InteractiveFly: GeneBrief

Dynein axonemal assembly factor 5: Biological Overview | References

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, a recently elucidated cilia transcriptional RFX-FOX code was used to identify novel PCD candidate genes. This study reports characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with outer arm dynein intermediate chain (DNAI2; see dynein, axonemal, intermediate chain 2), but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme (Diggle, 2014).

Cilia and flagella are small microtubule-based projections on the cell surface, where they perform diverse sensory, and in some cases motility functions. These highly conserved organelles are found across species from protozoa to mammals, and are believed to have evolved from flagellar structures found in the last eukaryotic common ancestor (LECA). At their core is an axoneme composed of a peripheral arrangement of 9 microtubule doublets. Extension and maintenance of cilia involves a conserved microtubule-based process of motor-driven intraflagellar transport (IFT) that traffics protein cargo from the ciliary base to the tip, and back again. Despite the functional and structural diversity that has arisen among cilia, they retain key elements. Axonemes of motile cilia, which usually have an additional central pair of singlet microtubules in a '9+2' arrangement, possess inner (IDA) and outer dynein arms (ODA) attached to the peripheral outer doublet A microtubule. These orchestrate the ATP-dependent sliding of the doublets relative to each other, enabling motility. Recent genomic and proteomic studies have compiled a 'motile ciliome', now consisting of several hundred centrosomal and ciliary components. However, how these components are assembled into different structural and functional cilia types remain largely unknown (Diggle, 2014).

Disorders specifically arising from dysfunction of motile cilia are called Primary Ciliary Dyskinesias. As a result of defective airway mucociliary clearance, individuals with PCD typically present in the first year of life with recurrent infections, rhinosinusitis and otitis media, resulting in a chronic respiratory condition which can progress to permanent lung damage (bronchiectasis). Around half of PCD patients also have laterality defects as a result of embryonic nodal cilia dysfunction that lead to randomization of the left-right body axis, most commonly situs inversus totalis (Kartagener syndrome) or more rarely heterotaxy defects affecting the heart. Fertility defects are also reported in individuals with PCD. PCD is genetically heterogeneous, and 27 causative loci have been identified to date, but these still account for only a fraction of total cases. Diagnosis of PCD typically involves identification of ultrastructural defects of the motile ciliary axoneme, over 70% of which involve the loss of ODA. Many cases of PCD can be attributed to mutations in genes encoding ultrastructural components of motile cilia such as dynein subunits or proteins involved in their docking and targeting, central pair microtubules, radial spokes and nexin-dynein regulatory complex. Less well functionally characterized are a growing group of cytoplasmic factors that are putatively involved in trafficking or stability of dynein arm components (DNAAF1/LRRC50, DNAAF2/KTU, DNAAF3/PF22, DNAAF4/DYX1C1, HEATR2, LRRC6, SPAG1, ZMYND10, CCDC103, C21ORF59). Several of these Dynein Axonemal Assembly Factors, such as the case for DNAAF1-4, work directly within a heat-shock protein (HSP)-based molecular chaperone complexes to direct the proper folding of axonemal dynein subunits (Diggle, 2014).

Given the complexity of the motile cilia assembly and function, it is possible that there is an underlying conserved transcriptional programme that could be used to identify further PCD candidate genes? Expression of the core ciliogenic programme, including components of the IFT and BBSome machinery, is regulated at the transcriptional level in part by the regulatory factor X (RFX) family of transcription factors. RFX proteins are essential for ciliogenesis in C. elegans and D. melanogaster. Of the eight paralogues that exist in mammals, Rfx2 and Rfx3 have been implicated in vertebrate motile ciliogenesis. These proteins contain a highly conserved DNA-binding domain, which directly interacts with a consensus sequence, the X-box motif. Identification of X-boxes in promoters of genes transcriptionally activated during ciliogenesis has been previously used for identification of putative ciliopathy candidates (Diggle, 2014).

It has been proposed that the diversity of cilia function could arise from elaboration of a core ciliary transcriptome through additional transcriptional controls. FOXJ1 (HFH4), a forkhead/winged-helix transcription factor, has been shown to activate gene expression required for motile cilia formation. FOXJ1 is highly expressed in tissues with motile cilia and only motile cilia are affected in Foxj1 null mice. Overexpression of FoxJ1 is sufficient to confer some motile functions on primary cilia in both D. renio and X. tropicalis. Functional diversification of specific subsets of motile cilia involves other transcription factors including the homeobox NOTO for nodal cilia, as well as MYB and nuclear MULTICILIN for multiciliated epithelia (Diggle, 2014).

