the Hh pathway downstream of Smo and upstream of Gli1 with the exception of the hindbrain, where Gli1-independent activities are present. Gli2a and Gli3 transduce Hh signaling chiefly through transcriptional regulation of gli1, but are also weak activators of other Hh targets (). We cannot rule out the possibility that Gli1 and/or Gli3 activity is also increased, independently of excess Gli2a activity.
Model for Rest interaction with the Hh pathway
The expansion of the gli2a domain into regions of active Hh signaling in rest morphants provides a mechanism to account for the effects on Hh signaling by Rest. The expression pattern of gli2a in rest morphants runs counter to alterations in other Hh pathway genes tested, including gli3 and gli2b, because enhanced Hh signaling normally results in decreased gli2a expression.
In addition, derepression of gli2a also accounts for enhanced repression of Hh targets by Rest knockdown in yot mutants, in which only the dominant repressor form of Gli2a is produced. Finally, restoration of Hh signaling in the hindbrain of dtr/gli1 mutants with compromised Rest function is blocked by Gli2a knockdown. This demonstrates a requirement for Gli2aA in Rest mediated enhancement of Hh signaling. Together, our results support a model in which Gli2a is the principal point of Rest interaction within the Hh pathway (). Following ectopic activation of the Hh pathway (shh or dnPKA mRNA treatment), conversion of Gli2a to Gli2aA predominates resulting in synergistic enhancement of Hh target gene expression. In CyA treated rest morphants, the excess Gli2a would be converted to Gli2aR, which would repress Hh target gene expression.
expression is most evident in the midline and ventral domains in rest
morphants (), and we were not able to discern any consistent differences from control embryos in the dorsolateral domains. In a wild-type embryo, this repression may serve to dampen the response to high levels of signaling. While Rest function in the developing embryo tempers the cellular response to Hh, the modest alteration in target gene expression seen in rest
morphants may be due to redundant regulation of Hh signaling (Dessaud et al., 2007
; Jeong and McMahon, 2005
). Subtle phenotypes in zebrafish arise from mutations in negative regulators of the pathway including ptc2
(Koudijs et al., 2008
; Koudijs et al., 2005
Although the absence of strong RE1 sites near or within the gli2a
locus does not rule out direct regulation by Rest, the transcriptional upregulation of gli2a
in response to reduced levels of Rest may indeed be indirect. In addition to Hh, other signaling pathways including the FGF, Notch and Wnt pathways also regulate Glis (Alvarez-Medina et al., 2008
; Brewster et al., 2000
; Ke et al., 2005
). A model in which Rest knockdown activates one of these pathways, which in turn enhances transcription of one or more gli
genes is consistent with our observations.
Rest function during development
Here, we show that reduced levels of Rest during zebrafish development leads to alterations in the progenitor domains responsible for generation of distinct neural subtypes. In addition to a role for REST in repressing neural genes in non-neural cells, REST has been implicated in the control of neurogenesis at multiple steps (Ballas et al., 2005
; Bergsland et al., 2006
; Otto et al., 2007
; Su et al., 2004
). Outside the developing nervous system, Rest has been placed upstream of the network controlling pancreatic islet development (Johnson et al., 2007
; Kemp et al., 2003). This is an interesting finding as the Hh pathway regulates both neural and pancreatic development. We also find that Rest knockdown enhances expression of nkx2.2a
in the developing zebrafish pancreas (, Supplemental Fig. 3
). The wide range of potential activities proposed for REST underscores the importance of considering the unique cellular environment in which REST is acting.
REST mutant mice undergo widespread apoptosis beginning at day E 9.0 and die by day E11.5 (Chen et al., 1998
). In contrast, we observed that the rest
MO treated zebrafish present with a much subtler phenotype, ventralization of the neural tube. The expansion of ventral cell types in rest
morphants, is unlikely to be produced by increased apoptosis. However, there are key differences in the mouse and fish experiments. Primarily, our treatments produce a knockdown, not a knock-out of Rest. In addition, zebrafish rest
is supplied as a maternal transcript, which may allow for adequate Rest activity during early stages.
REST has been identified as both a tumor suppressor (Coulson et al., 2000
; Westbrook et al., 2008
; Westbrook et al., 2005
) and an oncogene (Lawinger et al., 2000
; Su et al., 2006
). It will be important to determine whether REST regulates Shh signaling in transformed cells. It is perplexing that REST downregulation results in differentiation in some cell populations and proliferation in others. For example, β-TRCP dependent degradation of REST allows differentiation in neural stem cell culture, but proliferation in human mammary epithelial cell culture (Westbrook et al., 2008
). How loss of REST allows activation of such different pathways is not well understood, but it is clear that differential target regulation depends on cellular context. For example, in neural progenitors, cell cycle progression relies on degradation of REST during the G2 phase for optimal expression of mad2
, a direct REST target (Guardavaccaro et al., 2008
). Needless to say, the full repertoire of genes under the control of REST is not activated during the G2 phase of mitotic cells. Differential target regulation may depend on many factors, including preexisting epigenetic modifications and the unique combinations of co-repressors and/or transcriptional co-regulators present. Unique combinations of such factors determine and are determined by the unique cellular context, allowing a small number of signaling pathways to affect a wide array of transcriptional networks and produce diverse outcomes.
Our studies reveal a novel and unexpected interaction between Rest and the Hh pathway. We find that Rest acts as a modulator of the Hh signal by regulation of gli2a. Rest likely fine-tunes the response of cells to Hh signaling by controlling transcription levels of gli2a and possibly additional factors. Transcriptional repression by Rest may thus be an additional limiting factor for Hh signal transduction, independent of antagonistic pathway components such as Ptc and Hip. Hh signaling plays a key role in regulation of progenitor cell proliferation and differentiation in many places within the developing nervous system and other tissues. Regulation of Hh signaling by Rest may be critical in many of these domains. These findings have broad implications for regulation of signaling in the many places where Hh acts and provide an avenue for future studies into Hh-mediated cell fate decisions.