In neurogenesis, neural cell fate specification is generally triggered by proneural transcription factors. While the role of proneural factors in fate specification is well studied, the link between neural specification and the cellular pathways that ultimately must be activated to construct specialised neurons is usually obscure. High-resolution temporal profiling of gene expression reveals the events downstream of atonal proneural gene function during the development of Drosophila chordotonal (mechanosensory) neurons. Among other findings, this reveals the onset of expression of genes required for construction of the ciliary dendrite, a key specialisation of mechanosensory neurons. It was determined that atonal activates this cellular differentiation pathway in several ways. Firstly, atonal directly regulates Rfx, a well-known highly conserved ciliogenesis transcriptional regulator. Unexpectedly, differences in Rfx regulation by proneural factors may underlie variations in ciliary dendrite specialisation in different sensory neuronal lineages. In contrast, fd3F encodes a novel forkhead family transcription factor that is exclusively expressed in differentiating chordotonal neurons. fd3F regulates genes required for specialized aspects of chordotonal dendrite physiology. In addition to these intermediate transcriptional regulators, it was shown that atonal directly regulates a novel gene, dilatory, that is directly associated with ciliogenesis during neuronal differentiation. This analysis demonstrates how early cell fate specification factors can regulate structural and physiological differentiation of neuronal cell types. It also suggests a model for how subtype differentiation in different neuronal lineages may be regulated by different proneural factors. In addition, it provides a paradigm for how transcriptional regulation may modulate the ciliogenesis pathway to give rise to structurally and functionally specialised ciliary dendrites (Cachero, 2011).
Numerous genetic and misexpression analyses in a range of organisms have shown that proneural factors influence a neuron's ultimate phenotype (including its subtype identity) at an early stage in its development. However, the nature of this influence on the cell biological processes of neuronal differentiation has remained obscure. This study bridges the gap between early specification by the proneural factor, ato, and the differentiation of Ch neurons. The current model in both Drosophila and vertebrates is that proneural factors activate two types of target gene during neural precursor specification: a common target set for shared neuronal properties and a unique target set for subtype-specific properties. The data suggest that such neuronal subtype differences are ultimately controlled by proneural factors in several ways: by the differential regulation of both specific and common intermediate transcription factors, which in turn regulate genes for aspects of neuronal structural and functional differentiation, and by direct regulation of potential differentiation genes (Cachero, 2011).
The proneural factors ato and sc commit cells to similar but distinct neural precursor fates: Ch and ES neurons are evolutionarily related cell types with similar but distinct structural and physiological properties. Notably, both are characterised by the possession of specialised ciliary-based dendrites. Thus, ciliogenesis is a key pathway that must ultimately be activated in sensory neurons subsequent to proneural factor function. However, there are important differences between the dendrites of Ch and ES neurons. Ch dendrites have a more prototypically organised axonemal structure and possess a characteristic ciliary dilation - a specialisation that separates the Ch ciliary dendrite into functionally distinct zones. Moreover, there is evidence for an active 'beat' of Ch cilia during sensory transduction. In general, ES dendrites appear reduced in structure: although a basal body and short axoneme are present, the tip of the dendrite consists of a 'tubular body' of irregularly packed microtubules. Thus the basic ciliogenesis pathway must be modulated differently in Ch and ES differentiation, and ultimately this must reflect a difference in function between ato and sc proneural factors. The ciliogenic regulator Rfx is expressed and required for both ES and Ch lineages, but it is more strongly and more persistently expressed in Ch lineages (the Ch-enriched pattern). This modulation of Rfx expression is at least partly due to differences in its regulation by proneural factors, since it appears to be a direct target of ato but not sc. This study hypothesises that differences in Rfx regulation by the proneural factors lead to differences in implementation of a core cilia biogenesis program, thereby directly linking early proneural factor function with key differences of neuronal morphology. Consistent with this idea, the data show that several known or predicted ciliogenesis genes also exhibit this Ch-enriched pattern, and some of these are predicted or known Rfx targets (Cachero, 2011).
In this view, the subtype differences between Ch and ES neurons are partly produced by quantitative differences in timing or level of expression of a common differentiation process, which ultimately depends on a qualitative difference in Rfx regulation by the proneural factors. A possible example of this is CG6129. This gene is a predicted Rfx target gene and is expressed in a Ch-enriched pattern (Laurenon, 2007). The homologous mouse protein (Rootletin) localises to the ciliary rootlet and is required for its formation. Thus Ch-enriched expression of CG6129 explains the presence of the ciliary rootlet in Ch neurons but not ES neurons. One prediction of this hypothesis is that overexpression of Rfx in ES neurons will upregulate Ch-enriched genes, and this is borne out by preliminary experiments that show an increase in CG6129 expression in ES neurons upon Rfx overexpression. It is notable that differences in IFT activity are proposed to underlie differences in ciliary morphology while RFX class factors have been associated with regulating genes for IFT in a variety of organisms. This work suggests that variations in Rfx expression level and timing should be explored as a possible factor in cilium diversity (Cachero, 2011).