Unlike their wide distribution and diverse types in vertebrates, cilia in D. melanogaster are very restricted. The only somatic cells with cilia in flies are sensory neurons, which have specialized ciliary dendrites for sensory reception. Only a subset of these, the proprioceptive and auditory chordotonal (Ch) neurons, possess cilia that are motile. As a result, genes encoding axonemal dynein subunits and other motility components are uniquely expressed in Ch neurons and spermatocytes. In the Drosophila antenna, Ch neuron ciliary motility is proposed to be part of a mechanical amplification process involved in transducing sound vibrations through the interplay of motors and transduction channels. This simplicity of cilia diversity is recapitulated at the transcriptional level. A single Drosophila Rfx member controls expression of core ciliogenic targets in all ciliated sensory neurons (Vandaele, 2001). Although the existence of a Drosophila FOXJ1 orthologue had been questioned, it was recently demonstrated that the diverged Fox gene fd3F is required for Ch neuron function (Cachero, 2011). Fd3F protein cooperates with Rfx to control expression of Ch-specific genes, including those encoding many structural components of the motility machinery such as ODA subunits (dynein heavy chain Dhc93AB, homolog of DNAH9/11), and IDA subunits (Dhc16F, homolog of DNAH6, and CG6971, homolog of DNALI1). In addition, Fd3F was found to regulate several unknown or poorly characterized genes that shared a similar Ch neuron-specific transcriptional profile. Some of these have subsequently been found to encode cytoplasmic proteins that are implicated in the assembly and/or transport of the dynein arm apparatus (tilB/LRRC6, dtr/DNAAF1), and their mutation in humans leads to PCD. In the majority of these target genes, conserved Fox and Rfx consensus binding motifs could be found in their promoters in close proximity to the transcriptional start site. It is hypothesized that this transcriptional fingerprint could be used to predict novel components of the motile cilia machinery as a route to finding additional human orthologues potentially involved in PCD (Diggle, 2014).

This study presents studies on one such candidate, CG31320/HEATR2, which plays a conserved role in the assembly and/or stability of ODA and IDA in humans and in flies. A novel splice-acceptor HEATR2 mutation was identified in a PCD family associated with respiratory and laterality defects. CG31320 mutant flies exhibit aberrant proprioception, deafness and immotile sperm due to ciliary/flagella motility defects, which correlates with a lack of ODA and IDA in the cilia. Similar to the RFX- and FdF3- regulated expression of CG31320 in Ch neurons, this study shows higher levels of cytoplasmic HEATR2 early in mammalian motile ciliogenesis within differentiating cells that also express high RFX3 and FOXJ1. Human HEATR2 immunoprecipitation data is presented showing interaction with outer arm dynein intermediate chain DNAI2, but not other DNAAFs or HSPs. It is therefore proposed that HEATR2 is unique amongst known DNAAFs as it acts in the early stages of cytoplasmic dynein preassembly but not in a classic chaperone/client function. It is suggested HEATR2 functions as a flexible scaffold for stabilizing interactions between dynein subunits, like DNAI2, during cytoplasmic pre-assembly (Diggle, 2014).

An extended UK-Pakistani family was identified with three affected children presenting with PCD. The proband (IV:4) presented at the age of 3 with chronic respiratory infections, middle ear disease and chronic nasal discharge. She had also experienced neonatal respiratory distress and dextrocardia was apparent on chest X-ray. Nasal ciliary biopsy confirmed the PCD diagnosis by video microscopy, and written reports of transmission electron microscopy revealed absence of both IDA and ODA. Her cousins subsequently presented at the age of 2 (IV:1) and 4 (IV:10) with chronic chest infections and nasal discharge. The diagnosis of PCD was confirmed in both these latter cases by nasal ciliary biopsy, again electron microscopy confirmed absence of both inner and outer dynein arms (Diggle, 2014).

Whole genome SNP autozygosity mapping in this consanguineous family identified a single concordant homozygous region of 2.6 Mb on chromosome 7∶46,239-3,179,991 (GRCh37) shared by all three affected and not seen in unaffected sibs. A custom oligonucleotide array was designed to capture all exons as well as intron/exon boundaries within the interval, and these were analyzed by Next Generation Sequencing. This identified a single potentially pathogenic variant c.2432-1G>C in the splice acceptor site of the final exon of HEATR2 (NM_017802). Sanger sequencing confirmed the segregation of the variant with the phenotype within the family. The change which affects the highly conserved AG of the consensus U2-type splice acceptor is absent in 176 ethnically matched control individuals as well in 6503 European and African American subjects in the Exome Variant Server (NHLBI GO Exome Sequencing Project (ESP), Seattle, WA. All HEATR2 exons were sequenced in an additional 23 PCD patients but no further mutations were detected. However, another PCD-causing HEATR2 allele has been recently reported among a Midwest American Amish pedigree which contains a missense Leu795Pro mutation in a highly conserved residue in exon 12. Together, these studies indicate that independent mutations in HEATR2 contribute to a small proportion of PCD cases (Diggle, 2014).

