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Keywords:

  • basal body;
  • ciliogenesis;
  • cilium;
  • FOXJ1;
  • microtubule;
  • RFX;
  • transcription factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

Cilia and flagella have essential functions in a wide range of organisms. Cilia assembly is dynamic during development and different types of cilia are found in multicellular organisms. How this dynamic and specific assembly is regulated remains an important question in cilia biology. In metazoans, the regulation of the overall expression level of key components necessary for cilia assembly or function is an important way to achieve ciliogenesis control. The FOXJ1 (forkhead box J1) and RFX (regulatory factor X) family of transcription factors have been shown to be important players in controlling ciliary gene expression. They fulfill a complementary and synergistic function by regulating specific and common target genes. FOXJ1 is essential to allow for the assembly of motile cilia in vertebrates through the regulation of genes specific to motile cilia or necessary for basal body apical transport, whereas RFX proteins are necessary to assemble both primary and motile cilia in metazoans, in particular, by regulating genes involved in intraflagellar transport. Recently, different transcription factors playing specific roles in cilia biogenesis and physiology have also been discovered. All these factors are subject to complex regulation to allow for the dynamic and specific regulation of ciliogenesis in metazoans.


Abbreviations used:
AWB

amphid wing B

BBS

Bardet—Biedl syndrome

ChIP

chromatin immunoprecipitation

CRT1

constitutive RNR transcription 1

DAF

abnormal dauer formation

DNAH and Dnahc

dynein, axonemal, heavy chain

efhc

EF-hand-domain-(C-terminal)-containing

FGF

fibroblast growth factor

fgfr

FGF receptor

FKH-2

forkhead 2

FOXJ1

forkhead box J1

HNF

hepatocyte nuclear factor

ier

immediate early response

IFT

intraflagellar transport

KV

Kupfer's vesicle

miRNA

micro-RNA

Pkhd1

polycystic kidney and hepatic disease 1

RFX

regulatory factor X

RSHL

radial spoke-head-like

RSPH

radial spoke head

sak1

suppressor of A-kinase

SHH

Sonic Hedgehog

SPAG

sperm-associated antigen

WDR

WD repeat domain

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

Cilia and flagella are cellular organelles that protrude in the external environment of cells and have major functions in various organisms. They are highly conserved structures, found from protozoa to mammals, and are likely to have evolved from a common flagellated unicellular ancestor. Cilia and flagella are formed from a stereotyped assembly of microtubules, called the axoneme, which is anchored on the basal body, a modified centriolar-based structure. Two types of cilia can be distinguished by their axonemal ultrastructure: 9+2 axonemal arrangements which are generally motile and comprise almost all types of flagella; and 9+0 arrangements of microtubules that are generally non-motile. Cilia and flagella assembly in most organisms relies on a bidirectional transport of particles along the axoneme, called IFT (intraflagellar transport). In protozoa, flagella play a fundamental role in cell motility, but are also necessary for sensory and mating behaviour. In animals, cilia have major sensory functions as many neurons mediate sensation through ciliated structures. In vertebrates, in addition to a conserved function in perception in various specialized sensory cells, cilia are found on almost every cell type where they play major roles in signal transduction, as, for example, in the SHH (Sonic Hedgehog) pathway (for reviews, see Eggenschwiler and Anderson, 2007; Wong and Reiter, 2008). The fundamental biological role of cilia is highlighted by the recent discovery that defects in cilia cause a broad spectrum of human diseases which are described as ciliopathies (for reviews, see Baker and Beales, 2009; Marshall, 2008a; Cardenas-Rodriguez and Badano, 2009).

Cilia and flagella are dynamic structures that can grow and resorb in different physiological conditions, during the cell cycle and throughout development. For example, flagella in the green algae Chlamydomonas are resorbed after mating and before division, and reassemble thereafter. In vertebrate cells, cilia disappear during mitosis and are reassembled while entering the G1/G0 stages. In several tissues ciliary growth is highly dynamic; for example, multiple cilia of the cerebral ventricles and of the upper airways epithelia grow at precise developmental stages (Sorokin, 1968; Spassky et al., 2005; Jain et al., 2010). Epithelial cells of both tissues first harbour a primary cilium and switch to multiple ciliary growth under specific developmental programmes. Sperm flagella are likewise assembled at precise and controlled developmental stages. In addition, primary cilia differ widely between cells in one given organism, even though they apparently share a similar ultrastructure. Altogether, these observations imply that organisms must have developed regulatory mechanisms to ensure proper and specific subtypes of cilia assembly in a time- and tissue-restricted manner. Several lines of evidence suggest that transcriptional regulation of ciliary gene expression is one of them. We will first review the evidence that supports transcriptional control of ciliary genes. We will then focus on the major molecular players that have been described as governing ciliary gene expression, and discuss how different types of cilia are determined in metazoans.

Transcriptional control of ciliary gene expression

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

Historically, transcriptional control of ciliary gene expression was first documented in Chlamydomonas and sea urchins, in which deflagellation or deciliation can be induced by different chemical or mechanical stresses. In Chlamydomonas, regrowth of the flagella starts immediately after experimentally induced deflagellation and takes approx. 90 min (Rosenbaum et al., 1969). The flagella are assembled using a pre-existing pool of proteins that is sufficient to reconstitute a half-length flagellum. However, to accomplish complete ciliary growth, flagellar gene transcription must be turned on (Lefebvre et al., 1978, 1980; Silflow et al., 1982). Using nuclear run-on experiments, it has been demonstrated that accumulation of α- and β-tubulin mRNA, after experimentally induced deflagellation, results from increased transcription of new mRNAs (Keller et al., 1984), although changes in tubulin mRNA stability are also involved (Baker et al., 1986). More recently, significant variations of mRNA levels of many ciliary genes during Chlamydomonas flagellar resorbtion or reassembly were revealed by microarray hybridization, but changes in either mRNA transcription or stability could account for these variations (Pazour et al., 2005; Stolc et al., 2005; Chamberlain et al., 2008). Correlations between ciliary gene transcription and ciliary growth have also been documented in a model of experimentally induced reciliation in sea urchin embryos (Harlow and Nemer, 1987; Norrander et al., 1995). A formal demonstration of transcription regulation in physiological conditions of development was performed in the study of embryonic development of the marine limpet, Patella vulgata (Damen et al., 1994).

