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1.
eNeuro ; 8(5)2021.
Artículo en Inglés | MEDLINE | ID: mdl-34535504

RESUMEN

The hedgehog signaling pathway is best known for its role in developmental patterning of the neural tube and limb bud. More recently, hedgehog signaling has been recognized for its roles in growth of adult tissues and maintenance of progenitor cell niches. However, the role of hedgehog signaling in fully differentiated cells like neurons in the adult brain is less clear. In mammals, coordination of hedgehog pathway activity relies on primary cilia and patients with ciliopathies such as Bardet-Biedl and Alström syndrome exhibit clinical features clearly attributable to errant hedgehog such as polydactyly. However, these ciliopathies also present with features not clearly associated with hedgehog signaling such as hyperphagia-associated obesity. How hedgehog signaling may contribute to feeding behavior is complex and unclear, but cilia are critical for proper energy homeostasis. Here, we provide a detailed analysis of the expression of core components of the hedgehog signaling pathway in the adult mouse hypothalamus with an emphasis on feeding centers. We show that hedgehog pathway genes continue to be expressed in differentiated neurons important for the regulation of feeding behavior. Furthermore, we demonstrate for the first time that pathway activity is regulated at the transcriptional level by fasting. These data suggest that hedgehog signaling is involved in the proper functioning of brain regions that regulate feeding behavior and that hedgehog pathway dysfunction may play a role in the obesity observed in certain ciliopathies.


Asunto(s)
Ayuno , Proteínas Hedgehog , Animales , Cilios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Hipotálamo/metabolismo , Ratones , Transducción de Señal
2.
Genesis ; 59(7-8): e23438, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34124835

RESUMEN

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherry MCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

3.
J Vis Exp ; (171)2021 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-33999029

RESUMEN

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.


Asunto(s)
Inteligencia Artificial , Cilios , Animales , Microtúbulos , Receptores Acoplados a Proteínas G , Transducción de Señal
4.
Dev Dyn ; 2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33728725

RESUMEN

BACKGROUND: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. RESULTS: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. CONCLUSION: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

5.
Hum Mol Genet ; 30(3-4): 234-246, 2021 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-33560420

RESUMEN

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss.


Asunto(s)
Síndrome de Bardet-Biedl/metabolismo , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Mutación , Proteínas de Unión a Fosfato/genética , Hipófisis/anomalías , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Síndrome de Bardet-Biedl/fisiopatología , Proteínas del Citoesqueleto/metabolismo , Masculino , Ratones , Fenotipo , Proteínas de Unión a Fosfato/metabolismo , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo
6.
Curr Biol ; 31(6): 1141-1153.e7, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33400922

RESUMEN

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.

7.
Semin Cell Dev Biol ; 110: 43-50, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32466971

RESUMEN

An emerging number of rare genetic disorders termed ciliopathies are associated with pediatric obesity. It is becoming clear that the mechanisms associated with cilia dysfunction and obesity in these syndromes are complex. In addition to ciliopathic syndromic forms of obesity, several cilia-associated signaling gene mutations also lead to morbid obesity. While cilia have critical and diverse functions in energy homeostasis including their roles in centrally mediated food intake as well as in peripheral tissues, many questions remain. Here, we briefly discuss the syndromic ciliopathies and monoallelic cilia signaling gene mutations associated with obesity. We also describe potential ways cilia may be involved in common obesity. We discuss how neuronal cilia impact food intake potentially through leptin signaling and changes in ciliary G protein-coupled receptor (GPCR) signaling. We highlight several recent studies that have implicated the potential for cilia in peripheral tissues such as adipose and the pancreas to contribute to metabolic dysfunction. Then we discuss the potential for cilia to impact energy homeostasis through their roles in both development and adult tissue homeostasis. The studies discussed in this review highlight how a comprehensive understanding of the requirement of cilia for the regulation of diverse biological functions will contribute to our understanding of common forms of obesity.