HEATR2 cDNA was sequenced from patients to investigate the consequences of the G-to-C transversion mutation on HEATR2 transcripts. The PCD transversion mutation (ENST00000297440:c.2432-1G>C) affects the terminal G of the AG at the end of intron 12/13 located within the exon 13 splice acceptor consensus sequence. In patients, the first two coding bases of exon 13, also AG, create a cryptic splice site immediately adjacent which is utilised, resulting in a two base pair deletion of HEATR2 transcript and a consequent coding frameshift within exon 13. The mutation does not affect mutant HEATR2 transcript stability as shown by RT-qPCR and RT-PCR from parental control and patient cDNA, with the heterozygous parental samples containing approximately equal levels of each splicing variant. RT-PCR products spanning the splice acceptor mutation, exon 11-13 and exon 12-3'UTR showed no significant alterations in size, consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients and loss of just 2 bp. Moreover, ribonuclease protection assay (RPA) using riboprobes containing portions of exons 12 and 13 from unaffected control and patient cDNA confirmed with high sensitivity and specificity these splicing events are occurring in mutant HEATR2 transcripts (Diggle, 2014).

This PCD mutation and consequent frameshift (c.2432-2433delAG) was predicted to replace the final 44 amino acids of HEATR2 protein with 77 novel amino acids (pGlu811GlyfsTer78). This mutation would disrupt the last of ten highly conserved HEAT repeats and alter the C-terminus of the ARM-type fold superfamily domain. This simple array of repeating motifs is found in α-solenoid proteins and is best characterized by β-importin with 19 HEAT repeats. They are believed to create a highly flexible macromolecule with large surface area key for mediating protein-protein binding, both in terms of cargo selection and interactions with cell transport machinery. Western blot analysis confirmed the PCD HEATR2 mutation (pGlu811GlyfsTer78) resulted in a slight shift in mobility consistent with the predicted 3 kDa size increase. More striking, however, was a striking reduction in total HEATR2 levels in the patients. Parental samples, heterozygous for the mutation, showed a ~50% reduction in β actin-normalized HEATR2 levels compared to unrelated controls whilst the homozygous patient samples were ~3% of control levels. This suggests the mutation (pGlu811GlyfsTer78) resulted in pathogenic changes in the amino acid sequence and C-terminal structure of HEATR2 protein resulting in its instability. This study supports the recent report (Horani, 2012) of HEATR2's contribution to the genetic heterogeneity underlying Primary Ciliary Dyskinesia (Diggle, 2014).

Interest in transcriptional targets of the ciliary motility programme (Newton, 2012; Cachero, 2011) independently led this study to the Drosophila HEATR2 orthologue CG31320. High-resolution temporal gene expression profiling during Drosophila neural development suggested that CG31320 is expressed in differentiating Ch neurons prior to cilium formation (Cachero, 2011). This study proceeded to confirm the embryonic expression pattern of CG31320 by RNA in-situ hybridization. CG31320 mRNA was restricted to Ch neurons with expression beginning from about stage 12, after neuronal specification but preceding cilium formation, and continuing throughout neuronal differentiation (stage 14). Co-staining with anti-Rfx confirmed co-expression in late stage developing Ch neurons (Diggle, 2014).

Given its expression in early differentiating Ch neurons, whether CG31320 is regulated by ciliary transcription factors was investigated. CG31320 expression was found to be Rfx-dependent, since CG31320 expression is lost in Rfx mutant Ch neurons. CG31320 expression in Ch neurons was also fd3F-dependent, as it was absent in fd3F mutants and could be induced ectopically by forced Fd3F expression in all the Rfx-positive non-motile ciliated sensory neurons. These studies demonstrate that CG31320 expression is restricted in a Rfx- and fd3F-dependent manner to the neural cells that form specialized motile cilia. Outside the Ch lineage, CG31320 expression was also detected transiently in early stage 12 non-ciliated mesoderm and in the adult testes; its transcriptional control in these lineages remains unclear (Diggle, 2014).

To determine whether CG31320 is required for ciliary motility, CG31320 mutant flies were generated by imprecise excision of an associated P element in the line CG31320^EY06677. The resultant excision line CG31320²⁷ had a 992-bp deletion of the 5' end of the gene, including the transcriptional and translational start sites, and CG31320 mRNA was absent in homozygous CG31320²⁷ embryos. Visual inspection of deletion mutants showed a complete lack of surviving homozygote adult flies while homozygote larvae were smaller but with no obvious morphological defects. Since Ch neuron dysfunction is not lethal, this suggests a vital non-cilial role for CG31320 correlating with its expression in the developing midgut, which may affect nutrition. To bypass this lethality and focus on the cilial role, a Gal4-inducible RNAi line (P{KK102625}VIE-260B) was used to generate knock-downs lacking CG31320 specifically in embryonic and adult developing sensory neurons (scaGal4 UAS-Dcr2/UAS-CG31320-KK102625 RNAi). RNAi generated knock-down embryos showed a strong reduction in neural CG31320 mRNA. These knock-down flies are viable but uncoordinated, performing poorly in climbing assays, suggesting that Ch neurons are defective as these are required for proprioception during coordinated locomotion. Ch neurons are also required for hearing, and so larval auditory function was tested. Control larvae contract abruptly when exposed to a 1-kHz tone, due to auditory reception by Ch neurons. In contrast, CG31320 knock-down larvae failed to respond to the tone (Diggle, 2014).

