The Role of Sonic Hedgehog in Human Holoprosencephaly and Short-Rib Polydactyly Syndromes.

The Hedgehog (HH) signalling pathway is one of the major pathways controlling cell differentiation and proliferation during human development. This pathway is complex, with HH function influenced by inhibitors, promotors, interactions with other signalling pathways, and non-genetic and cellular factors. Many aspects of this pathway are not yet clarified. The main features of Sonic Hedgehog (SHH) signalling are discussed in relation to its function in human development. The possible role of SHH will be considered using examples of holoprosencephaly and short-rib polydactyly (SRP) syndromes. In these syndromes, there is wide variability in phenotype even with the same genetic mutation, so that other factors must influence the outcome. SHH mutations were the first identified genetic causes of holoprosencephaly, but many other genes and environmental factors can cause malformations in the holoprosencephaly spectrum. Many patients with SRP have genetic defects affecting primary cilia, structures found on most mammalian cells which are thought to be necessary for canonical HH signal transduction. Although SHH signalling is affected in both these genetic conditions, there is little overlap in phenotype. Possible explanations will be canvassed, using data from published human and animal studies. Implications for the understanding of SHH signalling in humans will be discussed.

Usher Syndrome: Genetics of a Human Ciliopathy.

Usher syndrome (USH) is an autosomal recessive syndromic ciliopathy characterized by sensorineural hearing loss, retinitis pigmentosa and, sometimes, vestibular dysfunction. There are three clinical types depending on the severity and age of onset of the symptoms; in addition, ten genes are reported to be causative of USH, and six more related to the disease. These genes encode proteins of a diverse nature, which interact and form a dynamic protein network called the "Usher interactome". In the organ of Corti, the USH proteins are essential for the correct development and maintenance of the structure and cohesion of the stereocilia. In the retina, the USH protein network is principally located in the periciliary region of the photoreceptors, and plays an important role in the maintenance of the periciliary structure and the trafficking of molecules between the inner and the outer segments of photoreceptors. Even though some genes are clearly involved in the syndrome, others are controversial. Moreover, expression of some USH genes has been detected in other tissues, which could explain their involvement in additional mild comorbidities. In this paper, we review the genetics of Usher syndrome and the spectrum of mutations in USH genes. The aim is to identify possible mutation associations with the disease and provide an updated genotype-phenotype correlation.

Exocrine pancreas proteases regulate ß-cell proliferation in zebrafish ciliopathy models and in murine systems.

Pancreatic ß-cells are a critical cell type in the pathology of diabetes. Models of genetic syndromes featuring diabetes can provide novel mechanistic insights into regulation of ß-cells in the context of disease. We previously examined ß-cell mass in models of two ciliopathies, Alström Syndrome (AS) and Bardet-Biedl Syndrome (BBS), which are similar in the presence of metabolic phenotypes, including obesity, but exhibit strikingly different rates of diabetes. Zebrafish models of these disorders show deficient ß-cells with diabetes in AS models and an increased ß-cells absent diabetes in BBS models, indicating ß-cell generation or maintenance that correlates with disease prevalence. Using transcriptome analyses, differential expression of several exocrine pancreas proteases with directionality that was consistent with ß-cell numbers were identified. Based on these lines of evidence, we hypothesized that pancreatic proteases directly impact ß-cells. In the present study, we examined this possibility and found that pancreatic protease genes contribute to proper maintenance of normal ß-cell numbers, proliferation in larval zebrafish, and regulation of AS and BBS ß-cell phenotypes. Our data suggest that these proteins can be taken up directly by cultured ß-cells and ex vivo murine islets, inducing proliferation in both. Endogenous uptake of pancreatic proteases by ß-cells was confirmed in vivo using transgenic zebrafish and in intact murine pancreata. Taken together, these findings support a novel proliferative signaling role for exocrine pancreas proteases through interaction with endocrine ß-cells.

Ciliopathic micrognathia is caused by aberrant skeletal differentiation and remodeling.