8.
Mol Genet Genomic Med ; 9(1): e1566, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33306870

RESUMEN

BACKGROUND: Senior-Loken syndrome is a rare genetic disorder that presents with nephronophthisis and retinal degeneration, leading to end-stage renal disease and progressive blindness. The most frequent cause of juvenile nephronophthisis is a mutation in the nephronophthisis type 1 (NPHP1) gene. NPHP1 encodes the protein nephrocystin-1, which functions at the transition zone (TZ) of primary cilia. METHODS: We report a 9-year-old Senior-Loken syndrome boy with NPHP1 deletion, who presents with bilateral vision decrease and cystic renal disease. Renal function deteriorated to require bilateral nephrectomy and renal transplant. We performed immunohistochemistry, H&E staining, and electron microscopy on the renal sample to determine the subcellular distribution of ciliary proteins in the absence of NPHP1. RESULTS: Immunohistochemistry and electron microscopy of the resected kidney showed disorganized cystic structures with loss of cilia in renal tubules. Phosphoinositides have been recently recognized as critical components of the ciliary membrane and immunostaining of kidney sections for phosphoinositide 5-phosphatase, INPP5E, showed loss of staining compared to healthy control. Ophthalmic examination showed decreased electroretinogram consistent with early retinal degeneration. CONCLUSION: The decreased expression of INPP5E specifically in the primary cilium, coupled with disorganized cilia morphology, suggests a novel role of NPHP1 that it is involved in regulating ciliary phosphoinositide composition in the ciliary membrane of renal tubular cells.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Ciliopatías/genética , Proteínas del Citoesqueleto/genética , Enfermedades Renales Quísticas/genética , Amaurosis Congénita de Leber/genética , Atrofias Ópticas Hereditarias/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Niño , Cilios/metabolismo , Ciliopatías/metabolismo , Ciliopatías/patología , Eliminación de Gen , Humanos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales Quísticas/metabolismo , Enfermedades Renales Quísticas/patología , Amaurosis Congénita de Leber/metabolismo , Amaurosis Congénita de Leber/patología , Masculino , Atrofias Ópticas Hereditarias/metabolismo , Atrofias Ópticas Hereditarias/patología , Monoéster Fosfórico Hidrolasas/genética
9.
Artículo en Inglés | MEDLINE | ID: mdl-33384763

RESUMEN

This study explores whether integrating multicultural content within a genetics laboratory course affected students' awareness of diversity and their perceptions of scientists' identities. Genetics laboratory curricula typically focus on content and experimental procedures, with cursory references to the scientists who made these discoveries. The resulting poor racial and gender representation in the curricula propagate biases about the abilities and contributions of scientists from underrepresented groups, which may adversely affect the retention and success of students in these groups. Initially, students completed a pre-test in which they were asked to recall the names of geneticists and their scientific contributions. Later students created a mock magazine issue featuring a diverse set of experts in genetics, specifically members of traditionally underrepresented gender/sexuality and/or racial/ethnic groups. To facilitate this assignment, students were randomly assigned a geneticist from a pool of active research scientists, spanning a wide range of scientific and cultural backgrounds and identities. Each student wrote a 500-word biography of their assigned geneticist and read biographies composed by peers. Then, in groups, the students categorized biographies based on student-selected unifying themes into a table of contents. On the final exam, the pre-test was repeated as a post-test. In the pre-test, scientists listed by students were 94% male and 6% female, with no members of other underrepresented groups included. In the post-test, scientists listed by students shifted to 84% male and 16% female with 18% from underrepresented groups. These data suggest that this intervention increases awareness of the multicultural nature of scientists.

10.
Front Cell Neurosci ; 13: 266, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31249512

RESUMEN

Primary cilia dysfunction has been associated with hyperphagia and obesity in both ciliopathy patients and mouse models of cilia perturbation. Neurons throughout the brain possess these solitary cellular appendages, including in the feeding centers of the hypothalamus. Several cell biology questions associated with primary neuronal cilia signaling are challenging to address in vivo. Here we utilize primary hypothalamic neuronal cultures to study ciliary signaling in relevant cell types. Importantly, these cultures contain neuronal populations critical for appetite and satiety such as pro-opiomelanocortin (POMC) and agouti related peptide (AgRP) expressing neurons and are thus useful for studying signaling involved in feeding behavior. Correspondingly, these cultured neurons also display electrophysiological activity and respond to both local and peripheral signals that act on the hypothalamus to influence feeding behaviors, such as leptin and melanin concentrating hormone (MCH). Interestingly, we found that cilia mediated hedgehog signaling, generally associated with developmental processes, can influence ciliary GPCR signaling (Mchr1) in terminally differentiated neurons. Specifically, pharmacological activation of the hedgehog-signaling pathway using the smoothened agonist, SAG, attenuated the ability of neurons to respond to ligands (MCH) of ciliary GPCRs. Understanding how the hedgehog pathway influences cilia GPCR signaling in terminally differentiated neurons could reveal the molecular mechanisms associated with clinical features of ciliopathies, such as hyperphagia-associated obesity.