Previous to this study, little was known about how ato/sc proneural genes control the acquisition of Ch/ES subtype identity, except that regulation of the Cut homeodomain transcription factor is involved. Mutant and misexpression analyses show that cut is a fate selector switch for ES identity downstream of sc, but nothing is known of its mode of action or targets. Whereas Rfx and fd3F functions are likely to be confined to neuronal morphology, cut affects the identity of support cells too. As a fate switch in the entire lineage, it appears likely that cut is involved in high-level fate specification (like proneural genes) rather than regulating aspects of differentiation directly. However, it is also possible that cut may repress ciliogenesis genes in ES neurons, either directly or by repressing Rfx expression. It will be important to integrate cut into the Ch/ES gene regulatory network in the future (Cachero, 2011).
In the temporal expression profiling data, there is a steady increase in the number of known or suspected differentiation genes expressed in developing Ch cells. Many more are not expressed until after the analysis ends. Ciliogenesis is a highly intricate cellular process requiring the coordination of perhaps hundreds of genes and differences in expression onset may indicate prerequisite steps in the process of differentiation and ciliogenesis. A surprising observation was the significant number of ciliogenesis and differentiation genes that are expressed even at the earliest profiling time point. This is unexpected, since the earliest time point is predicted to be not only before differentiation but also even before cell divisions have generated the neurons. It is suggested that further analysis of expression timing may lead to insights into the cell biology of ciliogenesis. The early activation of differentiation genes may reflect the rapid pace of development in the Drosophila embryo. Thus, early expression of ciliogenesis genes may provide components that prime cells for rapid cilium assembly later once differentiation has been triggered. Along these lines, the findings mirror striking observations of retinal ganglion cells, whose rapid differentiation within 15 minutes of the exit from mitosis has been taken to imply that genes required in postmitotic cells must be transcribed before cell division. A more intriguing possibility is that early expression reflects an orderly time course for ciliogenesis that begins many hours before the final cell division. For example, unc is thought to be required for the conversion of the mitotic centriole to ciliogenic basal body, but this stuyd found that the mRNA and fusion protein are expressed even in SOPs, several cell divisions before terminal differentiation. Interestingly, in mammals newly replicated centrioles mature over two cell cycles. It is conceivable that the sensory neuron basal body might similarly need time to mature (Cachero, 2011).
Since Rfx and some ciliogenesis genes are expressed in SOPs, what prevents ciliogenesis from being activated in the non-neuronal support cells? One possibility would be an extension of model recently proposed for the generation of support cell differences, in which Notch signalling between daughter cells confines the function of genes to one branch of the lineage. This would predict that ciliogenesis genes and/or Rfx are Notch target genes. Another possibility is that some of the gene products are asymmetrically segregated. Thirdly, ciliogenesis may not be triggered until one or more key gene products are produced in the neuronal cell (Cachero, 2011).
As a corollary, it will be important to explore further the gene regulatory network underlying the temporal and cell-type differences in ciliogenesis genes. Some early expressed differentiation genes are known or predicted Rfx targets (Laurenon, 2007). This gives a rationale for the early regulation of Rfx by ato in Ch lineages. However, in both C. elegans and D. melanogaster, Rfx regulates only a subset of ciliogenesis genes (notably, it does not regulate IFT-A genes). Further studies on ato target genes and the ciliogenesis regulatory network in sensory neurons will identify other important regulators. It remains to be determined how many differentiation genes are, like dila, direct targets of ato. Interestingly, vertebrate proneural factors are hypothesised to regulate directly the transition from cycling neural progenitor (or neural stem cell) to postmitotic differentiating neuron. Perhaps ato has retained some part of an ancestral proneural factor function in direct regulation of terminal differentiation despite the subsequent evolution of SOPs that must undergo several divisions before differentiating (Cachero, 2011).
Regulatory factor X (RFX) transcription factors play a key role in ciliary assembly in nematode, Drosophila and mouse. Using the tremendous advantages of comparative genomics in closely related species, novel genes were identified regulated by dRFX in Drosophila.
A subset of known ciliary genes in Caenorhabditis elegans and Drosophila are regulated by dRFX and have a conserved RFX binding site (X-box) in their promoters in two highly divergent Drosophila species. An X-box consensus sequence was designated (GYTRYY N1-3 RRHRAC) and a genome wide computer screen was carried out to identify novel genes under RFX control. 412 genes were identified that share a conserved X-box upstream of the ATG in both species, with 83 genes presenting a more restricted consensus. 25 of these 83 genes were analyzed, 16 of which are indeed RFX target genes. Two of them have never been described as involved in ciliogenesis. In addition, reporter construct expression analysis revealed that three of the identified genes encode proteins specifically localized in ciliated endings of Drosophila sensory neurons. It is concluded that this X-box search strategy led to the identification of novel RFX target genes in Drosophila that are involved in sensory ciliogenesis. A highly valuable Drosophila cilia and basal body dataset was established. These results demonstrate the accuracy of the X-box screen and will be useful for the identification of candidate genes for human ciliopathies, as several human homologs of RFX target genes are known to be involved in diseases, such as Bardet-Biedl syndrome (Laurençon, 2007).
