Interplay of RFX transcription factors 1, 2 and 3 in motile ciliogenesis.

Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo.

Altered GLI3 and FGF8 signaling underlies acrocallosal syndrome phenotypes in Kif7 depleted mice.

Acrocallosal syndrome (ACLS) is a rare genetic disorder characterized by agenesis or hypoplasia of corpus callosum (CC), polydactyly, craniofacial dysmorphism and severe intellectual deficiency. We previously identified KIF7, a key ciliary component of the Sonic hedgehog (SHH) pathway, as being a causative gene for this syndrome, thus including ACLS in the group of ciliopathies. In both humans and mice, KIF7 depletion leads to abnormal GLI3 processing and over-activation of SHH target genes. To understand the pathological mechanisms involved in CC defects in this syndrome, we took advantage of a previously described Kif7-/- mouse model to demonstrate that in addition to polydactyly and neural tube closure defects, these mice present CC agenesis with characteristic Probst bundles, thus recapitulating major ACLS features. We show that CC agenesis in these mice is associated with specific patterning defects of the cortical septum boundary leading to altered distribution of guidepost cells required to guide the callosal axons through the midline. Furthermore, by crossing Kif7-/- mice with Gli3Δ699 mice exclusively producing the repressive isoform of GLI3 (GLI3R), we demonstrate that decreased GLI3R signaling is fully responsible for the ACLS features in these mice, as all phenotypes are rescued by increasing GLI3R activity. Moreover, we show that increased FGF8 signaling is responsible in part for CC defects associated to KIF7 depletion, as modulating FGF8 signaling rescued CC formation anteriorly in Kif7-/- mice. Taken together our data demonstrate that ACLS features rely on defective GLI3R and FGF8 signaling.

Genetic specification of left-right asymmetry in the diaphragm muscles and their motor innervation.

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

RFX2 Is a Major Transcriptional Regulator of Spermiogenesis.

Spermatogenesis consists broadly of three phases: proliferation of diploid germ cells, meiosis, and finally extensive differentiation of the haploid cells into effective delivery vehicles for the paternal genome. Despite detailed characterization of many haploid developmental steps leading to sperm, only fragmentary information exists on the control of gene expression underlying these processes. Here we report that the RFX2 transcription factor is a master regulator of genes required for the haploid phase. A targeted mutation of Rfx2 was created in mice. Rfx2-/- mice are perfectly viable but show complete male sterility. Spermatogenesis appears to progress unperturbed through meiosis. However, haploid cells undergo a complete arrest in spermatid development just prior to spermatid elongation. Arrested cells show altered Golgi apparatus organization, leading to a deficit in the generation of a spreading acrosomal cap from proacrosomal vesicles. Arrested cells ultimately merge to form giant multinucleated cells released to the epididymis. Spermatids also completely fail to form the flagellar axoneme. RNA-Seq analysis and ChIP-Seq analysis identified 139 genes directly controlled by RFX2 during spermiogenesis. Gene ontology analysis revealed that genes required for cilium function are specifically enriched in down- and upregulated genes showing that RFX2 allows precise temporal expression of ciliary genes. Several genes required for cell adhesion and cytoskeleton remodeling are also downregulated. Comparison of RFX2-regulated genes with those controlled by other major transcriptional regulators of spermiogenesis showed that each controls independent gene sets. Altogether, these observations show that RFX2 plays a major and specific function in spermiogenesis.

A novel function of Huntingtin in the cilium and retinal ciliopathy in Huntington's disease mice.

Huntington's disease (HD) is a neurodegenerative disorder caused by the toxic expansion of polyglutamine in the Huntingtin (HTT) protein. The pathomechanism is complex and not fully understood. Increasing evidence indicates that the loss of normal protein function also contributes to the pathogenesis, pointing out the importance of understanding the physiological roles of HTT. We provide evidence for a novel function of HTT in the cilium. HTT localizes in diverse types of cilia--including 9 + 0 non-motile sensory cilia of neurons and 9 + 2 motile multicilia of trachea and ependymal cells--which exert various functions during tissue development and homeostasis. In the photoreceptor cilium, HTT is present in all subciliary compartments from the base of the cilium and adjacent centriole to the tip of the axoneme. In HD mice, photoreceptor cilia are abnormally elongated, have hyperacetylated alpha-tubulin and show mislocalization of the intraflagellar transport proteins IFT57 and IFT88. As a consequence, intraflagellar transport function is perturbed and leads to aberrant accumulation of outer segment proteins in the photoreceptor cell bodies and disruption of outer segment integrity, all of which precede overt cell death. Strikingly, endogenous mouse HTT is strongly reduced in cilia and accumulates in photoreceptor cell bodies, suggesting that HTT loss function contributes to structural and functional defects of photoreceptor cilia in HD mouse. Our results indicate that cilia pathology participates in HD physiopathology and may represent a therapeutic target.

The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating the patterning of prethalamus and ventral telencephalon.

Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3(-/-) corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3(-/-) VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development.

Novel VCAN mutations and evidence for unbalanced alternative splicing in the pathogenesis of Wagner syndrome.