Although the transcriptional regulation of ciliary gene expression in experimentally induced deflagellated Chlamydomonas has been documented, the mechanisms underlying this regulation are largely unknown in this organism. Studies have mainly been aimed at identifying the promoter regulatory sequences that control tubulin gene expression, but no unified consensus sequences on tubulin gene promoters has emerged and no specific molecular players have been isolated so far. Nevertheless, searches for genes that are specifically induced (at either the transcriptional or the post-transcriptional level) during flagellar regeneration in Chlamydomonas have proven to be highly instructive in identifying many ciliary structural components and regulators of their assembly (Pazour et al., 2005; Stolc et al., 2005).

In metazoans, in addition to the few studies that formally demonstrated that inhibition of transcription impaired cilia assembly (Damen et al., 1994), the dynamic expression profile of genes encoding ciliary proteins during development of ciliated tissues led to the assumption that these genes may in many cases be under transcriptional regulation. This was confirmed by independent genetic studies in different organisms, through the identification of the key players controlling the transcription of ciliary genes. Two major families of proteins were identified: the FOXJ1 (forkhead box J1) and RFX (regulatory factor X) transcription factors.

FOXJ1 transcription factors and the motile ciliogenic programme

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

The function of FOXJ1 (HFH4) was described more than 10 years ago. FOXJ1 is a member of the forkhead/winged-helix family of transcription factors, and orthologues of FOXJ1 have been found in vertebrates and many invertebrates, including sea urchins and the planerian model Shmidtea mediterrannea, an emerging powerful model for motile ciliogenesis studies. No orthologues of FOXJ1 have been found in ecdyzozoa such as Caenorhabditis elegans and Drosophila melanogaster (Mazet et al., 2003).

Several arguments support the hypothesis that FOXJ1 is necessary for motile ciliogenesis. In mammals, Foxj1 is expressed in several tissues with motile cilia (Hackett et al., 1995; Lim et al., 1997; Blatt et al., 1999; Brody et al., 2000; Jain et al., 2010). For example, Foxj1 expression in the lung is clearly correlated with the growth of motile cilia (Blatt et al., 1999), but not with non-motile primary cilia (Jain et al., 2010). Mice null for Foxj1 show left—right asymmetry defects and hydrocephalus, two phenotypes known to result from defective motile cilia. Indeed, Foxj1−/− mice have a complete absence of motile 9+2 cilia in the nasal epithelium and ventricular cells (Jacquet et al., 2009). Interestingly, in Foxj1-deficient multi-ciliated tissues, basal bodies are amplified, but fail to dock at the apical plasma membrane (Brody et al., 2000; Huang et al., 2003). No obvious ultrastructural defects of embryonic nodal cilia have been observed, suggesting that only their motility may be affected in accordance with left—right asymmetry defects observed in Foxj1−/− animals (Brody et al., 2000). No ultrastructural defects of olfactory 9+0 primary cilia have been observed in these mice (Brody et al., 2000). Hence, FOXJ1 appears to be specific for the biogenesis of motile cilia.

Recent data support this conclusion and show that FOXJ1 is a master regulator of the motile ciliogenic programme in zebrafish and Xenopus. Indeed, ectopic expression of Foxj1 in the entire zebrafish or in the Xenopus epidermis was sufficient to induce motile ciliary growth in various tissues of the zebrafish or in Xenopus epidermis (Stubbs et al., 2008; Yu et al., 2008). However, Foxj1 overexpression was not sufficient to drive motile ciliary growth in mouse cell cultures (You et al., 2004), even though overexpression of Foxj1 during lung development led to ectopic β-IV tubulin expression in a few bronchiolar cells, suggesting that FOXJ1 induced ectopic cilia assembly in these cells (Tichelaar et al., 1999). Additionally, overexpression of high levels of Foxj1 in Xenopus embryos was not sufficient to induce ectopic cilia in all cells. Finally, overexpression of FOXJ1 induces the growth of only a small number of cilia on each cell and limited centriole proliferation. Altogether, these data suggest that additional factors may be necessary to complete the motile ciliogenic programme in addition to FOXJ1. It has been suggested that the level of expression of CP110 (centrosomal protein of 110 kDa), a centriolar-associated protein that blocks the ciliary programme, may be important to allow FOXJ1 to induce the biogenesis of motile cilia (Stubbs et al., 2008). Other candidate factors include the RFX family of transcription factors. Indeed, RFX3 has been shown to be necessary for motile cilia biogenesis of ependymal cells in vitro (El Zein et al., 2009). In addition, RFX3 and FOXJ1 share some common direct target genes, suggesting that both types of transcription factors co-operate to govern a specific motile ciliogenic programme (Stubbs et al., 2008; Yu et al., 2008; El Zein et al., 2009; see below).