Ciliopathies represent a growing class of diseases caused by defects in microtubule-based organelles called primary cilia. Approximately 30% of ciliopathies are characterized by craniofacial phenotypes such as craniosynostosis, cleft lip/palate and micrognathia. Patients with ciliopathic micrognathia experience a particular set of difficulties, including impaired feeding and breathing, and have extremely limited treatment options. To understand the cellular and molecular basis for ciliopathic micrognathia, we used the talpid2 (ta2 ), a bona fide avian model for the human ciliopathy oral-facial-digital syndrome subtype 14. Histological analyses revealed that the onset of ciliopathic micrognathia in ta2 embryos occurred at the earliest stages of mandibular development. Neural crest-derived skeletal progenitor cells were particularly sensitive to a ciliopathic insult, undergoing unchecked passage through the cell cycle and subsequent increased proliferation. Furthermore, whereas neural crest-derived skeletal differentiation was initiated, osteoblast maturation failed to progress to completion. Additional molecular analyses revealed that an imbalance in the ratio of bone deposition and resorption also contributed to ciliopathic micrognathia in ta2 embryos. Thus, our results suggest that ciliopathic micrognathia is a consequence of multiple aberrant cellular processes necessary for skeletal development, and provide potential avenues for future therapeutic treatments.

Variants in RABL2A causing male infertility and ciliopathy.

Approximately 7% of men worldwide suffer from infertility, with sperm abnormalities being the most common defect. Though genetic causes are thought to underlie a substantial fraction of idiopathic cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia and Bardet-Biedl syndrome, also suffer from infertility because cilia and sperm flagella share several characteristics. Here, we identified two deleterious alleles of RABL2A, a gene essential for normal function of cilia and flagella. Our in silico predictions and in vitro assays suggest that both alleles destabilize the protein. We constructed and analyzed mice homozygous for these two single-nucleotide polymorphisms, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), and found that they exhibit ciliopathy-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects and hydrocephalus. Our study provides a paradigm for triaging candidate infertility variants in the population for in vivo functional validation, using computational, in vitro and in vivo approaches.

Divergent roles of the Wnt/PCP Formin Daam1 in renal ciliogenesis.

Kidneys are composed of numerous ciliated epithelial tubules called nephrons. Each nephron functions to reabsorb nutrients and concentrate waste products into urine. Defects in primary cilia are associated with abnormal formation of nephrons and cyst formation in a wide range of kidney disorders. Previous work in Xenopus laevis and zebrafish embryos established that loss of components that make up the Wnt/PCP pathway, Daam1 and ArhGEF19 (wGEF) perturb kidney tubulogenesis. Dishevelled, which activates both the canonical and non-canonical Wnt/PCP pathway, affect cilia formation in multiciliated cells. In this study, we investigated the role of the noncanoncial Wnt/PCP components Daam1 and ArhGEF19 (wGEF) in renal ciliogenesis utilizing polarized mammalian kidney epithelia cells (MDCKII and IMCD3) and Xenopus laevis embryonic kidney. We demonstrate that knockdown of Daam1 and ArhGEF19 in MDCKII and IMCD3 cells leads to loss of cilia, and Daam1's effect on ciliogenesis is mediated by the formin-activity of Daam1. Moreover, Daam1 co-localizes with the ciliary transport protein Ift88 and is present in cilia. Interestingly, knocking down Daam1 in Xenopus kidney does not lead to loss of cilia. These data suggests a new role for Daam1 in the formation of primary cilia.

Pharmacological cholesterol depletion disturbs ciliogenesis and ciliary function in developing zebrafish.

Patients with an inherited inability to synthesize sufficient amounts of cholesterol develop congenital malformations of the skull, toes, kidney and heart. As development of these structures depends on functional cilia we investigated whether cholesterol regulates ciliogenesis through inhibition of hydroxymethylglutaryl-Coenzyme A reductase (HMG-CoA-R), the rate-limiting enzyme in cholesterol synthesis. HMG-CoA-R is efficiently inhibited by statins, a standard medication for hyperlipidemia. When zebrafish embryos are treated with statins cilia dysfunction phenotypes including heart defects, left-right asymmetry defects and malformation of ciliated organs develop, which are ameliorated by cholesterol replenishment. HMG-CoA-R inhibition and other means of cholesterol reduction lowered ciliation frequency and cilia length in zebrafish as well as several mammalian cell types. Cholesterol depletion further triggers an inability for ciliary signalling. Because of a reduction of the transition zone component Pi(4,5)P2 we propose that cholesterol governs crucial steps of cilium extension. Taken together, we report that cholesterol abrogation provokes cilia defects.

Primary Cilia Reconsidered in the Context of Ciliopathies: Extraciliary and Ciliary Functions of Cilia Proteins Converge on a Polarity theme?