11.
FASEB J ; 33(1): 1440-1455, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30133325

RESUMEN

The transition zone (TZ) is a domain at the base of the cilium that is involved in maintaining ciliary compartment-specific sensory and signaling activity by regulating cilia protein composition. Mutations in TZ proteins result in cilia dysfunction, often causing pleiotropic effects observed in a group of human diseases classified as ciliopathies. The purpose of this study is to describe the importance of the TZ component Meckel-Grüber syndrome 6 ( Mks6) in several organ systems and tissues regarding ciliogenesis and cilia maintenance using congenital and conditional mutant mouse models. Similar to MKS, congenital loss of Mks6 is embryonic lethal, displaying cilia loss and altered cytoskeletal microtubule modifications but only in specific cell types. Conditional Mks6 mutants have a variable cystic kidney phenotype along with severe retinal degeneration with mislocalization of phototransduction cascade proteins. However, other phenotypes, such as anosmia and obesity, which are typically associated with cilia and TZ dysfunction, were not evident. These data indicate that despite Mks6 being a core TZ component, it has tissue- or cell type-specific functions important for cilia formation and cilia sensory and signaling activities. Lewis, W. R., Bales, K. L., Revell, D. Z., Croyle, M. J., Engle, S. E., Song, C. J., Malarkey, E. B., Uytingco, C. R., Shan, D., Antonellis, P. J., Nagy, T. R., Kesterson, R. A., Mrug, M. M., Martens, J. R., Berbari, N. F., Gross, A. K., Yoder, B. K. Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.


Asunto(s)
Cilios/metabolismo , Proteínas del Citoesqueleto/genética , Mutación , Acetilación , Animales , Trastornos de la Motilidad Ciliar/genética , Citoplasma/metabolismo , Encefalocele/genética , Femenino , Genes Letales , Enfermedades Renales Quísticas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Trastornos del Olfato/genética , Fenotipo , Enfermedades Renales Poliquísticas/genética , Degeneración Retiniana/genética , Retinitis Pigmentosa/genética , Tubulina (Proteína)/metabolismo , Aumento de Peso/genética
12.
Genesis ; 56(8): e23217, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29806135

RESUMEN

The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.


Asunto(s)
Hormonas Hipotalámicas/fisiología , Melaninas/fisiología , Hormonas Hipofisarias/fisiología , Receptores de Somatostatina/fisiología , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Integrasas , Melaninas/metabolismo , Ratones , Ratones Transgénicos , Modelos Animales , Neuronas/metabolismo , Neuropéptidos/metabolismo , Hormonas Hipofisarias/metabolismo , Receptores de Somatostatina/metabolismo , Transducción de Señal , Tamoxifeno
13.
Glia ; 66(5): 987-998, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29380422

RESUMEN

Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis.


Asunto(s)
Astrocitos/metabolismo , Diferenciación Celular/fisiología , MicroARNs/metabolismo , Células-Madre Neurales/metabolismo , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Immunoblotting , Hibridación in Situ , Ratones Endogámicos C57BL , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Xenopus laevis
14.
Artículo en Inglés | MEDLINE | ID: mdl-28096262

RESUMEN

The ciliopathies Bardet-Biedl syndrome and Alström syndrome cause obesity. How ciliary dysfunction leads to obesity has remained mysterious, partly because of a lack of understanding of the physiological roles of primary cilia in the organs and pathways involved in the regulation of metabolism and energy homeostasis. Historically, the study of rare monogenetic disorders that present with obesity has informed our molecular understanding of the mechanisms involved in nonsyndromic forms of obesity. Here, we present a framework, based on genetic studies in mice and humans, of the molecular and cellular pathways underlying long-term regulation of energy homeostasis. We focus on recent progress linking these pathways to the function of the primary cilia with a particular emphasis on the roles of neuronal primary cilia in the regulation of satiety.


Asunto(s)
Síndrome de Alstrom/fisiopatología , Síndrome de Bardet-Biedl/fisiopatología , Cilios/fisiología , Obesidad/patología , Síndrome de Alstrom/patología , Animales , Síndrome de Bardet-Biedl/patología , Metabolismo Energético , Humanos , Ratones , Obesidad/genética , Respuesta de Saciedad
15.
Methods Mol Biol ; 1454: 203-14, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27514924

RESUMEN

Neuroscientists have been captivated by cilia ever since these slender, microtubule-based projections on the cell body were found to play critical roles in neuronal specification, maintenance, and function. In mammals, the most common cilia marker, acetylated α-tubulin, is extremely difficult to detect in neuronal cilia. Here, we describe methods to detect neuronal cilia in culture, in fixed sections, and in vivo, taking advantage of transgenic mice carrying fluorescently tagged cilia proteins.