Several target genes of FOXJ1 proteins have been described. In mice, Dnahc11 (dynein, axonemal, heavy chain 11), encoding an axonemal dynein essential for cilia motility, was the first gene shown to be down-regulated in Foxj1-deficient lungs (Chen et al., 1998). The calpastatin gene was also shown to be down-regulated in Foxj1-deficient lungs (Gomperts et al., 2004). It is not known if Dnahc11 or the calpastatin gene are direct targets of FOXJ1. Nevertheless, calpastatin has been shown to act on the amount of apical ezrin at the surface of lung epithelial cells. The reduction in ezrin apical localization may, in part, explain the defective apical transport of the basal bodies observed in Foxj1−/− lungs (Brody et al., 2000; Huang et al., 2003).

Recently, several other FOXJ1 target genes have been identified in Xenopus, zebrafish and mice (Stubbs et al., 2008; Yu et al., 2008; Jacquet et al., 2009), among which many are involved in ciliary motility. For instance, approx. 100 genes were found to be up-regulated more than 10-fold by the overexpression of Foxj1 in Xenopus epidermis, among which one-third are found in databases of ciliary proteins (Gherman et al., 2006; Inglis et al., 2006; Arnaiz et al. 2009). Many encode proteins found to be exclusive to motile axonemal structures such as subunits of axonemal dyneins [DNAH9 (dynein, axonemal, heavy chain 9), DNAH8 and DNAI1 (dynein, axonemal, intermediate chain 1)], WDR16 (WD repeat domain 16) (Hirschner et al., 2007), a central pair complex component [SPAG6 (sperm-associated antigen 6)] and various radial spoke proteins [RSHL2 (radial spoke-head-like 2), RSHL3 and RSPH1 (radial spoke head 1)]. In zebrafish, five genes, all involved in ciliogenesis, were also shown to be regulated by Foxj1a (encoded by one of the two foxj1 orthologues): dnah9, centrin2, tektin1, wdr78 and efhc1 [EF-hand-domain-(C-terminal)-containing 1]. In addition, ChIP (chromatin immunoprecipitation) experiments in zebrafish, showed that Foxj1a associates with the promoters of the dnah9 and the wdr78 genes in vivo, indicating that these two genes are direct targets of FOXJ1. Recently, FOXJ1 target genes have been identified by microarray analysis of RNA extracted from the mouse ventricular zone (Jacquet et al., 2009). Approx. 200 genes were found to be statistically altered more than 1.5-fold in Foxj1−/− samples, among which 55 had known function in the brain ependymal zone. Almost half of them encode known ciliary proteins or cytoskeleton-associated molecular motors. Some of these proteins could explain the defect in apical basal body transport in ventricular cells observed in Foxj1−/− mouse brains (Jacquet et al., 2009), even though their function in this process is not demonstrated.

Interestingly, 16 of the targets identified in Foxj1−/− brain ependymal cells (Table 1) are also targets of Foxj1 in Xenopus, such as dnah9, Spag6, efhc1 and dnaic1 (dynein, axonemal, intermediate chain 1). Only three out of these 16 conserved FOXJ1 target genes in vertebrates have not been associated with ciliogenesis so far.

Table 1.  Genes regulated by FOXJ1 in both Xenopus and mouseOnly the mouse gene name is reported.
Gene nameDefinition
Dnahc9Dynein, axonemal, heavy chain 9
IqcaIQ-motif-containing with AAA domain
Wdr66WD repeat domain 66
Efhc1EF-hand-domain-(C-terminal)-containing 1
Tekt4Tektin-4
Spata17Spermatogenesis-associated 17
Spa17Sperm-autoantigenic protein 17
Dynlrb2Dynein light chain roadblock-type 2
Dnaic1Dynein, axonemal, intermediate chain 1
Syne1Synaptic nuclear envelope 1
Spag6Sperm-associated antigen 6
Dnali1Dynein, axonemal, light intermediate polypeptide 1
Tm4sf1Transmembrane protein/tetraspanin family
Kif9Kinesin family member 9
Rsph4aRadial spoke head 4 homologue A (Chlamydomonas)
NEK5NimA-related protein kinase 5

Altogether, these observations demonstrate that FOXJ1 proteins are important players in regulating genes necessary for motile cilia function in vertebrates. As well, no genes encoding proteins known to be involved in the process of IFT were identified as FOXJ1 target genes in either mice or Xenopus.

As mentioned above, no orthologue of Foxj1 can be found in C. elegans and Drosophila. As there are no motile cilia in C. elegans, this supports the hypothesis that Foxj1 is necessary for motile cilia biogenesis. However, in Drosophila, sperm flagella are motile and chordotonal cilia have outer dynein-like arms, a hallmark of motile cilia (Lee et al., 2008). Several lines of functional evidence also suggest that chordotonal cilia are motile, despite the lack of central pairs (Gopfert and Robert 2003; Gopfert et al., 2005; Lee et al., 2008). Thus cilia motility in Drosophila is not dependent on Foxj1, and other factors must substitute to control motile cilia biogenesis in this organism. RFX protein is one possible candidate to fulfill this function (see below). Other forkhead proteins could also have evolved to play such a role in Drosophila.

The above observations also raise the question of the function of FOXJ1 being restricted to the motile ciliogenic programme. In mice, genetic evidence suggests that FOXJ1 plays a role in cells that have so far been described as harbouring no motile cilia (astrocytes) or even no cilia at all (circulating lymphocytes). In the immune system, FOXJ1 regulates T-cell activation and prevents autoimmunity. FOXJ1 also causes restrained B-cell activation by antagonizing NF-κB (nuclear factor κB) and IL-6 (interleukin 6) (Lin et al., 2004, 2005). Both B- and T-cells do not harbour primary cilia (Alieva and Vorobjev, 2004). In the mouse brain, FOXJ1 has also been shown to be necessary for the differentiation of astrocytes (Jacquet et al., 2009). It would be of particular interest to understand how FOXJ1 target-gene specificity is governed in these systems. One way of driving promoter specificity is the combined action of several transcription factors, as shown in mouse orofacial morphogenesis where PITX2 (pituitary homeobox 2) and FOXJ1 co-operate (Venugopalan et al., 2008).