Ciliogenic RFX regulatory networks are conserved between C. elegans and D. melanogaster. Based on these first observations, the genomic screens that were conducted combined with functional and in vivo gene analyses led to the identification of at least 11 novel genes that had never been described as RFX targets in any biological model. In addition, this screen allowed identification of at least two novel genes specifically expressed in ciliated sensory neurons in Drosophila that are potentially involved in sensory ciliogenesis. These results validate the accuracy of the screens. This work thus provides a new set of candidate genes for further functional studies in ciliogenesis (Laurençon, 2007).
The presence of a conserved X-box upstream of genes in both D. melanogaster and D. pseudoobscura is thus a good prognostic factor to predict novel dRFX target genes. The genome of both Drosophila species was screened for the presence of X-boxes. All possible matches were sought to a defined motif sequence using a Perl based algorithm. The most degenerated consensus RYYNYY N1-3 RRNRAC found 50,000 hits throughout the entire genome of D. melanogaster and, therefore, could not be used within the experimental framework. Five different more restricted consensus motifs were chosed that cover X-boxes of the entire set of known target genes at the time. Four (RYYVYY N1-3 RRHRAC, GYTNYY N1-3 RRNRAC, GYTDYY N1-3 RRNRAC, GYTRYY N1-3 RRHRAC) were searched in a 1 kb window upstream of the ATG, and the less degenerated one, RTNRCC N1-3 RGYAAC, in a 3 kb window (Laurençon, 2007).
Under these conditions, 4,726 non-redundant genes in D. melanogaster and 3,848 in D. pseudoobscura with an X-box upstream of the start codon were selected. Based on a best hit reciprocal search between the two coding sequence (CDS) lists, 1,462 homologous genes were identified having an X-box in their 5' region in both species. This first set of 1,462 genes was further restricted by selecting only genes that share an X-box with no more than 4 bases different (out of the 12 nucleotides recognized by the protein on either side of the spacer) between each species and in a conserved position upstream of the ATG (500 bp difference at most). The list was thus restricted to a subset of 412 genes. An even more restricted subset of genes was selected using the X-box motif GYTRYY N1-3 RRHRAC, which was found upstream of most known target RFX genes at the beginning of this work, leading to a list of 83 genes. Indeed, among the identified dRFX target genes for which a conserved X box was found in both Drosophila species, the highest percentage of target genes (50%, 8 out of 16) was found in this list of 83 genes. The remaining 50% of known RFX target genes were not selected by the X-box screen and thus represent false negatives (Laurençon, 2007).
The Drosophila genome wide X-box screen led to the identification of 83 X-box genes among which 11 novel RFX targets were identified. Combined with the genes identified by comparisons to C. elegans or to other genomic studies in Drosophila, 35 genes regulated by dRFX are reported in this study. Most of these genes can be classified based on their described function. Many of the RFX target genes are involved in IFT, which is necessary for cilium assembly and function. Remarkably, a second class of genes regulated by dRFX includes all the Drosophila homologs of Bardet-Biedl Syndrome (BBS) genes. Similarly, most C. elegans BBS genes are regulated by DAF-19. This strong dependence of BBS genes on RFX control may thus be conserved in mammals. Hence, RFX proteins may be involved in BBS in humans. Interestingly, two of the three Drosophila genes coding for proteins with B9 domains are also controlled by dRFX (tectonic, CG14870). One human B9 domain protein, MKS1, is known to be involved in the human Meckel-Gruber syndrome. The molecular function of this domain is unknown and work in Drosophila suggested that these two B9 domain containing proteins are likely involved in ciliogenesis. Several of the novel dRFX target genes that were identified in this study encode known components of the ciliary axoneme and associated structures, such as axonemal dyneins or rootletin. Other genes encode different types of proteins likely involved in sensory transduction (CG4536/osm-9/TRPV4 or MIP-T3). A last class includes genes for which the function is either not described or poorly understood, such as CG31036 and CG13125. However, functional studies strongly suggest that they are also probably involved in sensory ciliogenesis in Drosophila as well. Thus, RFX target genes play various roles in ciliary structure and function and the X-box search strategy has proven to be useful to identify novel ciliogenic genes (Laurençon, 2007).