Once dismissed as vestigial organelles, primary cilia have garnered the interest of scientists, given their importance in development/signaling, and for their implication in a new disease category known as ciliopathies. However, many, if not all, "cilia" proteins also have locations/functions outside of the primary cilium. These extraciliary functions can complicate the interpretation of a particular ciliopathy phenotype: it may be a result of defects at the cilium and/or at extraciliary locations, and it could be broadly related to a unifying cellular process for these proteins, such as polarity. Assembly of a cilium has many similarities to the development of other polarized structures. This evolutionarily preserved process for the assembly of polarized cell structures offers a perspective on how the cilium may have evolved. We hypothesize that cilia proteins are critical for cell polarity, and that core polarity proteins may have been specialized to form various cellular protrusions, including primary cilia.

The cholangiocyte primary cilium in health and disease.

Cholangiocytes, like most cells, express primary cilia extending from their membranes. These organelles function as antennae which detect stimuli from bile and transmit the information into cells regulating several signaling pathways involved in secretion, proliferation and apoptosis. The ability of primary cilia to detect different signals is provided by ciliary associated proteins which are expressed in its membrane. Defects in the structure and/or function of these organelles lead to cholangiociliopathies that result in cholangiocyte hyperproliferation, altered fluid secretion and absorption. Since primary cilia dysfunction has been observed in several epithelial tumors, including cholangiocarcinoma (CCA), primary cilia have been proposed as tumor suppressor organelles. In addition, the loss of cilia is associated with dysregulation of several molecular pathways resulting in CCA development and progression. Thus, restoration of the primary cilia may be a potential therapeutic approach for several ciliopathies and CCA.

Cilia and sensory signaling: The journey from "animalcules" to human disease.

Nearly all cell types in mammals contain cilia, small rod-like or more elaborate structures that extend from the cell surface. Cilia house signaling proteins that allow the cell to sample their environment and respond appropriately. Mutations in ciliary genes alter the functions of a broad range of cell and tissue types, including sensory and central neurons, and underlie a collection of heterogeneous human disorders called ciliopathies. Here, I highlight the critical contributions of nearly three centuries of research in diverse organisms to our current knowledge of cilia function in sensory signaling and human disease.

Discovery, Diagnosis, and Etiology of Craniofacial Ciliopathies.

Seventy-five percent of congenital disorders present with some form of craniofacial malformation. The frequency and severity of these malformations makes understanding the etiological basis crucial for diagnosis and treatment. A significant link between craniofacial malformations and primary cilia arose several years ago with the determination that ∼30% of ciliopathies could be primarily defined by their craniofacial phenotype. The link between the cilium and the face has proven significant, as several new "craniofacial ciliopathies" have recently been diagnosed. Herein, we reevaluate public disease databases, report several new craniofacial ciliopathies, and propose several "predicted" craniofacial ciliopathies. Furthermore, we discuss why the craniofacial complex is so sensitive to ciliopathic dysfunction, addressing tissue-specific functions of the cilium as well as its role in signal transduction relevant to craniofacial development. As a whole, these analyses suggest a characteristic facial phenotype associated with craniofacial ciliopathies that can perhaps be used for rapid discovery and diagnosis of similar disorders in the future.

The ophthalmic phenotype of IFT140-related ciliopathy ranges from isolated to syndromic congenital retinal dystrophy.

BACKGROUND: Conorenal syndrome is a systemic skeletal ciliopathy characterised by skeletal and renal findings and caused by biallelic mutations in the gene intraflagellar transport 140 Chlamydomonas homologue (IFT140). Most studies have focused on syndromic features and are by non-ophthalmologists. We highlight the ophthalmic phenotype. METHODS: Retrospective consecutive case series (2010-2014). RESULTS: Twelve subjects with confirmed homozygous mutations were identified (11 consanguineous families; 7 boys; assessed at age 10 months to 20 years, average and median age 6.5 and 4 years). All were homozygous for the same IFT140 mutation (c.1990G>A; p.Glu664Lys) except one who was homozygous for c.1541_1542delinsAA. All had poor vision and nystagmus since birth, with visual acuity after 5 years old of hand motions or light perception. In early childhood, nine were noted to stare at lights, four were noted to have a happy demeanour, high hyperopia was typical, and electroretinography was non-recordable. Fundus appearance was grossly normal before the age of 1 year but thereafter appeared dystrophic. Eight children had developmental delay, two had short stubby fingers, and one had renal disease, but four had no evident extraocular disease, including one aged 18 years who also had two older affected siblings in their twenties who remained non-syndromic and were excelling academically. CONCLUSIONS: Recessive IFT140 mutations cause a severe congenital retinal dystrophy with high hyperopia and often early photophilia. Developmental delay is common but not universal and not all patients have obvious extraocular findings. The c.1990G>A mutation represents a founder effect or mutational hotspot on the Arabian Peninsula.