RFX family of transcription factors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

The second major type of transcription factors which have been shown to play key roles in ciliogenesis control in C. elegans, Drosophila and vertebrates are the RFX proteins. They were first discovered by their ability to bind to the so-called X-box motif that is conserved in MHC class II gene promoters (Reith et al., 1990). These proteins share a highly conserved DNA-binding domain that belongs to the winged-helix family of transcription factors (Gajiwala et al., 2000). A particularity of the RFX subgroup of winged-helix proteins is that they use their so-called recognition motif to bind the minor groove of DNA (Gajiwala et al., 2000). Seven members of the family have been identified in mammals, whereas only one has been described in yeast and C. elegans (Emery et al., 1996a; Aftab et al., 2008) (Figure 1A). Two subgroups can be distinguished, on the basis of their structural features: a first subgroup includes the yeast protein, DAF-19 (abnormal dauer formation 19) in C. elegans, RFX in Drosophila, and RFX1–RFX4 and RFX6 in vertebrates. A second subgroup comprises the second Rfx gene in Drosophila (CG9727; B. Durand and A. Laurençon, unpublished data) and RFX5 and RFX7 in vertebrates. Note that a third Drosophila Rfx gene has been published (Otsuki et al., 2004), but is not found in the Drosophila genome and is related to RFXs from fungi (B. Durand, unpublished data). All members of the first subgroup harbour a dimerization domain that allows homodimerization and/or heterodimerization, whereas members of the second subgroup do not (Figure 1B). They also differ in their DNA-binding specificities. For example, RFX5 has been shown to be devoted to MHC class II gene regulation in both mice and humans (Steimle et al., 1995; Clausen et al., 1998). Binding of RFX5 to HLA class II promoters requires additional factors, and the X-box motif recognized by RFX5 is different from the X-box motif defined for RFX1–RFX3 (Steimle et al., 1995; Emery et al., 1996a, 1996b; Durand et al., 1997; Masternak et al., 1998).

image

Figure 1. Phylogeny and structure of RFX transcription factors

(A) Phylogenetic tree of the RFX family of transcription factors. Two major subgroups can be recognized. A first subgroup includes the only RFX genes of C. elegans and yeast, one RFX gene in Drosophila and five RFX genes (RFX1–RFX4, RFX6) in vertebrates. The second subgroup includes the second Drosophila gene (CG9227) and two genes in vertebrates. (B) Structure of the different RFX members from yeast to mammals. The X-box motif was defined in mammals for RFX1 (Emery et al., 1996b). A, B and C, conserved domains of unknown function; DBD, DNA-binding domain; DE, acidic-amino-acid-rich region; DIM, dimerization domain; P, proline-rich region; PQ, proline- and glutamine-rich region; Q, glutamine-rich region. Ensembl accession numbers are as follows. For RFX5: ENSG00000143390, ENSMUSG00000005774, ENSXETG00000027541, ENSDARG00000063258; RFX7: ENSG00000181827, ENSMUSG00000037674, ENSXETG00000024544, (RFX7a) ENSDARG00000077237, (RFX7b), ENSDARG00000074522; Drosophila: FBgn0037445 (CG9727); RFX6: ENSG00000185002, ENSMUSG00000019900, ENSDARG00000041702; RFX4: ENSG00000111783, ENSMUSG00000020037, ENSXETG00000014145, ENSDARG00000026395; RFX1: ENSG00000132005, ENSMUSG00000031706, ENSXETG00000005567; RFX3: ENSG00000080298, ENSMUSG00000040929, ENSXETG00000011698, ENSDARG00000014550; RFX2: ENSG00000087903, ENSMUSG00000024206, ENSXETG00000021877, ENSDARG00000013575; Drosophila RFX: FBgn0020379; DAF-19: F33H1.1. GenBank accession number for S. pombe Sak1: NM_001019499. C.e., C. elegans; D.m., Drosophila melanogaster; M.m., Mus musculus; S.p., S. pombe.

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Pioneering work demonstrating the involvement of members of the first subgroup of RFX proteins in ciliogenesis control was first carried out on C. elegans. Swoboda et al. (2000) showed that daf-19 is required for sensory cilia assembly in C. elegans. In addition, this study was the first to identify several DAF-19 target genes that are involved in cilia assembly in C. elegans (Swoboda et al., 2000). This work paved the way for several subsequent studies that used the X-box motif to identify novel genes involved in ciliogenesis in C. elegans and in other organisms (Haycraft et al., 2001, 2003; Schafer et al., 2003; Avidor-Reiss et al., 2004; Li et al., 2004; Blacque et al., 2005; Efimenko et al., 2005; Winkelbauer et al., 2005; Chen et al., 2006; Laurençon et al., 2007). On the basis of recognition algorithms and genomic comparisons, these studies were fundamental in identifying novel genes involved in ciliogenesis. In addition, the comparison of the transcriptome of wild-type or daf-19-deficient worms also allowed for the compilation of lists of candidate ciliary proteins (Blacque et al., 2005; Efimenko et al., 2005; Chen et al., 2006).