This full set of dRFX target genes in Drosophila is of crucial importance, since it is now possible to more precisely define X-box sequences and the promoter context required for dRFX control. This will be particularly useful for further database mining of dRFX target genes in Drosophila. In fact, several genes that are under dRFX control, for example CG4525, CG17599) for which an X-box can be identified did not come out in the whole genome X-box screen. Several reasons can explain this result. First, homologs were not all annotated in CDS listings that were available at the time of the search (for example, CG18631, CG9595, nompB in D. pseudoobsura). Second, annotation of both Drosophila databases is incomplete, as sometimes the start codon is not properly defined for all genes. The X-box search algorithm keeps only genes for which the X-box match is upstream of the ATG. For example, for CG15666/GA13881, it was clearly predicted that the correct ATG should be considered 75 bp downstream of the currently defined ATG, based on evolutionarily conserved sequences. This definition clearly excludes the homologous genes CG15666 and GA13881 from the dataset. However, the X-box consensus cannot define a clearly conserved X-box match in the two Drosophila species for genes that appear to be down-regulated in a dRfx mutant, while several individual X-boxes are found separately in each organism. This could either reflect that these genes are not direct dRFX targets but are shut down by a feedback control loop that is not dependent on a X-box motif, or that the X-box is only loosely conserved in some promoter contexts. Notably, homologs of these genes in C. elegans are under RFX (DAF-19) control and have a well defined X-box (for example, CG9333/che-2, CG13691/bbs-8), which argues in favor of the second possibility. Interestingly, the expression levels were also quantified in control and dRfx deficient Drosophila of several genes of the DCBB dataset that did not come out of the X-box genome-wide motif search. It allowed identification of several novel genes that are indeed down-regulated in dRfx mutants, but for which no conserved X-box can be recognized based on the initial consensus motif. Altogether, these observations clearly highlight the difficulties encountered in motif definition in promoters. Similar conclusions were deduced from a parallel approach performed in C. elegans, which has led to the identification of several novel DAF-19 target genes. Interestingly, in that study the in silico search was associated with microarray analysis of transcripts in wild-type and daf-19 mutant worms. The in silico search allowed the identification of 93 X-box genes. Yet, among the 466 genes that were shown to be down-regulated at least two-fold in microarray hybridization experiments, only 25 were also represented in the 93 in silico X-box gene list. Thus, in silico searches on isolated motifs are likely hampered by a high level of false negatives. In order to improve the screening efficiency, the use of combinatorial motif searches would probably greatly enhance the accuracy of the screen as proposed by other studies. Even though, since conserved X-boxes that were identified are rarely associated with highly conserved surrounding sequences, it is reasonable to assume that other conserved nearby motifs, still to be identified, could help to discriminate between false positives and false negatives (Laurençon, 2007).
35 genes were identified that are transcriptionally down-regulated in dRfx mutants. RFX regulatory networks are conserved between C. elegans and Drosophila as most of the genes controlled by DAF-19 in C. elegans are also under dRFX control in D. melanogaster. Interestingly, the results show that only certain subsets of ciliogenic genes are regulated by RFX proteins. For example, in assay conditions all the genes known to be involved in IFT-A complexes are not regulated by dRFX, whereas all IFT-B homologous proteins are regulated by dRFX. In addition, retrograde motors are also regulated by dRFX (CG15148/btv and CG3769), whereas anterograde motors seem not to be. Indeed, in addition to CG10642/KIF3A, the main described anterograde motor in several organisms, it was shown that two other kinesin subunits, CG17461/Kif3C/osm-3 and CG7293/Klp68D, are invariantly expressed in wild-type and dRfx-deficient Drosophila. It is also interesting to note that all the BBS gene homologs in D. melanogaster are under dRFX control (Laurençon, 2007).
The biological significance of these observations is unclear. It could reflect the fact that IFT-B proteins, BBS proteins and the dyneins involved in IFT are dedicated to ciliogenesis and, therefore, need to be turned on concomitantly only when the cilium is formed, whereas IFT-A complexes or anterograde transport kinesin II share more complex regulatory controls as they might be necessary also for other cellular functions. This is the case for kinesin II motors, but does not seem to be true for IFT-A complexes as these proteins are proposed to be specific for ciliated organisms. In C. elegans, the ciliary IFT machinery works in modular fashion, and it is tempting to speculate that RFX-dependent proteins could be involved in specialized ciliogenic transport modules (Laurençon, 2007).
Genes necessary for centriole biogenesis or replication, such as the recently described DSas-6, DSas-4 or sak genes are not present in the screen and no conserved X-box can be found upstream of these genes. Thus, dRFX does not seem to regulate centriole biogenesis and appears to be restricted to cilia assembly only (Laurençon, 2007).
To find which transcription factors are responsible for governing other sets of ciliary proteins will certainly be one track to follow. Based on the data, it would be of particular interest to compare promoter sequences of genes, either regulated by dRFX, or not. It may allow discovery og novel regulatory motifs and protein modules that are necessary to coordinate ciliogenesis control. So far, only a few transcription factors have been shown to be involved in the control of ciliogenesis: the RFX proteins, Foxj1, and HNF1-beta. However, these have no obvious homologs in Drosophila. Thus, this work strongly suggests that novel transcription factors necessary for ciliogenesis still need to be discovered (Laurençon, 2007).