Wagner syndrome (WS) is an autosomal dominant vitreoretinopathy affecting various ocular features and is caused by mutations in the canonical splice sites of the VCAN gene, which encodes the large chondroitin sulfate proteoglycan, versican. We report the identification of novel splice acceptor and donor-site mutations (c.4004-1G>C and c.9265+2T>A) in two large WS families from France and the United Kingdom. To characterize their pathogenic mechanisms we performed qRT-PCR experiments on RNA from patient-derived tissues (venous blood and skin fibroblasts). We also analyzed RNA from the original Swiss family reported by Wagner (who has the previously reported c.9265+1G>A mutation). All three mutations resulted in a quantitative increase of transcript variants lacking exons 7 and/or 8. However, the magnitude of the increase varied between tissues and mutations. We discuss altered balance of VCAN splice variants in combination with reduction in glycosaminoglycan protein modifications as possible pathogenic mechanisms.

The ciliogenic transcription factor RFX3 regulates early midline distribution of guidepost neurons required for corpus callosum development.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

Transcriptional control of genes involved in ciliogenesis: a first step in making cilia.

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.

RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies.

Cilia are cellular organelles that play essential physiological and developmental functions in various organisms. They can be classified into two categories, primary cilia and motile cilia, on the basis of their axonemal architecture. Regulatory factor X (RFX) transcription factors have been shown to be involved in the assembly of primary cilia in Caenorhabditis elegans, Drosophila and mice. Here, we have taken advantage of a novel primary-cell culture system derived from mouse brain to show that RFX3 is also necessary for biogenesis of motile cilia. We found that the growth and beating efficiencies of motile cilia are impaired in multiciliated Rfx3(-/-) cells. RFX3 was required for optimal expression of the FOXJ1 transcription factor, a key player in the differentiation program of motile cilia. Furthermore, we demonstrate for the first time that RFX3 regulates the expression of axonemal dyneins involved in ciliary motility by binding directly to the promoters of their genes. In conclusion, RFX proteins not only regulate genes involved in ciliary assembly, but also genes that are involved in ciliary motility and that are associated with ciliopathies such as primary ciliary dyskinesia in humans.

New phenotype associated with an Arg116Cys mutation in the CRYAA gene: nuclear cataract, iris coloboma, and microphthalmia.

OBJECTIVE: To describe a new phenotype with an arginine-to-cysteine mutation at position 116 (Arg116Cys) in the CRYAA gene. METHODS: We investigated a 4-generation French family with autosomal dominant cataract and performed a genetic linkage analysis using microsatellite DNA markers encompassing 15 known cataract loci. Exons 1, 2, and 3 and flanking intronic sequences of the CRYAA gene were amplified and analyzed using direct sequencing. RESULTS: All of the affected individuals had nuclear cataract and iris coloboma. Genetic analysis revealed the previously described Arg116Cys mutation in the CRYAA gene in the heterozygous state in all of the affected members of the family but not in unaffected individuals. CONCLUSION: To our knowledge, this is the first case to date in which an Arg116Cys mutation in the CRYAA gene was associated with nuclear cataract and iris coloboma. CLINICAL RELEVANCE: This study indicates that an Arg116Cys mutation in the CRYAA gene could be associated with an unusual phenotype in affected individuals. In this family, the clinical observation of iris coloboma allows for the possibility of identifying individuals carrying the mutation. Iris coloboma is particularly important in terms of perinatal diagnosis because its detection in the newborn requires a careful and regular examination of the lens.

Physical and transcript map of the autosomal dominant colobomatous microphthalmia locus on chromosome 15q12-q15 and refinement to a 4.4 Mb region.

Congenital microphthalmia is a developmental disorder characterized by shortened axial length of the eye. We have previously mapped the gene responsible for autosomal dominant colobomatous microphthalmia in a 5-generation family to chromosome 15q12-q15. Here, we set up a physical and transcript map of the 13.8 cM critical region, flanked by loci D15S1002 and D15S1040. Physical mapping and genetic linkage analysis using 20 novel polymorphic markers allowed the refinement of the disease locus to two intervals in close vicinity, namely a centromeric interval, bounded by microsatellite DNA markers m3-m17, and a telomeric interval, m76-m24, encompassing respectively 1.9 and 2.5 Mb. Moreover, we excluded three candidate genes, CKTSF1B1, KLF13 and CX36. Finally, although a phenomenon of anticipation was suggested by phenotypic and pedigree data, no abnormal expansion of three trinucleotide repeats mapping to the refine interval was found in affected individuals.

Localization of a non-syndromic X-linked mental retardation gene (MRX80) to Xq22-q24.

Isolated mental retardation is clinically and genetically heterogenous and may be inherited in an autosomal dominant, autosomal recessive, or X-linked manner. We report here a linkage analysis in a large family including 15 members, 6 of whom presenting X-linked non-syndromic mental retardation (MRX). Two-point linkage analysis using 23 polymorphic markers covering the entire X chromosome demonstrated significant linkage between the causative gene and DXS8055 with a maximum LOD score of 2.98 at theta = 0.00. Haplotype analysis indicated location for the disease gene in a 23.1 cM interval between DXS1106 and DXS8067. This MRX localization overlaps with 7 XLMR loci (MRX23, MRX27, MRX30, MRX35, MRX47, MRX53, and MRX63). This interval contains two genes associated with non-syndromic mental retardation (NSMR), namely the PAK3 gene, encoding a p21-activated kinase (MRX30 and MRX47) and the FACL4 gene encoding a fatty acyl-CoA ligase (MRX63). As skewed X-inactivation, an apparently constant feature in FACL4 carrier females was not observed in an obligate carrier belonging to the MRX family presented here, the PAK3 gene should be considered as the strongest candidate for this MRX locus.