In other metazoans, several RFX proteins of the first subgroup have a conserved function in ciliogenesis control. In Drosophila, RFX is necessary for ciliogenesis in sensory neurons (Dubruille et al., 2002). In mice, RFX3 is necessary for the growth of primary cilia in the embryonic node and in the endocrine pancreas (Bonnafe et al., 2004; Ait-Lounis et al., 2007). RFX3 is also necessary for proper completion of the motile ciliogenic programme, in agreement with strong expression of RFX3 in motile ciliated cells (El Zein et al., 2009; L. Morlé and S. Sagnol, unpublished data). In mice, Rfx4 appears to be dynamically expressed during brain development and governs the growth of primary cilia in regions of the central nervous system expressing this gene (Ashique et al., 2009). Rfx4-deficient mutants show dorsal—ventral patterning defects in the telencephalon and in the spinal cord, resulting from defective SHH signalling (Ashique et al., 2009). In zebrafish, Rfx2 was shown to be involved in the motile ciliogenic programme (Liu et al., 2007). The other RFX transcription factors, for which mutant animal models are available, are apparently not devoted to ciliogenesis (Smith et al., 2010; Soyer et al., 2010).

Conversely to what has been observed in invertebrates, loss of only one RFX protein does not lead to complete ciliary loss in any tissues examined so far in mice. This may be the consequence of redundant RFX functions. This hypothesis is strengthened by the observation that no differences in binding specificity between RFX −1, −2 and −3 have been described yet. On the contrary, complementary functions may also occur. For instance, subcommissural organ developmental defects observed in the brain of Rfx4+/− mice can also be observed in Rfx3-deficient brains, suggesting a concerted action of these factors in this organ (Blackshear et al., 2003; Baas et al., 2006). Rfx6- and Rfx3-deficient mice also share common developmental defects of the pancreas (Ait-Lounis et al., 2007; Smith et al., 2010).

RFX target genes have been identified extensively in C. elegans and Drosophila and some targets have also been described in mice (Figure 2). All the RFX proteins that have been shown to have a function in ciliogenesis share at least one conserved target, which is IFT172/OSM-1 (osmotic avoidance abnormal)/OSEG2 (outer segment 2), a component of IFTB particles. Remarkably, in the worm and the fly, RFX proteins control the expression of all IFTB-associated components and all BBS (Bardet—Biedl syndrome) proteins, which have been shown to be involved in co-ordinating IFT transport and regulating ciliary trafficking. RFX proteins also regulate at least one subunit of the IFT retrograde motor, Dync2li1 (dynein cytoplasmic 2 light intermediate chain 1), and a protein involved in regulating tubulin polyglutamylation in C. elegans (DYF-1) and zebrafish (Fleer), a process shown to be important for axonemal integrity and IFT transport (Ou et al., 2005; Redeker et al., 2005; Pathak et al., 2007). Conversely, none of IFTA components nor the anterograde kinesin motors are targets of RFX in Drosophila, and only IFT122 (daf-10) has been described to be under DAF-19 control in C. elegans (Efimenko et al., 2005). Thus RFX proteins regulate IFT transport by controlling the expression of IFTB components, BBS components and retrograde motors. In addition, RFX factors control the expression of proteins associated with the basal body or the transition zone of cilia, such as several orthologues of genes involved in Meckel syndrome, a human ciliopathy, and, in particular, the members of the B9-domain family of proteins (Bialas et al., 2009). In C. elegans, DAF-19 regulates nph-1 (nephronophthisis 1) and nph-4, encoding proteins associated with the transition zone above the basal body (Winkelbauer et al., 2005). Their orthologues are involved in nephronophtisis in humans, another ciliopathy. Finally, in Drosophila, RFX regulates rootletin, a major component of the ciliary rootlet that links the two centrioles and anchors the basal body in the cell cytoskeleton (Yang et al., 2002, 2006; Bahe et al., 2005). The rootletin gene is involved in long-term cilia maintenance in mouse (Yang et al., 2005). Figure 2 summarizes RFX target genes that have been published in C. elegans, Drosophila and mice.

image

Figure 2. RFX target genes known to be involved in ciliogenesis

Simplified scheme of a primary cilium and its basal body. The basal body is derived from the mother centriole of the cell and harbours specialized structures, such as the basal foot. The basal body is anchored to the plasma membrane by the transition fibres. The region between the basal body and the axoneme is the transition zone. For a comprehensive description of the basal body, see Marshall (2008b) or Seeley and Nachury (2010). In mammalian cells the daughter centriole is orthogonal to the basal body as drawn. Note that in Drosophila ciliated sensory neurons, the daughter centriole is aligned under the basal body and is surrounded by the ciliary rootlet. In motile cilia, axonemal dynein arms ensure motility. In C. elegans and Drosophila, RFX proteins control all genes encoding components of the IFTB complex and all BBS orthologues. RFX regulates the dynein motor of retrograde IFT. RFXs also regulate genes involved in the control of tubulin polyglutamylation, which are shown to be important for IFT transport in C. elegans and zebrafish. RFX proteins regulate genes involved in basal body anchoring (rootletin and transition zone protein). Finally, RFX proteins regulate channels found to be localized on cilia and several proteins known to be associated with cilia function, but of which the mechanism is still to be understood. For example, the tubby family of proteins have been found to be associated with cilia function or signalling, but their mode of action is still unclear. Bold: genes found to be controlled by RFX in the different organisms. Grey: orthologues not tested in the different organisms. &1: Ift172 is down-regulated in the Rfx3−/− node, the pancreas and in Rfx4−/− nervous system. &2: Ift88 is down-regulated in the Rfx3−/− pancreas, but not in the node of E7.5 (embryonic day 7.5) embryos (Bonnafe et al., 2004; Ait-Lounis et al., 2010). &3: Bbs4 was not found to be regulated by RFX3 in ependymal cells (El-Zein et al., 2009).