Some of the novel RFX target genes found in Drosophila were unexpected. For example, several proteins were identified that are proposed to be involved in flagella or cilia motility, such as dynein heavy chains (CG17150/Dhc93AB). Recently, a CG13125 homolog has also been shown to function as a motility factor in T. brucei (TbCMF46). Sensory cilia are thought not to be motile in general. However, it has been shown that Drosophila chordotonal neurons of the antenna generate motion that depends on the integrity of proteins encoded by genes such as CG15148/btv (cytoplasmic dynein heavy chain) or CG14620/tilB (LRRC6 homolog), described to affect the axonemal structure. In addition, cilia of the chordotonal neurons of the grasshopper bend upon vibration stimulation. Thus, proteins involved in axonemal motility might be important for motion generation of the cilium in response to mechanical stimulation. It will be of high interest to determine whether flies defective in these 'motility' genes are affected in hearing and, more specifically, in the motility of the mechanosensory cilium that amplifies hearing vibrations. Interestingly, CG13125/TbCMF46 does not seem to be expressed in fly testis, where the spermatozoa are the only cell type with a motile flagellum in flies. This suggests that like CG15148/btv, CG13125/TbCMF46 function could be restricted to the sensory cilium and, more specifically, in allowing these cilia to mechanically respond to auditory vibrations. Thus, the data suggest that in the fly, possible axonemal motility could be regulated by different subsets of proteins in sperm flagella and in mechanosensory cilia. This is of particular interest with regard to hearing in mammals, which is dependent on hair cell motility. It will be very interesting to determine whether the CG13125/TbCMF46 homolog in mammals does have a specific function in those cell types (Laurençon, 2007).
Three genes (CG6054/Su(fu), CG13415/Cby, CG33038/Ext(2)) were identified that are known to be involved in the hedgehog or wingless signaling pathways in Drosophila. Su(fu) and Ext(2) are involved in the Hedgehog pathway and Su(fu) is localized to cilia in mammalian cells. However, Su(fu) and Ext(2) do not appear to be under dRfx control according to real-time PCR quantification results and may be false positives in the screen. This result argues in favor of the generally accepted observation that the Hedgehog signaling pathway does not seem to depend on ciliogenic proteins in Drosophila. Only Chibby (Cby) is statistically down-regulated two-fold in a dRfx deficient background. Cby was isolated in a two-hybrid screen for armadillo/beta-catenin interactors. RNAi knock-down of Cby in Drosophila embryos leads to ectopic activation of the wingless pathway. Cby is also described to antagonize the Wnt/beta-catenin pathway in mammalian cells. However, the expression pattern of Cby in Drosophila is not documented, so it is not known if the variations of expression observed in the dRfx deficient background are connected to dRfx expression and, thus, if it is biologically significant (Laurençon, 2007).
Among the 83 genes with conserved X-boxes between D. melanogaster and D. pseudoobscura, several genes were hardly detectable by quantitative RT-PCR. Hence it was not possible to determine by this approach if they are under dRFX control. This could reflect that these genes are expressed only in a subset of sensory neurons and, thus, difficult to detect by quantitative RT-PCR. Nevertheless, several genes are interesting as potential ciliogenic or RFX target genes. For example, CG14079 is homologous to a mouse protein that appears to be specific to testis. CG11356 is homologous to mammalian arl13, which has just been isolated in an ethyl-nitroso-urea screen for neural tube defects in mouse. Indeed, mutation of arl13 affects ciliary architecture and Sonic-Hedgehog signaling in mouse. This gene, CG11356, was not found in any previous ciliogenesis study, again illustrating the accuracy of the screen. Functional studies in Drosophila will be of particular importance to demonstrate the role of this gene in sensory ciliogenesis (Laurençon, 2007).
This study has identified more than 30 dRFX target genes in Drosophila by exploiting the efficiency of the X-box promoter motif search by using two divergent Drosophila species in a comparative approach. These full sets of RFX dependent or independent ciliary genes are of particular importance for studies of X-box promoter motifs and associated promoter contexts in Drosophila. More remarkably, the screen allowed the identification of at least two novel genes specific to sensory ciliary architecture in D. melanogaster and provides several new RFX target gene candidates potentially involved in ciliogenesis. This is of particular importance with regard to the growing number of human diseases that are being associated with ciliary defects (Laurençon, 2007).
Rfx expression was analyzed during embryogenesis. Rfx mRNAs are detected only in the peripheral nervous system and in the brain of the embryo (Durand, 2000).
Drosophila Rfx immunoreactivity is distributed in type I sensory neuron lineage of the peripheral nervous system throughout Drosophila development and thus represents the first described type I lineage characteristic marker in Drosophila. In addition, Rfx is also detected in the brain throughout development and in spermatids in adult flies (Vandaele, 2001).
