artificial-intelligence-approaches-to-assessing-primary-cilia

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. ...

Indiana University-Purdue University Indianapolis

Indiana University

Indiana University School of Medicine

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. 

Trafficking of ciliary G protein-coupled receptors.

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.

Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

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.

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

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.

Methods for Visualization of Neuronal Cilia.

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.

Cilia and Obesity.

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.

MicroRNA-31 is required for astrocyte specification.

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.

A CreER mouse to study melanin concentrating hormone signaling in the developing brain.

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.

Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.

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 . 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.

Hedgehog Pathway Activation Alters Ciliary Signaling in Primary Hypothalamic Cultures.

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.

Cilia signaling and obesity.

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.

Using a Student-Generated Mock Magazine Issue To Improve Students' Awareness of Diverse Scientists.

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.

Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin.

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 preweaning 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 P7 indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity independent of the age of Bbs5 loss.

A mouse model of BBS identifies developmental and homeostatic effects of BBS5 mutation and identifies novel pituitary abnormalities.

Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited.

Article	Year
Trafficking of ciliary G protein-coupled receptors. Methods in cell biology\| PubMed ID: 26928538	2016
Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse. Developmental biology\| PubMed ID: 26945718	2016
Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease. PLoS genetics\| PubMed ID: 27472056	2016
Methods for Visualization of Neuronal Cilia. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27514924	2016
Cilia and Obesity. Cold Spring Harbor perspectives in biology\| PubMed ID: 28096262	2017
MicroRNA-31 is required for astrocyte specification. Glia\| PubMed ID: 29380422	2018
A CreER mouse to study melanin concentrating hormone signaling in the developing brain. Genesis (New York, N.Y. : 2000)\| PubMed ID: 29806135	2018
Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 30133325	2019
Hedgehog Pathway Activation Alters Ciliary Signaling in Primary Hypothalamic Cultures. Frontiers in cellular neuroscience\| PubMed ID: 31249512	2019
Cilia signaling and obesity. Seminars in cell & developmental biology\| PubMed ID: 32466971	2021
Using a Student-Generated Mock Magazine Issue To Improve Students' Awareness of Diverse Scientists. Journal of microbiology & biology education\| PubMed ID: 33384763	2020
Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin. Current biology : CB\| PubMed ID: 33400922	2021
A mouse model of BBS identifies developmental and homeostatic effects of BBS5 mutation and identifies novel pituitary abnormalities. Human molecular genetics\| PubMed ID: 33560420	2021
A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 33728725	2021

Nicolas F. Berbari

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