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Altogether, RFX transcription factors appear to regulate a particular subset of ciliary proteins that are involved in molecular transport which is important for cilia assembly and function. In addition, RFX proteins regulate a specific set of proteins that are involved in basal body anchoring in the cell cytoskeleton (rootlet) or in the apical membrane (transition zone proteins). None of these particular proteins seem to be regulated by FOXJ1. A last class of genes regulated by RFX proteins, such as Dnahc9, is involved in ciliary motility and is also regulated by FOXJ1.

Not all RFX proteins are involved in ciliogenesis control. In mammals, RFX5 is involved in HLA class II gene regulation. In vertebrates, Rfx6 has recently been shown to be involved in pancreatic development in the zebrafish and in mice and humans (Smith et al., 2010; Soyer et al., 2010), but is not necessary for ciliary growth in this organ. In addition, RFX proteins have been identified in organisms that do not have cilia, such as yeasts. In the budding yeast, CRT1 (constitutive RNR transcription 1) has a major function in the DNA damage checkpoint as a negative regulator of genes involved in nucleotide metabolism (Huang et al., 1998). Potential novel Crt1 target genes in yeast have been described, that are involved either in sugar metabolism or in secretory pathways, but confirmation of these genes as true Crt1 targets is still required (Zaim et al., 2005). In the fission yeast, sak1 (suppressor of A-kinase) appears to regulate cell cycle exit (Wu and McLeod, 1995).

These observations raise the question of whether the function of the ciliogenic RFX transcription factors (daf-19 in C. elegans, Rfx in Drosophila, Rfx3 and Rfx4 in mouse, and rfx2 in zebrafish) is restricted to ciliogenesis. Of all the RFX target genes identified by X-box motif searches, some have not been shown to be involved in ciliogenesis. In C. elegans, different isoforms of DAF-19 have been identified (Senti and Swoboda, 2008). One is necessary for ciliated sensory neurons, whereas the others are necessary for synaptic stability in all other non-ciliated sensory neurons. The non-ciliated specific isoforms cannot rescue ciliary defects observed in daf-19 mutants, which suggests different target specificities between isoforms, even though they share the same DNA binding domain. Hence, it is possible that, as in C. elegans, specific RFX isoforms have different functions in mammals and that defects observed in Rfx3- or Rfx4-deficient mice is a combination of ciliary and non-ciliary defects. For instance, RFX3 regulates both the growth of primary cilia in the mouse endocrine pancreas and the expression of the glucokinase gene required for glucose-stimulated insulin secretion (Ait-Lounis et al., 2007, 2010).

Functional relationships between the FOXJ1 and RFX transcription factors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

In mice, we have shown that RFX3 regulates axonemal dynein genes, including Dnahc9, a target of FOXJ1 (El Zein et al., 2009). Thus, in mice, RFX proteins and FOXJ1 are likely to co-operate to regulate a subset of genes necessary for cilia motility. Among the 50 target genes identified in Xenopus and mice, and that have an orthologue in Drosophila and C. elegans, 10 are also likely targets of RFX in Drosophila (F. Soulavie and A. Laurençon, unpublished data). Most are involved in cilia motility itself, such as axonemal dyneins. Thus, as no Foxj1 orthologue has been identified in Drosophila, RFX could be sufficient to drive the expression of genes necessary for chordotonal cilia motility in this organism.

Besides sharing common target genes, several observations also suggest a possible cross-regulation between FOXJ1 and RFX transcription factors in vertebrates. In zebrafish, rfx2 appears to be in part regulated by Foxj1, whereas the opposite does not seem to be true (Yu et al., 2008). In mice, RFX3 appears to modulate Foxj1 expression in cultured ependymal cells (El Zein et al., 2009), but the complete in vivo relationships between these two transcription factors need to be more thoroughly studied in order to define their relationships precisely.

Other transcription factors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

Besides the two major transcriptional factors, FOXJ1 and RFX proteins, several other players have been described as controlling genes involved in ciliary assembly or function. Only those for which evidence suggests a direct role in ciliary gene expression are described in this section. Interestingly, these factors appear to be important for the assembly of only a subset of primary cilia among the different types of cilia found in the same organism.

The HNF1β (hepatocyte nuclear factor 1β) transcription factor has been shown to play a crucial part in postnatal liver, pancreas and kidney homoeostasis (Igarashi et al., 2005). Interestingly, HNF1β-deficient mice show severe polycystic kidneys, a hallmark of many ciliopathies in humans (Gresh et al., 2004). Efficient ChIP approaches have identified several ciliary genes, including Pkhd1 (polycystic kidney and hepatic disease 1), Pkd2 (polycystic kidney disease 2), Ift88 and collectrin, as direct targets of HNF1β, suggesting that HNF1β is an important player of ciliogenesis in the kidneys (Gresh et al., 2004; Hiesberger et al., 2005). No data suggest a function of HNF1β in ciliogenesis in other tissues. This could illustrate the requirement for fine regulation of ciliogenesis and account for the different types of cilia found in one organism. However, some indirect data based on the observation of Hnf6-deficient mice suggest that HNF1β could regulate genes encoding ciliary proteins in the pancreas as well. Hnf6−/− mice show kidney and pancreatic duct cysts associated with lack of cilia, lack of Hnf1β expression in cells lining the cysts and reduced Pkhd1 expression. Because HNF6-binding sites were found in the Hnf1β gene promoter, it was proposed that Pkhd1 down-regulation is a consequence of the lack of HNF1β in the Hnf6−/− pancreas (Pierreux et al., 2006). Hence, HNF1β is important in the genetic programme that governs cilia assembly and function in the kidneys and probably in the pancreatic ducts.