Despite these functional defects, the specification and gross differentiation of Ch neurons in CG31320 RNAi flies was unaffected. Moreover, loss of CG31320 does not grossly disrupt formation of Ch neuron cilia or their functional compartmentalization, as shown by immunofluorescence for a neuronal marker and the ion channel NompC/TRPN1, which marks the distal non-motile cilium tip. Ch neuron ultrastructure in the antenna of CG31320 knock-down adult flies was examined by transmission electron microscopy (TEM). This revealed that the normal 9+0 arrangement of microtubule doublets in the proximal (motile) zone of the Ch neuron cilium was present but the axoneme lacked both ODA and IDA. Structures consistent with remnant dynein arms were only ever detected on a small minority of doublets. Thus, CG31320 is required specifically for the presence of the Ch ciliary motility apparatus (Diggle, 2014).

CG31320 is also expressed in the adult testes, which contains the only other motile cilium-like structure in Drosophila, the sperm flagellum. Transcriptional control of ciliary motility in testes is unclear. Although Rfx is expressed in spermatids, Rfx mutant spermatozoa are motile but males are too uncoordinated to mate. Similarly, fd3F mutant males are fertile. Moreover, extension and maintenance of the Drosophila sperm flagella is IFT-independent, such that IFT-B mutant sperm are motile and structurally normal. Thus the assembly of a 9+2 sperm axoneme with ODA and IDA occurs by an alternate cytosolic assembly mechanism. To address whether CG31320 function is also required for sperm motility, testes-specific CG31320 RNAi mutant males (Bam-VP16-Gal4; UAS-Dcr2/UAS-CG31320-KK102625 RNAi) were generated. Although such males can mate, they are sterile. Sperm development was examined in control and mutant testes. Normally spermatogonial germ cells at the apical tip of testes go through 4 synchronous mitotic amplifications with incomplete cytokinesis to produce a cyst of 16 interconnected spermatogonia (or primary spermatocytes). Eventually all 16 spermatocytes undergo meiosis I and II, to form a cyst of 64 inter-connected primary spermatids. Formation of sperm axonemes yields a bundle of extremely long spermatids that stretch almost the entire length of the testis. The final step in spermatogenesis is a highly complex process of membrane remodelling called individualisation to yield 64 individual sperm that are then transferred to the seminal vesicle (SV) in a process which appears to be dependent on sperm motility. CG31320 knock-down testes appeared to develop normally and mature sperm were clearly present, but no motile sperm were observed in the SV (Diggle, 2014).

To investigate the cellular defect underlying CG31320 mutant sperm immotility, TEM analysis was carried out of spermatid cysts. While wild type cysts contain 64 spermatids produced by division of a single precursor, CG31320 knock-down cysts contained an average of 60.7 identifiable spermatids. These had a normal axonemal '9+2' arrangement, but dynein arms were generally not visible, consistent with the lack of sperm motility. In addition, a proportion of axonemes showed separation of some doublets from the core, suggesting defects in motility-associated nexin links. In addition some 'A' sub-tubules of the doublets contained electron-dense cores, which are normally only seen in the accessory and central pair microtubules. This constellation of ultrastructural defects has been previously reported for tilB (LRRC6) and CG11253 (ZMYND10) mutant sperm (Moore, 2013; Eberl, 2000). Despite the fundamental differences between how Ch neuron and sperm flagella axonemes are built, similar ODA and IDA defects in both demonstrate CG31320 plays a core role in assembly of the ciliary motility apparatus (Diggle, 2014).