In embryos, Rfx is first detected at the onset of segmentation in the sensory organ precursors (SOPs) located in the gnathal segment and in the two first SOPs of each thoracic and abdominal hemisegments described as the A (anterior) and the P (posterior) SOPs. At stages 12-14 of embryogenesis, when A and P SOPs have generated new precursors by division or recruitment, Rfx is detected in these secondary precursors. At stage 15, Rfx is found predominantly in nuclei of all chordotonal (ch) and external sensory (es) organ neurons, and, at a lower level, in the accessory sister cells resulting from the last asymmetric division of the precursors. At stage 16 of embryogenesis, Rfx progressively disappears in the accessory sister cells and is only maintained in neuron nuclei at the end of embryogenesis. These results are in agreement with Rfx mRNA expression described previously (Durand, 2000) and provide new important information on Rfx stability. Whereas Rfx messenger RNAs disappear in all cells of the peripheral nervous system except ch neurons at the end of embryogenesis, the protein still persists in es neurons. Two types of sensory neurons can be distinguished in Drosophila: (1) type I neurons that innervate es and ch organs bear a ciliated sensory process at the tip of a unique dendrite and are surrounded by specialized supporting cells, and (2) type II neurons are nonciliated multidendritic neurons. In the embryo, both types of neurons are also characterized by their relative position and shape. Using an antibody that labels all sensory neurons (mAb 22C10), Rfx is found to be restricted to type I neurons of the thoracic and abdominal segments. In the head, Rfx is detected in all sensory neurons, except Bolwig's organ, which represents the future larval visual organ (Vandaele, 2001).
An accumulation of Rfx in the brain is also observed starting from stage 12 of embryogenesis in two small bilateral cell clusters in the procephalic neurogenic region. From stage 14 to 17 of embryonic development, Rfx persists in a small number of cells abutting the optic lobe invagination. Rfx is detected in the brain throughout larval and pupal development in a restricted number of cells in each brain lobe (Vandaele, 2001).
During larval and pupal life, Rfx is absent in early SOPs of the imaginal discs, but appears later in development, i.e. after puparium formation (APF). Rfx is found in all adult type I sensory organ lineage analyzed (antennae, leg and wing discs). No expression is detected in the eye disc. Expression is first detected in the leg discs, at the beginning of eversion, in the innermost SOPs of the femoral ch organ, that are ready to differentiate. Similarly, Rfx is detected in the Johnston organs of the second antennal segment of the antennal discs only once they are fully everted (around 24 h APF) (Vandaele, 2001).
Finally, Rfx is very transiently present in adult males during spermatogenesis in syncitial bundles of 64 spermatids. According to their shape and position within the testis lumen, these spermatids are in the elongation phase of their flagellum. The protein is not detectable in spherical spermatid nuclei nor in late condensing spermatid nuclei. Rfx was not detected in the female germline and in other tissues of whole stained dissected larvae (Vandaele, 2001).
Rfx is located on the third chromosome at 86A1-2 and is flanked by two genes transcribed on the opposite strand: jumeaux (also described as Domina) and CG17100. CG17100 has been suggested to be affected in stich1 mutants. Available deficiencies were investigated in the 86A-B domain that would uncover Rfx. Df(3R)hth uncovers Rfx, as visualized by polytene chromosome in situ hybridization with a Rfx probe. Moreover, no trace of RFX protein is detected in Df(3R)hth homozygous embryo tissues by immunostaining. All together with complementation results these data indicate that Df(3R)hth is a deficiency uncovering the entire 85F-86C region (Dubruille, 2002).
A P-element inserted 5' of Rfx (A143.1F3) was mobilized to generate more aberrations affecting the Rfx locus. Several chromosomes were selected by molecular screening, including Rfx49, a small deficiency uncovering the 5' end of Rfx. Molecular characterization of Rfx49 breakpoints revealed that the deletion does not uncover the adjacent jumu gene. Indeed, Rfx49 complements the lethal phenotype of all jumu lethal alleles tested. Moreover, no RFX immunostaining was found in the peripheral nervous system of Rfx49 mutant embryos compared with wild-type embryos (Vandaele, 2001; Dubruille, 2002).
Complementation analysis with Rfx49 and Df(3R)hth chromosomes identifies two complementation groups in the 86A1-2 region that have been identified as jumu and Rfx. All stich1 alleles fail to complement jumu loss of function alleles. S143702 is actually a deficiency uncovering both jumu and Rfx loci. All together these results imply that stich1 mutations affect the jumu gene but not CG17100 (Dubruille, 2002).
The more distal breakpoint of Rfx49 maps 100 bp upstream of the 5' end of CG17100. It is therefore theoretically possible that Rfx49 also affects this gene. An EMS non-complementation screen was undertaken to isolate other Rfx alleles. The Rfx253 chromosome was isolated that defines the same complementation group as Rfx49. Sequencing analysis revealed a mutation causing an amino acid substitution, S435F, in the DNA-binding domain. Serine at position 65 of the DBD is replaced by a phenylalanine. Remarkably, the mutated serine is absolutely conserved in all RFX proteins described from yeast to mammals and, in a crystallographic structure of the mammalian RFX1 DBD, is located in the 'wing' loop that mediates most DNA contacts (Gajiwala, 2000). Electromobility shift assay confirmed that Rfx253 is no longer able to bind its target sequence. Thus, based on its lack of DNA binding, Rfx253 represents a hypomorphic or null allele of Rfx (Dubruille, 2002).