The existence of such cell-specific programmes necessary to build a subset of cilia was remarkably well illustrated by a genetic study on C. elegans. In the nematode, different types of cilia can be morphologically distinguished. For example, a particular type of olfactory neuron, the AWB (amphid wing B) neuron, shows a characteristic wing cilium with two ciliary branches of different lengths. Interestingly, FKH-2 (forkhead 2), another member of the forkhead family of transcription factors, has been described to be necessary for the specification of only this particular type of sensory cilium (Mukhopadhyay et al., 2007). FKH-2 regulates IFT transport in these neurons by controlling the level of expression of kap-1 (kinesin-associated protein), encoding a kinesin-II motor subunit, specifically in the AWB neurons. Thus, whereas DAF-19 is necessary to govern the formation of all types of cilia, FKH-2 is only required to specify a particular ciliary subtype.

Hence, factors involved in the specification of a subtype of cilia begin to emerge in the literature and it is likely that a number of other factors are still to be discovered.

Developmental control of the regulators

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

All of the above-mentioned transcription factors show dynamic and tissue-specific expression patterns involving complex regulation. Some of the regulators have been characterized. The noto homeobox gene is expressed in the mouse embryonic node and has been shown to control ciliogenesis. In noto-deficient mice, Foxj1 and Rfx3 expression in the embryonic node is lost, showing that noto acts upstream of these ciliogenic transcription factors (Beckers et al., 2007). It is not known whether they are direct targets of NOTO. The expression of several ciliary genes was also significantly reduced in noto−/− embryonic node [Dnahc5/Dnahc9/Dncl2b (dynein light chain 2B)/Dnchc2 (dynein cytoplasmic heavy chain 2)/Nphp3 (nephronophthisis 3)], but all these genes are also targets of FOXJ1 or RFX3 and their down-regulation could thus result from the reduction in Foxj1 and Rfx3. In medaka fish, the expression of the floating head gene, the orthologue of noto, requires the function of a member of the polycomb group of nuclear proteins OLEED (EED in mice). oleed morphants can be rescued by floating head gene expression (Arai et al., 2009), suggesting a regulatory cascade that goes from oleed to noto to foxj1/rfx transcription factors.

All these factors are probably under the control of developmental signals. SHH signalling has been shown to regulate the expression of foxj1 in the ventral neural tube of zebrafish, but not in the pronephric duct or in the KV (Kupfer's vesicle). Foxj1a neural expression was either decreased or increased when SHH signalling was impaired or enhanced respectively (Yu et al., 2008). FGF (fibroblast growth factor) signalling has been shown to be an important player in controlling the expression of the ciliogenic programme. FGF signalling has been known to affect left—right asymmetry in mice, chicks and zebrafish. Two studies on zebrafish demonstrated the implication of an FGF signalling cascade in ciliogenesis during development. The first study aimed at identifying genes that were up-regulated in response to FGF signalling by microarray analysis. This led to the identification of ier2 (immediate early response 2), encoding a nuclear protein, as an important player for the growth of motile cilia in the KV in zebrafish. Morpholino knockdown of either ier2 or of its interacting partner, fibp1 (FGF-interacting binding protein 1), led to left—right asymmetry defects associated with shorter cilia in the KV (Hong and Dawid, 2009). The second study showed that impaired fgf8/fgf24-fgfr1 (FGF receptor 1) signalling in zebrafish or Xenopus also led to reduced ciliary length in the KV or gastrocoel roof-plate respectively. In addition, both ciliogenic transcription factors Foxj1 and Rfx2 were down-regulated in fgfr1 morphants, suggesting that FGF controls ciliogenesis during development by modulating foxj1 and rfx2 expression (Neugebauer et al., 2009). Other potential regulators of the regulators are likely to operate, such as E2F4 in the development of ciliated epithelial cells, but the pathway that goes from this gene to cilia assembly has not been described (Danielian et al., 2007). Figure 3 summarizes the different factors that control the expression of genes necessary for cilia assembly or function.

image

Figure 3. Summary of the different transcription factors described to regulate genes encoding proteins necessary for cilia assembly or function in different organisms

FOXJ1 has the specific function of governing motile cilia assembly, at least in vertebrates. RFX is necessary to regulate cilia assembly, from C. elegans to mammals. Additional transcription factors have been shown to play a role in the specification of only a subset of primary cilia; HNF1β regulates ciliary genes in the mouse kidneys and probably in the pancreas. HNF6 regulates HNF1β expression in the mouse pancreas. FKH2 regulates the assembly of a subset of cilia in the nematode. Foxj1 regulates rfx2 in zebrafish, whereas RFX3 appears to modulate Foxj1 expression in mouse ependymal cells. Foxj1 and Rfx3 expression is controlled by the NOTO transcription factor in the mouse embryonic node. FGF signalling regulates foxj1 and rfx2 expression in the zebrafish. SHH signalling regulates foxj1a expression in zebrafish neural tube.