These studies indicated that CG31320 in Drosophila is associated with ciliary motility function and identification of human HEATR2 PCD disease mutations suggested this function is conserved. Co-distribution of CG31320 orthologues with representative components of axonemal dynein across the eukaryotic evolutionary tree supported a specific role in the assembly or stability of structures required for motile cilia function. CG31320/HEATR2 orthologues, as well as components of both ODA and IDA, were found to be absent from all non-ciliated lineages such as yeast but importantly also absent from lineages possessing only non-motile cilia such as nematodes. CG31320/HEATR2 orthologues and components of both IDA and ODA were identified in all lineages that have motile cilia or flagella at some point in their life cycle, with a few conspicuous exceptions. A CG31320/HEATR2 orthologue was found in the marine centric diatom Thalassiosira pseudonana, which has motile sperm whose 9+0 axonemes bear only ODAs. Conversely, a CG31320/HEATR2 orthologue was also identified in the moss species Physcomitrella patens, which has motile sperm with axonemes bearing only IDAs. This supports the above finding that CG31320/HEATR2 is functionally required for the correct assembly of both types of dynein arms. Surprisingly, a conserved CG31320/HEATR2 orthologue was identified in the non-motile green algae Chlorella variabilis, which is assumed to be asexual. However, the existence of several meiosis-specific and flagellar genes in this organism including subunits of outer arm dyneins, has led to the suggestion that some flagellar-derived structure involved in sexual reproduction may have been retained. This evolutionary pattern of co-conservation of CG31320/HEATR2 with the ciliary motility machinery, together with the loss of IDA and ODA observed in CG31320 mutant axonemes, suggests CG31320 is an ancient component of the ciliary/flagellar motility programme required for both IDA and ODA (Diggle, 2014).

CG31320/HEATR2 orthologues are found in species with cilia/flagella that have motile function and retain elements of the axonemal dyneins required for this motility. Species which have no cilia (ie. amoebozoans, flowering plants, yeast) or those which lack motile cilia (i.e., nematodes) have lost HEATR2 orthologues as well as the axonemal dynein genes. Interestingly, unusual species with variant motility programmes still retain HEATR2 orthologues. These include T. pseudonana whose male gametes have motile axonemes without inner arm dyneins, and P. patens, whose male gametes have motile flagella without outer arm dyneins. Similarly, P. falciparum which assembles its flagella intracytosolically through an IFT-independent programme, retains a HEATR2 orthologue. This suggests CG31320/HEATR2 is an essential element of an ancient programme required for ciliary/flagellar motility.

Whether FOX/RFX ciliary motility transcriptional signature used to identify CG31320 was also conserved amongst orthologues was further investigated. Analysis of promoter sequences of CG31320/HEATR2 orthologues in several vertebrate genomes revealed the presence of highly conserved X-boxes situated within 500 bp upstream of the transcriptional start site. In all species examined, very close to the start of Heatr2 transcription, this study identified most often in the same position (-16), one palindromic RFX (X-box) binding site extremely well-matched in both the 5' and 3' half sites. Interestingly, a second sometimes more degenerate X box was also identified in close proximity (15-162 bp away, relative position more variable between species, where the 5' half-site was more degenerate. This second 'relaxed' motif has previously been reported in several RFX targets that are components of the motile machinery in both flies and mammals and suggests the existence of multiple alternative DNA-recognition modes among members of the RFX family of proteins. Although the significance of multiple X-box motifs in regulating target gene expression is unclear, it has been proposed that they may 'fine-tune' the level and spatial expression of targets. Initial analysis using a stringent FOXJ1 consensus site (WDTGTTTGTTTA or KTTTGTTGTTKTW) revealed no upstream sites in close proximity to the transcriptional start in all vertebrate promoters, however using a less rigorous consensus core motif used by the majority of forkhead proteins (RYMAAYA ) and used in Drosophila studies, it was possible to identify conserved motifs in this 500 bp upstream regulatory region. These were similar to motifs for FOXJ1 recently defined by protein-binding microarray. These studies suggest that the co-operative transcriptional control of CG31320/HEATR2 by FOX and RFX factors as part of the ciliary motility programme may also be widely conserved (Diggle, 2014).

To examine the physiological relevance of these putative binding sites in the regulation of Heatr2 expression, an RFX3 ChIP-seq data set was examined from differentiated mouse primary ependymal cell cultures which bear motile, multicilia . ChIP-seq analysis identified a single unique RFX3-peak within 5 kb of the transcriptional start site of Heatr2, a region that contained both the predicted X-boxes. This is similar to peaks for the known RFX3-target gene Dync2li1, with a canonical conserved X-box in close proximity to the transcriptional start site and strongly suggests that the X-boxes are occupied during motile ciliogenesis. ChIP-qPCR validation revealed strongly enriched RFX3 occupancy at the Heatr2 promoter comparable to or greater than validated target Dync2li1 target sequence. Consistent with these data, Heatr2 expression in Rfx3^-/- ependymal cells is decreased ~55% compared to controls, similar to that which is observed for known targets Dyn2li1 and Bbs5. Together, these analyses indicate that mouse Heatr2, as in Drosophila, is directly regulated in part in an RFX-dependent manner (Diggle, 2014).