Rfx is necessary for olfactory and gustatory perception in Drosophila larvae. Under standard rearing conditions, most Rfx49 and Rfx253 homozygotes die as larvae. Mutant pupae could not be observed on the walls of the culture vials, regardless of allelic combination. However, in uncrowded conditions, a small proportion of homozygous pupae were detected on vials, indicating that a few larvae can pupate. Approximately 15% of first instar larvae give rise to pupae. Mutant larvae lag their heterologous siblings by more than 3 days in development to pupae. Close observation of larvae show a peculiar foraging behavior with weak food digging efficiency. This could result from an altered peripheral sensitivity, a hypothesis that was suggested by the fact that Rfx is expressed in sensory neurons during embryogenesis (Vandaele, 2000). The gustatory and olfactory-driven behavior of mutant larvae was therefore investigated (Dubruille, 2002).
An olfactory test was performed using two pure odors (butanol and n-octyl-acetate) that induce characteristic attractive and repulsive responses. Whereas control larvae responded efficiently, mutant larvae showed no sensitivity to either odor. Also, repulsion was assayed by high NaCl concentrations: while control larvae are repulsed by concentrated salt, mutant larvae do not avoid this medium, showing that Rfx mutant larvae are defective in gustatory chemotaxis as well. To demonstrate that these responses are not due to a defect in the larval motility, phototaxis behavior was tested. This is based on the finding that Rfx is not expressed in the Bolwig organs, the larval visual system (Vandaele, 2001). Rfx mutant larvae should therefore not be affected in visual perception. This was clearly demonstrated by phototaxis behavioral assay. The phototaxis behaviors of Rfx mutant and control larvae are identical, demonstrating that Rfx mutants are not affected in their capacity to move. These data led to the conclusion that Rfx is necessary for olfactory and gustatory perception in Drosophila larvae (Dubruille, 2002).
Emerging adults are not able to walk and fly and thus stick to the medium and cannot be retrieved in culture vials. In order to recover adult mutants, third instar larvae and pupae were selected and raised until hatching on wet filter paper. The 15% to 30% of Rfx49/Rfx253 or S143702/Rfx253 adults that emerge from pupae, all present a characteristic uncoordinated phenotype. Mutant flies cannot stand or walk: legs are crossed and wings are held up. However, mutants do react to light stimulation by moving legs and wings in an effort to right themselves and walk, but uncoordination limits their movement to a few steps or a flip from one side to another. This transient activity indicates that general neuromuscular excitability is retained. To ascertain that Rfx is responsible for the observed phenotype, an Rfx cDNA was expressed in all neurons in Rfx49/Rfx253 mutants. The transgene expression indeed completely rescued the Rfx49/Rfx253 uncoordinated and low viability phenotypes (Dubruille, 2002).
The uncoordination of Rfx mutant flies is similar to the phenotype of mutants with defects in mechanosensory transduction. Type I external mechanosensory bristles include three support cells and one neuron. The innermost support cell (sheath cell) forms a tubular extension around the sensory process and produces an extracellular dendritic cap that covers the cilium tip. The two outer support cells (shaft and socket cells) construct the cuticular bristle socket and shaft and in mature organs generate the receptor lymph. To test if Rfx mutants also have defects in mechanosensory bristle electrophysiology, transepithelial potentials (TEPs) and currents were recorded from macrochaete bristles. Unstimulated wild-type bristles have a positive TEP, due to electrogenic K+ transport by sensory support cells into the restricted apical extracellular space. This creates the electrical driving force for a receptor current that is triggered by bristle deflection: current flows from the apical extracellular space into the sensory neuron, reducing the TEP and depolarizing the neuron so that it fires a series of action potentials. The response adapts to a sustained deflection (Dubruille, 2002).
The average resting TEP in Rfx mutants (12±9 mV) is significantly lower than in their heterozygous sibs (61±4 mV), although the mutant and control distributions overlap. More strikingly, no mechanically elicited changes in TEP were detected in any Rfx mutant bristle, even in bristles with TEP in the normal range. Holding the TEP constant while stimulating the bristle enables a mechanoreceptor current to be recorded. No receptor currents were elicited by mechanical stimulation in Rfx mutants, even when the TEP was clamped at a large positive value (+100 mV). Finally, no action potentials were observed in mutant neurons. Thus, the adult uncoordination phenotype probably reflects a failure of bristle mechanotransduction. Maintenance of the TEP requires both active ion transport by sensory support cells, and high cuticular and transepithelial resistance. The resistance at Rfx mutant bristles was found to be, on average, one-third that at control bristles, suggesting some reduction in the integrity of the intercellular junctions connecting the neuron and the surrounding support cells (Dubruille, 2002).
Most mechanosensory mutations affect chordotonal organs as well as bristles. Chordotonal organs are type I stretch receptors that transduce proprioceptive and auditory stimuli. To test chordotonal organ operation, extracellular sound-elicited potentials were recorded from the antennal nerve projecting from the auditory chordotonal organ (Johnston's organ) in wild-type and mutant antennae. Johnston's organ comprises over 100 individual sensory units or scolopidia located in the second antennal segment, that normally respond when distal antennal segments are vibrated by near-field sound. A series of sound pulses elicited a corresponding response from Rfx49/TM6B heterozygotes, but Rfx49/Rfx253 mutants showed no response to stimuli of the same or greater amplitude, indicating a complete loss of auditory chordotonal organ function (Dubruille, 2002).