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Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

In metazoans, ciliary gene expression is regulated by key transcription factors that have synergistic and complementary functions in the specification of different types of cilia. A growing body of evidence shows that the FOXJ1 and RFX transcription factors regulate a set of genes involved in specific functions of cilia biogenesis and physiology. Different sets of specific proteins are regulated by each transcription factor: FOXJ1 regulates genes involved in cilia motility and in the apical transport of basal bodies, whereas RFX proteins regulate genes encoding IFT proteins or regulating IFT and in basal body anchoring. In addition, RFX and FOXJ1 share common target genes involved in cilia motility. Yet many aspects of the functional interactions between these two transcription factors still need to be understood, along with the mechanisms that govern target specificity among the different proteins. However, the identification of their target genes in different cell systems has proved to be highly informative for identifying novel ciliary components. Several additional transcription factors are moreover involved in tuning the specific expression of ciliary genes, and new transcription factors are likely to be discovered to explain the variety of cilia that can be found in one given organism.

If transcriptional regulation of ciliary gene expression is an important step in controlling cilia assembly during metazoan development, not much is known about the importance or not of such a mechanism during the cell cycle, where assembly and disassembly of cilia occurs both in protozoans and metazoans. For instance, it is not known if oscillating expression of ciliary genes occurs during the cell cycle and if such a mechanism could play any role compared with the important described function of kinase signalling in cell-cycle control of cilia assembly (Pugacheva et al., 2007; for review see, Seeley and Nachury, 2010). It should be also noted that some important genes necessary for cilia assembly do not appear to be regulated by any of the yet-identified ciliogenic transcription factors. For example, it is not known how genes encoding IFTA particles are regulated. This applies also to genes governing centriole de novo amplification required for the growth of multiple cilia.

Several lines of evidence suggest that post-transcriptional mechanisms also control the expression levels of ciliary proteins in various physiological and experimental conditions. For example, in Chlamydomonas and sea urchins, post-transcriptional mechanisms are involved in tubulin gene regulation after experimentally induced deciliation (Baker et al., 1986). In mammals, recent observations illustrate the function of the small RNAi (RNA interference) pathway in governing the expression of polycystin 2 (Sun et al., 2009). An miRNA (micro-RNA) specific to sensory cells has also been described, but its function has not yet been demonstrated (Pierce et al., 2008). In ciliated cells of the mammalian inner ear, miR-96 mutations have been found to induce progressive hearing loss in humans and mice (Lewis et al., 2009; Mencia et al., 2009; Sacheli et al., 2009). Among miR-96 target genes, Odf2 (outer dense fibre of sperm tails 2), a component of the basal body and necessary for cilia assembly is found (Mencia et al., 2009), showing that the expression of ciliary proteins may be controlled by miRNAs. Altogether, these data suggest that the fine tuning of ciliary gene expression involves a complex network of factors and mechanisms. Understanding how ciliary genes are regulated is an important issue in the context of development of ciliated organisms.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References

Work in the group of B.D. is supported by the CNRS; University of Lyon; Fondation pour la Recherche Médicale (Equipe FRM 2009); Agence Nationale de le Recherche [grant numbers ANR-05-MRAR-022-01, ANR-09-GENO-004-01]; and Région Rhône-Alpes (Cible 2008).

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  2. Abstract
  3. Introduction
  4. Transcriptional control of ciliary gene expression
  5. FOXJ1 transcription factors and the motile ciliogenic programme
  6. RFX family of transcription factors
  7. Functional relationships between the FOXJ1 and RFX transcription factors
  8. Other transcription factors
  9. Developmental control of the regulators
  10. Conclusion
  11. Funding
  12. References
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Footnotes
  1. Different types of cilia: Cilia can be classified in terms of their axonemal architecture and their motile properties. The axoneme of primary cilia comprises 9 doublets of peripheral microtubules with no central pair (9+0 axoneme). Primary cilia are generally non-motile; one exception is the primary cilia of the mouse embryonic node. Most motile cilia are composed of an axoneme that comprises 9 outer doublets of microtubules with a central pair (9+2). Specific structures are associated with the 9+2 axoneme, such as radial spokes. Outer and inner dynein arms ensure the motility of cilia or flagella and can be found on 9+0 (embryonic node) or 9+2 axonemes. A variety of exceptions can be found to this stereotyped 9+0 and 9+2 axonemal architecture (see, for example, Mencarelli et al., 2008).

  2. Intraflagellar transport (IFT): This is a bidirectional transport of proteins along the axoneme that was first identified in Chlamydomonas (Kozminski et al., 1993) and has been shown to be conserved and required for the assembly of cilia in almost all organisms, with the exception of the Plasmodium falciparum and Drosophila flagella (Han et al., 2003; Briggs et al., 2004). Kinesin II drives the anterograde transport, whereas retrograde transport is ensured by the cytoplasmic dynein complex. IFT particles can be separated into two biochemical fractions: the A particles and the B particles, which have different functions. Hence, mutations in genes coding for A and B particles can also be phenotypically distinguished. Reviewed in Rosenbaum (2002), Pedersen and Rosenbaum (2008) and Scholey (2008).

  3. Winged-helix transcription factors: Transcription factors are classified on the basis of the structure of their DNA-binding domain. The term winged-helix was coined in 1993 on the basis of crystallographic studies of HNF3-γ, a member of the HNF-3 family of liver-specific transcription factors (Brennan, 1993; Clark et al., 1993). Proteins from this family share a highly conserved DNA-binding domain with the Drosophila homeotic forkhead proteins. Hence, winged-helix proteins are also referred to as belonging to the HNF-3/forkhead family. The winged-helix motif consists of two wings, three α-helices and three β-strands arranged in a specific order. Three-dimensional reconstruction of the HNF3 DNA-binding domain bound to the DNA strand looks like a butterfly. In the winged-helix family, two subgroups can be distinguished: the canonical subtype, in which the winged helix contacts the major groove of the DNA helix, and the RFX1 subtype, in which the winged helix contacts DNA in the minor groove (Gajiwala and Burley, 2000; Gajiwala et al., 2000).