Resting cells rely on the DNA helicase component MCM2 to build cilia.

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.

ATR promotes cilia signalling: links to developmental impacts.

Mutations in ATR(ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development.

Compound heterozygous GATA5 mutations in a girl with hydrops fetalis, congenital heart defects and genital anomalies.

GATA5 belongs to the GATA family of transcription factors characterized by highly evolutionarily conserved zinc-finger DNA-binding domains. Mouse models have implicated a role of GATA5 during mammalian embryogenesis, including proper heart development and gender-specific regulation of female genitourinary tract formation. Previous studies have found an association of heterozygous missense alterations in GATA5 with a broad variety of heart diseases; however, the clinical relevance of the identified susceptibility variants has remained unclear. Here, we report on a girl with hydrops fetalis, congenital heart defects, clitoromegaly and postnatally increased 17-hydroxyprogesterone levels. By trio whole-exome sequencing, we identified compound heterozygous missense mutations, p.Ser19Trp and p.Arg202Gln, in GATA5 as putative disease-causing alterations. The identified mutations fail to rescue the cardia bifida phenotype in a zebrafish model, mislocalize to subnuclear foci when transiently transfected in HEK293 cells and possess less transcriptional activity. In addition to demonstrating the pathogenicity of identified mutations, our findings show that GATA5 mutations, in addition to heart diseases, can result in congenital abnormalities of the female genitourinary tract in humans.

Left-right asymmetry in the light of TOR: An update on what we know so far.

The internal left-right (LR) asymmetry is a characteristic that exists throughout the animal kingdom from roundworms over flies and fish to mammals. Cilia, which are antenna-like structures protruding into the extracellular space, are involved in establishing LR asymmetry during early development. Humans who suffer from dysfunctional cilia often develop conditions such as heterotaxy, where internal organs appear to be placed randomly. As a consequence to this failure in asymmetry development, serious complications such as congenital heart defects (CHD) occur. The mammalian (or mechanistic) target of rapamycin (mTOR) pathway has recently emerged as an important regulator regarding symmetry breaking. The mTOR pathway governs fundamental processes such as protein translation or metabolism. Its activity can be transduced by two complexes, which are called TORC1 and TORC2, respectively. So far, only TORC1 has been implicated with asymmetry development and appears to require very precise regulation. A number of recent papers provided evidence that dysregulated TORC1 results in alterations of motile cilia and asymmetry defects. In here, we give an update on what we know so far of mTORC1 in LR asymmetry development.

Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of Origin recognition complex factors.

Meier-Gorlin syndrome (MGS) is a rare, congenital primordial microcephalic dwarfism disorder. MGS is caused by genetic variants of components of the origin recognition complex (ORC) consisting of ORC1-6 and the pre-replication complex, which together enable origin firing and hence genome replication. In addition, ORC1 has previously been shown to play a role in ciliogenesis. Here, we extend this work and investigate the function of ORC1 and two other members of the complex on cilia at an organismal level. Knockdown experiments in zebrafish confirmed the impact of ORC1 on cilia. ORC1-deficiency confers defects anticipated to arise from impaired cilia function such as formation of oedema, kidney cysts, curved bodies and left-right asymmetry defects. We found ORC1 furthermore required for cilium formation in zebrafish and demonstrate that ciliopathy phenotypes in ORC1-depleted zebrafish could not be rescued by reconstitution with ORC1 bearing a genetic variant previously identified in MGS patients. Loss-of-function of Orc4 and Orc6, respectively, conferred similar ciliopathy phenotypes and cilium shortening in zebrafish, suggesting that several, if not all, components of the ORC regulate ciliogenesis downstream to or in addition to their canonical function in replication initiation. This study presents the first in vivo evidence of an influence of the MGS genes of the ORC family on cilia, and consolidates the possibility that cilia dysfunction could contribute to the clinical manifestation of ORC-deficient MGS.

The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5.

G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5.