To further explore developmental transcriptional control of mammalian Heatr2 during formation of motile multiciliated cells (MMCs), RT-qPCR was used during mouse embryonic trachea and lung development. Ciliation in developing mouse airway epithelia occurs in a distinct spatial and temporal manner, from E14.0 when the first few FoxJ1-positive epithelial cells emerge in a proximal-distal sequence. Surface multicilia are subsequently detected from E16.5, becoming more abundant in the airway epithelia with longer cilia as development progresses. RT-qPCR analysis of E14.5 through to P2 mouse trachea and lungs revealed Dnah5, Dnali1 and Zmynd10 to have exponential expression curves during development (similar to FoxJ1), whilst Rfx3 and Heatr2 followed more linear increases in gene expression. Both Rfx3 and Heatr2 were detected prior to FoxJ1 expression, but a highly significant two fold increase in Heatr2 expression was observed during Foxj1-dependent differentiation. The spatial expression pattern of HEATR2, RFX3 and FOXJ1 were refined by immunofluorescence in the developing bronchial epithelium at E15.5, prior to multiciliation. During this proximal-to-distal wave of differentiation, only the larger proximal airways had interspersed cells expressing high levels of FOXJ1 and RFX3, as well as subunits of inner and outer arm dyneins DNALI1 and DNAI2. These same cells expressed high levels of HEATR2. At other stages and sites of MMC differentiation, HEATR2 is expressed in a 'salt-and pepper' pattern, including E18.5 epithelial cells of trachea and bronchus, as well as in ependymal cells lining the lateral ventricles of P5 brains and MMCs lining the adult oviduct ampulla. For comparative analysis of human cilia, asynchronous nasal brush epithelial cells from healthy controls were used, in which it was possible to distinguish both immature cells alongside terminally differentiated and fully ciliated mature cells. This revealed that HEATR2 expression was highest in immature cells in the process of extending multicilia, which were also those expressing higher nuclear RFX3 and FOXJ1 and predominantly cytoplasmic DNALI1. In adjacent fully mature MMCs, when DNALI1 was predominantly axonemal, comparatively lower levels of HEATR2 were observed, as well as lower RFX3 and FOXJ1 expression. Together, these results suggest that while Heatr2 expression in mammals has evolved more complex transcriptional control compared to flies, the conserved FOX/RFX ciliary motility signature that was identified is still used to drive dynamic high level expression at a developmental window when axonemal dynein pre-assembly is occurring in the cytoplasm (Diggle, 2014).

HEATR2 was an interesting candidate for functional characterization as it had not been identified in any of the ciliary axoneme or centrosome proteomic studies. This presumably was due to isolation techniques used in these studies focused on cilial/flagellar axonemes and precluded the identification of cytoplasmic components involved in ciliogenesis. Indeed, this study found that endogenous HEATR2 shows granular localization throughout the cytoplasm in human nasal epithelia, but is never detected in the ciliary axonemes even transiently in immature MMCs when the pool of outer (as represented by DNAH5 and DNAI2) and inner (as represented by DNALI1) arm dynein subunits are predominantly cytoplasmic and moving into the ciliary compartment. This extends recent findings reported by Horani (2012). Moreover, both tagged CG31320 and HEATR2 remained cytoplasmic, without any axonemal localization, in fly Ch neurons and mouse cells respectively. Together, these data define a temporospatial window of highest HEATR2/CG31320 expression, and likely function, during early cytoplasmic dynein pre-assembly (Diggle, 2014).

Given the loss of identifiable IDA and ODA in fly CG31320 and human HEATR2 mutant cilia, it was next asked how HEATR2 was affecting the localization of axonemal components of motile cilia. Similar to previous outer arm defects by DNAI1 immunofluorescence, no expression of outer arm dynein DNAH5 was detected by immunofluorescence in patient HEATR2 mutant cilia. Endogenous HEATR2 complexes were isolated by immunoprecipitation from terminally differentiated control human bronchial epithelial lysates and immunoblotted with antibodies against components of dynein arms (DNAH5, DNALI1, DNAI2), dynein assembly factors (KTU, DNAAF3, ZMYND10) as well as chaperones (HSP70, HSP90). In terminally differentiated cells, it was possible to establish that HEATR2 interacts with DNAI2, a critical component added in the initial step of ODA assembly (Fowkes, 1998), but not other axonemal dynein components, dynein assembly factors or chaperones. These findings are confirmed by previous studies showing that the Chlamydomonas DNAI2 orthologue DIC2/IC78 is near absent in htr2 RNAi mutant axonemal extracts, suggesting this interaction is conserved and of functional significance to the ODA loss phenotype in HEATR2-mutant cilia. Consistent with the absence of IDA from HEATR2 PCD patient and mutant fly axonemes observed by TEM, endogenous DNALI1 was not detected in HEATR2 PCD mutant cilia by immunofluorescence and in Drosophila a tagged orthologue CG6971::mVenus failed to enter axonemes in CG31320 knock-down Ch neurons. The mechanism for HEATR2 in controlling IDA assembly remains unclear as it was not possible to confirm interactions with DNALI1 or other DNAAFs in terminally differentiated MMCs (Diggle, 2014).