Taken together, the broad spectrum of behavioral and electrophysiological defects suggest that all type I organs are affected in Rfx mutants. This idea was further pursued using light and electron microscopy (Dubruille, 2002).
To examine the morphology of sensory neurons in Rfx mutants, a series of molecular markers for the peripheral nervous system (PNS) was used. With the 22C10 antibody, which stains a microtubule-associated protein in all sensory neurons, no differences were observed between mutant and wild-type embryos in the number and position of sensory neurons. By contrast, the anti-HRP antibody, which stains all neuron surfaces in wild-type embryos, revealed a very weak staining of the PNS in the mutant embryos regardless of the Rfx allelic combination used. An identical weak anti-HRP staining of sensory neurons was also reported for S143702 embryos (a deficiency mutation uncovering Rfx), confirming that this phenotype is specifically due to loss of Rfx function (Dubruille, 2002).
Anti-HRP staining in mutant embryos is, however, strong enough to visualize a specific dendrite defect, particularly visible in the lateral chordotonal organ clusters (1ch5). Indeed, this array of chordotonal organs is disorganized in Rfx embryos. Most dendrites are shorter, and vary in length within a cluster. The cilia present in wild type or control embryos are always either shorter or missing in Rfx mutants (Dubruille, 2002).
To visualize neuronal dendrites in more detail, mutant flies were engineered expressing a membrane-localized GFP in all neurons. Focus was placed on two types of adult sensory organs: campaniform sensilla of the wing and chordotonal organs of the femur (Dubruille, 2002).
Campaniform sensilla of the wing are composed of a single neuron and three accessory cells. The neuronal cell body and the dendrite are situated in the same focal plane and are thus easily visible when marked with GFP. The cilium at the tip of the dendrite is located exactly under the dome-shaped sensilla in wild-type or in rescued wings. In Rfx mutant wings, the dendrite does not reach the dome and the cilium is either absent, or shorter (Dubruille, 2002).
In the femoral chordotonal organ, each scolopidium is innervated by two ciliated neurons. Instead of external cuticular structures, a rigid scolopale structure encloses the ciliary outer segments. By confocal microscopy analysis of GFP distribution, three ciliary regions can be distinguished. These regions correlate with an ultrastuctural organization that has been described by transmission electron microscopy: the axoneme, the ciliary dilation and the tubular body enclosing the tubular bundle (a dense array of microtubules). These subregions are clearly distinguished in control or rescued Rfx mutant flies. By contrast, the cilium is always shorter and even absent in mutant flies. When present, no clear cilium subregions can be distinguished indicating that the architecture of the cilium is very disorganized. Moreover, the inner dendritic segment appears swollen. These defects are specifically rescued when the Rfx cDNA is expressed in all neurons, thus demonstrating that Rfx expression in the neuron is necessary for differentiation of the ciliated sensory ending in adult sense organs (Dubruille, 2002).
Investigation of the ultrastructure of sensitive neurons was carried out on the Johnston's organ in the second antennal segment. Each scolopidium of the Johnston's organ includes two neurons, a scolopale cell and an elongated conical dendritic cap that encloses the distal tips of the two cilia. The tips of a pair of cilia are attached via an extracellular cap to a distal attachment cell (cap cell), and their bases are anchored within the neurons by a long axial filament that extends through the neuronal dendrites and cell bodies. Cross-sections performed at different proximodistal positions reveal the three different typical ultrastructural subregions of chordotonal cilia. The sensory cilium is composed of 9+0 microtubule doublets. The ciliary dilation contains a paracrystalline inclusion and the tubular body is composed of microtubule arrays that are not assembled in a stereotyped fashion as in the cilium. The two cilia are surrounded by electron dense structures (scolopale rods) produced by the scolopale cell. The cilium at the tip of the dendrite contacts an extracellular matrix (dendritic cap) (Dubruille, 2002).
Examination of sectioned antennae of Rfx mutants by light microscopy showed no gross defects in Johnston's organ, but the ultrastructure of individual scolopidia was profoundly altered. Sections of several scolopidia at different proximodistal positions clearly show the 9+0 axonemal structure in wild-type antennae, but no axoneme profiles were found in serial sections of Rfx253/Rfx49 antennae. In longitudinal sections the anchoring axial filament can be clearly observed in wild type, but not in mutant scolopidia. Its absence indicates that Rfx mutant defects extend beyond the cilium proper and affect associated structures in the neuron. In addition, a non-neuronal defect is noted: the electron dense scolopale rods enclosing the cilium are not as regularly arrayed as in wild-type flies. By contrast, the dendritic cap, also produced by the scolopale cell appears equally well organized in wild type and in Rfx mutants. These results demonstrate that Rfx is necessary for proper cilium assembly in chordotonal organs (Dubruille, 2002).
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date revised: 15 April 2011
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