Browsing by Author "Berbari, Nicolas F."

Now showing 1 - 10 of 51
  • Thumbnail Image
    Item
    A 360-kb interchromosomal duplication of the human HYDIN locus
    (Elsevier, 2006) Doggett, Norman A.; Xie, Gary; Meincke, Linda J.; Sutherland, Robert D.; Mundt, Mark O.; Berbari, Nicolas F.; Davy, Brian E.; Robinson, Michael L.; Rudd, M. Katharine; Weber, James L.; Stallings, Raymond L.; Han, Cliff; Biology, School of Science
    The HYDIN gene located in human chromosome band 16q22.2 is a large gene encompassing 423 kb of genomic DNA that has been suggested as a candidate for an autosomal recessive form of congenital hydrocephalus. We have found that the human HYDIN locus has been very recently duplicated, with a nearly identical 360-kb paralogous segment inserted on chromosome 1q21.1. The duplication, among the largest interchromosomal segmental duplications described in humans, is not accounted for in the current human genome assembly and appears to be part of a greater than 550-kb contig that must lie within 1 of the 11 sequence gaps currently remaining in 1q21.1. Both copies of the HYDIN gene are expressed in alternatively spliced transcripts. Elucidation of the role of HYDIN in human disease susceptibility will require careful discrimination among the paralogous copies.
  • Thumbnail Image
    Item
    A Bbs5 mouse model reveals pituitary cilia contributions to developmental abnormalities
    (Cold Spring Harbor Laboratory, 2020-08-19) Bentley, Melissa R.; Engle, Staci E.; Haycraft, Courtney J.; Andersen, Reagan S.; Croyle, Mandy J.; Clearman, Kelsey R.; Rains, Addison B.; Berbari, Nicolas F.; Yoder, Bradley K.; Biology, School of Science
    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 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 congenital null and conditional allele of Bbs5. Bbs5 null mice develop a complex phenotype including craniofacial defects, skeletal shortening, ventriculomegaly, infertility, and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly, and pituitary abnormalities are only found when Bbs5 is mutated prior to P7 indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity independent of the age of Bbs5 loss. Compared to other animal models of BBS, Bbs5 mutant mice exhibit pathologies that suggest a specialized role for Bbs5 in ciliary function.
  • Thumbnail Image
    Item
    A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development
    (Wiley, 2022) Rockwell, Devan M.; O’Connor, Amber K.; Bentley-Ford, Melissa R.; Haycraft, Courtney J.; Croyle, Mandy J.; Brewer, Kathryn M.; Berbari, Nicolas F.; Kesterson, Robert A.; Yoder, Bradley K.; Biology, School of Science
    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.
  • Thumbnail Image
    Item
    Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin
    (Elsevier, 2021-03) McGrath, Jamis; Tung, Chun-Yu; Liao, Xiayi; Belyantseva, Inna A.; Roy, Pallabi; Chakraborty, Oisorjo; Li, Jinan; Berbari, Nicolas F.; Faaborg-Andersen, Christian C.; Barzik, Melanie; Bird, Jonathan E.; Zhao, Bo; Balakrishnan, Lata; Friedman, Thomas B.; Perrin, Benjamin J.; Biology, School of Science
    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.
  • Thumbnail Image
    Item
    An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue
    (BMC, 2013-07-03) O’Connor, Amber K.; Malarkey, Erik B.; Berbari, Nicolas F.; Croyle, Mandy J.; Haycraft, Courtney J.; Bell, P. Darwin; Hohenstein, Peter; Kesterson, Robert A.; Yoder, Bradley K.; Biology, School of Science
    Background: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. Results: To facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. Conclusions: The ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.
  • Thumbnail Image
    Item
    Artificial Intelligence Approaches to Assessing Primary Cilia
    (MyJove Corporation, 2021-05-01) Bansal, Ruchi; Engle, Staci E.; Kamba, Tisianna K.; Brewer, Kathryn M.; Lewis, Wesley R.; Berbari, Nicolas F.; Biology, School of Science
    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.
  • Thumbnail Image
    Item
    Artificial Intelligence Approaches to Assessing Primary Cilia
    (MyJove Corp., 2021-05-01) Bansal, Ruchi; Engle, Staci E.; Kamba, Tisianna K.; Brewer, Kathryn M.; Lewis, Wesley R.; Berbari, Nicolas F.; Biology, School of Science
    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.
  • Thumbnail Image
    Item
    Assessing neuronal ciliary localization of Melanin Concentrating Hormone Receptor 1 in vivo
    (2021-08) Kamba, Tisianna K.; Berbari, Nicolas F.; Mastracci, Teresa; Dai, Guoli
    Obesity is a growing pandemic that claims close to three hundred thousand lives per year in the United States alone. Despite strong interest and investment in potential treatments, obesity remains a complex and challenging disorder. In the study of obesity, mouse models have been excellent tools that help in understanding the function of different genes that contribute to this disease of energy homeostasis. However, it was surprising when disfunction in primary cilia was found to be linked to syndromic obesity. To understand the role of primary cilia in obesity, a growing subset of GPCRs have been identified to selectively localize to the organelle. Several of which have known roles in energy homeostasis. In some examples, ciliary GPCRs appear to dynamically localize to the organelle; such is the case of GPR161 and smoothened in the hedgehog signaling pathway. Thus, we were interested to see if other GPCRs dynamically localize to the primary cilia as part of their regulation of energy homeostasis. For example, the GPCR MCHR1 selectively localizes to the cilia and is involved in energy homeostasis. Although much is known about the expression of the receptor in the brain, how its ciliary subcellular localization impacts its roles in energy homeostasis is unknown. Observing neuronal cilia in vivo remains a difficult task as some of the available tools such as tagged alleles rely on overexpression of ciliary protein which may impact function. Additionally, most of the work is done in vitro, leaving much to be discovered about neuronal cilia in vivo. In this thesis, we show that using a newly constructed reporter allele mCherryMCHR1, we can see ciliary expression of MCHR1 in the brain of developing and adult mice; more specifically in the ARC and PVN. Subsequently, using a novel Artificial intelligence analysis approach, we measured the length and composition of MCHR1 positive cilia under physiological conditions associated with MCHR1 function. Although in this work we are reporting no changes in dynamic localization of MCHR1 in the hypothalamus specifically, we are not excluding the potential for changes in other regions of the brain or under other conditions; and we are suggesting that pharmacological approaches may help highlight potential ciliary GPCR dynamic localization.
  • Thumbnail Image
    Item
    Bardet–Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia
    (National Academy of Science, 2008) Berbari, Nicolas F.; Lewis, Jacqueline S.; Bishop, Georgia A.; Askwith, Candice C.; Mykytyn, Kirk; Biology, School of Science
    Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet–Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity.
  • Thumbnail Image
    Item
    Cilia and Obesity
    (Cold Spring Harbor Laboratory Press, 2017-07-05) Vaisse, Christian; Reiter, Jeremy F.; Berbari, Nicolas F.; Biology, School of Science
    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.