These human and fly data support a model in which HEATR2/CG31320 is a cytoplasmic factor, whose dynamic expression is elevated in an RFX- and FOX-dependent manner prior to assembly of motile cilia at the apical surface of MMCs and Ch neurons. Once mature motile cilia are assembled, HEATR2/CG31320 expression appears reduced. This corresponds to a shift from a predominantly cytoplasmic pool of precursor dynein protein subunits to assembled stable dynein complexes successfully docked on the microtubules of the axonemes. These observations suggest HEATR2 interactions are also likely very transient, as HEATR2 remains exclusively cytoplasmic throughout cilia assembly while components of dynein arms become enriched in the axonemal compartment. The interaction with DNAI2 identified in this study in human bronchial epithelial cultures by co-IP as well as the absence of outer dynein arms in HEATR2/CG31320 mutant axonemes and DNAH5 localization in patient cell lines, strongly suggest that HEATR2 is functioning in the earliest steps of outer arm dynein pre-assembly and stability. This is further supported by the reports of loss of IC2 in axonemes of htr2 mutants (Horani, 2012). Whether HEATR2 also functions in apical transport of dynein arm complexes to basal bodies is unclear, as no apical inclusions of dynein arm components were observed in the loss-of-function HEATR2 PCD cells (in this study and Horani, 2012). Moreover, no direct link to IFT is suggested by the fact that Drosophila CG31320 is required for dynein arms in both IFT-dependent (Ch neurons) and IFT-independent (sperm flagella) axonemal assembly (Diggle, 2014).

Although it was not possible to determine how HEATR2 may be regulating inner arm dynein pre-assembly, strong loss of IDA by was shown be TEM and DNALI1 IF in HEATR2 patients and mutant flies. In contrast, algal htr2 silenced strains showed incomplete loss of IDA by TEM and varying stability of different inner arm dynein subunits by axonemal immunoblots (Horani, 2012). This suggests the role for HEATR2 may vary in the preassembly of the seven major species of inner arm dyneins in algae, each associated with an intermediate chain/light chain complex. While the diversity of inner arm dyneins in modified motile cilia of other organisms is as yet poorly characterised, this study showed that the inner arm DNALI1, whose orthologue p28 is a component of several IDA species in algae, is destabilized in PCD patients and in Drosophila. In contrast, Horani reported that inner arm dynein heavy chain DNAH7 (a component of centrin-containing inner arm dynein species b/I3') is correctly localized in their HEATR2 PCD cilia. Consistent with this, htr2 silenced Chlamydomonas strains had increased levels of centrin (component of the light chain complex of subspecies b/I3', e/I2b and g/I3). Interestingly, while PCD patients with mutations in DNAAF2/KTU also show loss of light chain p28/DNALI1 immunostaining, expression levels of p28 were unchanged in the corresponding pf13 algal mutants. Defining the currently under-appreciated molecular complexity of inner arm dyneins in functionally diverse motile cilia may help reconcile differences between PCD studies and clarify the distinct and overlapping roles for individual disease genes within the dynein cytoplasmic assembly network. Interestingly, in CG31320 RNAi Ch axonemes, IDAs appear very cleanly absent in the majority of sections, more so than a recent report for ZMYND10 mutant flies (Moore, 2013). This suggests that despite overlapping gross phenotypes, important molecular subtleties with respect to IDA assembly may exist between these mutants, even within the highly modified cilium of Drosophila Ch neurons, which utilize a simplified ciliary motility machinery (Diggle, 2014).