Asunto(s)
Cilios/metabolismo , Imagen Molecular/métodos , Neuronas/metabolismo , Animales , Humanos , Coloración y Etiquetado
16.
PLoS Genet ; 12(7): e1006220, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27472056

RESUMEN

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.


Asunto(s)
Tipificación del Cuerpo/genética , Cilios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animales , Movimiento Celular/genética , Chlamydomonas/genética , Cilios/patología , Proteínas del Citoesqueleto , Citoesqueleto/genética , Modelos Animales de Enfermedad , Extremidades/crecimiento & desarrollo , Extremidades/patología , Predisposición Genética a la Enfermedad , Humanos , Síndrome de Kartagener/patología , Ratones , Microtúbulos/genética , Mutación , Tubo Neural/crecimiento & desarrollo , Tubo Neural/patología , Transducción de Señal/genética
17.
Dev Biol ; 412(2): 208-18, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26945718

RESUMEN

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans.


Asunto(s)
Fertilidad/genética , Proteínas/genética , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismo , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Cabeza del Espermatozoide/ultraestructura , Motilidad Espermática/genética , Cola del Espermatozoide/ultraestructura , Espermátides/crecimiento & desarrollo , Espermátides/metabolismo , Espermátides/ultraestructura , Espermatogénesis/genética , Espermatozoides/crecimiento & desarrollo , Espermatozoides/ultraestructura , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Tetrahymena thermophila/citología , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo
18.
Methods Cell Biol ; 132: 35-54, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26928538

RESUMEN

In the last decade highly conserved cellular appendages called cilia have enjoyed a renewed interest from basic, biomedical scientists, and clinicians alike. This interest has grown upon the elucidation that cilia throughout the body serve as important sensory and signaling centers in both development and adult homeostasis. Furthermore, the identification of several rare genetic disorders associated with cilia dysfunction has broadened the field. However, even though their potential role in human health and disease is now recognized many basic questions about their functions remain. This chapter seeks to explore the trafficking of cilia-specific G protein-coupled receptors (GPCRs) and discusses several model systems in which this has been explored. We open the chapter by briefly discussing cilia and GPCRs then begin discussing some aspects of rhodopsin trafficking, arguably the most well studied of cilia GPCRs. We continue with sections on neuronal cilia and olfactory cilia receptor trafficking. Finally, we conclude with the emerging area of dynamic ciliary GPCR trafficking and speculate about future directions and some of the questions that remain for ciliary GPCRs.


Asunto(s)
Cilios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Síndrome de Bardet-Biedl/metabolismo , Humanos , Neuronas/metabolismo , Transporte de Proteínas
19.
Oncotarget ; 6(19): 17805-16, 2015 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-26164206

RESUMEN

Glioblastomas (GBMs) are deadly tumors of the central nervous system. Most GBM exhibit homozygous deletions of the CDKN2A and CDKN2B tumor suppressors at 9p21.3, although loss of CDKN2A/B alone is insufficient to drive gliomagenesis. MIR31HG, which encodes microRNA-31 (miR-31), is a novel non-coding tumor suppressor positioned adjacent to CDKN2A/B at 9p21.3. We have determined that miR-31 expression is compromised in >72% of all GBM, and for patients, this predicts significantly shortened survival times independent of CDKN2A/B status. We show that miR-31 inhibits NF-κB signaling by targeting TRADD, its upstream activator. Moreover, upon reintroduction, miR-31 significantly reduces tumor burden and lengthens survival times in animal models. As such, our work identifies loss of miR-31 as a novel non-coding tumor-driving event in GBM.


Asunto(s)
Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/genética , MicroARNs/genética , Transducción de Señal/genética , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Glioblastoma/metabolismo , Xenoinjertos , Humanos , Ratones , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo
20.
PLoS One ; 9(9): e106576, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25184295

RESUMEN

It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia.


Asunto(s)
Cilios/metabolismo , Miedo , Aprendizaje por Laberinto , Neurogénesis/genética , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Cilios/genética , Depresión/genética , Depresión/patología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Esquizofrenia/genética , Esquizofrenia/patología , Proteínas Supresoras de Tumor/genética
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