This work supports a conserved role for HEATR2/CG31320 in the hierarchy of cytoplasmic factors involved in a multistep process of axonemal dynein pre-assembly In consequence, it has been agreed with the HUGO Gene Nomenclature Committee (HGNC) that HEATR2 should be referred to as DNAAF5. This group includes DNAAF1/LRRC50, DNAAF2/KTU, DNAAF3/PF22, DNAAF4/DYX1C1, LRRC6, SPAG1 and ZMYND10. While Chlamydomonas have only one outer arm dynein species comprised of a single complex of three dynein heavy chains (DHCs) with several intermediate and light chains, human studies suggest two types of double-headed HC ODA complexes vary in their composition along motile axonemes. Unlike DNAAF2/KTU, HEATR2 is required for formation of both types of ODA subtypes whereas in DNAAF2 mutant respiratory cilia only distal DNAH5⁺ DNAH9⁺ ODA species are affected. Unlike ZMYND10 and LRRC6, HEATR2 is exclusively cytoplasmic. Via its interaction with DNAI2, it is proposed that HEATR2 acts in the early stages of cytoplasmic dynein preassembly, before complexes are transferred to a loading zone around the basal body where IFT comes into play. Loss of DNAI2 in htr2 silenced algal axonemes and ODA intermediate chain DNAI1 and heavy chain DNAH5 immunofluorescence in PCD patient cells with two different HEATR2 mutations, strongly suggest HEATR2 may play a conserved role in stabilizing formation of the IC1/IC2 complex in the cytoplasm from the pool of precursors. Stability of Chlamydomonas DNAI1 (IC1) and DNAI2 (IC2) are mutually dependent, and necessary for subsequent cytoplasmic pre-assembly of outer arm heavy chains. A similar role in mammalian testes has been proposed for DNAAF2/KTU and the closely related PIH1D3 via interaction with DNAI2. However, this study has been unable to show an interaction between HEATR2 and HSP70 or HSP90 by co-IP in human mature MMCs, suggesting HEATR2 is not functioning in a classical client/chaperone manner. Given no cytoplasmic accumulations of axonemal dyneins are observed in HEATR2 PCD MMCs, it is instead proposed that the tandem arrays of HEAT repeats in HEATR2 are acting as flexible joints or scaffold stabilizing and facilitating interactions between subunits during assembly of dynein complexes (Diggle, 2014).

The dynamic cytoplasmic expression of HEATR2 during the period of motile ciliogenesis suggests that its interactions may be quite transient. In the terminally differentiated human airway cultures used in the co-IP experiments, only a small fraction of cells would be in early ciliogenesis with DNAI2 cytoplasmic rather than axonemal. This would explain why only a portion of DNAI2 is co-immunoprecipitated with HEATR2. Transient interactions may be a trend for dynein assembly factors, such as PIH1D3, which localizes to the cytoplasm of spermatogenic cells but is absent from differentiated spermatids or mature sperm (Diggle, 2014).

Intriguingly, unlike other cytoplasmic assembly PCD proteins which cause PCD when deficient, such as ZMYND10 and LRRC6, this study suggests the control of HEATR2/CG31320 expression has evolved more complexity, possibly due to independent recruitment during evolution of its HEAT repeat-dependent scaffolding functions in other non-cilia cytoplasmic assembly processes. Indeed, CG31320 was the only gene identified in the motility candidate screen in Drosophila whose expression is not confined to Ch neurons and testes, displaying transient, yet apparently vital, mesodermal expression during embryonic development. The role of HEATR2/CG31320 outside motile ciliated cells is currently unknown. Despite its expression in non-ciliated tissues, the clinical phenotypes caused by both human HEATR2 point mutations reported to date only manifest in cells expressing the highest levels of HEATR2, those with motile cilia, suggesting they may be most sensitive to profoundly reduced levels of HEATR2 protein (Diggle, 2014).

Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. This study identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations (Horani, 2012).

Search PubMed for articles about Drosophila HEATR2

Cachero, S., Simpson, T. I., Zur Lage, P. I., Ma, L., Newton, F. G., Holohan, E. E., Armstrong, J. D. and Jarman, A. P. (2011). The gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons. PLoS Biol 9: e1000568. PubMed ID: 21283833

Diggle, C. P., et al. (2014). HEATR2 plays a conserved role in assembly of the ciliary motile apparatus. PLoS Genet 10: e1004577. PubMed ID: 25232951

Eberl, D. F., Hardy, R. W. and Kernan, M. J. (2000). Genetically similar transduction mechanisms for touch and hearing in Drosophila. J Neurosci 20: 5981-5988. PubMed ID: 10934246

Fowkes, M. E. and Mitchell, D. R. (1998). The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits. Mol Biol Cell 9: 2337-2347. PubMed ID: 9725897

Horani, A., Druley, T. E., Zariwala, M. A., Patel, A. C., Levinson, B. T., Van Arendonk, L. G., Thornton, K. C., Giacalone, J. C., Albee, A. J., Wilson, K. S., Turner, E. H., Nickerson, D. A., Shendure, J., Bayly, P. V., Leigh, M. W., Knowles, M. R., Brody, S. L., Dutcher, S. K. and Ferkol, T. W. (2012). Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia. Am J Hum Genet 91: 685-693. PubMed ID: 23040496

Moore, D. J., Onoufriadis, A., Shoemark, A., Simpson, M. A., Zur Lage, P. I., et al. (2013). Mutations in ZMYND10, a gene essential for proper axonemal assembly of inner and outer dynein arms in humans and flies, cause primary ciliary dyskinesia. Am J Hum Genet 93: 346-356. PubMed ID: 23891471

Vandaele, C., Coulon-Bublex, M., Couble, P. and Durand, B. (2001). Drosophila regulatory factor X is an embryonic type I sensory neuron marker also expressed in spermatids and in the brain of Drosophila. Mech Dev 103: 159-162. PubMed ID: 11335126

date revised: 22 May 2023

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