Publications by authors named "Cesar P Canales"

11 Publications

  • Page 1 of 1

CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis.

Proc Natl Acad Sci U S A 2020 09 1;117(37):23073-23084. Epub 2020 Sep 1.

Department of Cell Biology and Human Anatomy, University of California, Davis, CA 95616;

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.
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http://dx.doi.org/10.1073/pnas.2002749117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502717PMC
September 2020

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations.

J Vis Exp 2016 08 4(114). Epub 2016 Aug 4.

The School of Medical Sciences, University of New South Wales Australia;

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.
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http://dx.doi.org/10.3791/54401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091712PMC
August 2016

RNA-Seq analysis of Gtf2ird1 knockout epidermal tissue provides potential insights into molecular mechanisms underpinning Williams-Beuren syndrome.

BMC Genomics 2016 06 13;17:450. Epub 2016 Jun 13.

Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia.

Background: Williams-Beuren Syndrome (WBS) is a genetic disorder associated with multisystemic abnormalities, including craniofacial dysmorphology and cognitive defects. It is caused by a hemizygous microdeletion involving up to 28 genes in chromosome 7q11.23. Genotype/phenotype analysis of atypical microdeletions implicates two evolutionary-related transcription factors, GTF2I and GTF2IRD1, as prime candidates for the cause of the facial dysmorphology.

Results: Using a targeted Gtf2ird1 knockout mouse, we employed massively-parallel sequencing of mRNA (RNA-Seq) to understand changes in the transcriptional landscape associated with inactivation of Gtf2ird1 in lip tissue. We found widespread dysregulation of genes including differential expression of 78 transcription factors or coactivators, several involved in organ development including Hey1, Myf6, Myog, Dlx2, Gli1, Gli2, Lhx2, Pou3f3, Sox2, Foxp3. We also found that the absence of GTF2IRD1 is associated with increased expression of genes involved in cellular proliferation, including growth factors consistent with the observed phenotype of extreme thickening of the epidermis. At the same time, there was a decrease in the expression of genes involved in other signalling mechanisms, including the Wnt pathway, indicating dysregulation in the complex networks necessary for epidermal differentiation and facial skin patterning. Several of the differentially expressed genes have known roles in both tissue development and neurological function, such as the transcription factor Lhx2 which regulates several genes involved in both skin and brain development.

Conclusions: Gtf2ird1 inactivation results in widespread gene dysregulation, some of which may be due to the secondary consequences of gene regulatory network disruptions involving several transcription factors and signalling molecules. Genes involved in growth factor signalling and cell cycle progression were identified as particularly important for explaining the skin dysmorphology observed in this mouse model. We have noted that a number of the dysregulated genes have known roles in brain development as well as epidermal differentiation and maintenance. Therefore, this study provides clues as to the underlying mechanisms that may be involved in the broader profile of WBS.
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http://dx.doi.org/10.1186/s12864-016-2801-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907016PMC
June 2016

The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation.

Hum Genet 2015 Oct 15;134(10):1099-115. Epub 2015 Aug 15.

Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, 2052, Australia.

GTF2IRD1 is one of the three members of the GTF2I gene family, clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities, mental retardation, visuospatial deficits and hypersociability of WBS. However, the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here, for the first time, we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner, yeast two-hybrid libraries were screened, isolating 38 novel interaction partners, which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function, as the isolated partners are mostly involved in chromatin modification and transcriptional regulation, whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain, behaviour and human disease.
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http://dx.doi.org/10.1007/s00439-015-1591-0DOI Listing
October 2015

The role of GTF2IRD1 in the auditory pathology of Williams-Beuren Syndrome.

Eur J Hum Genet 2015 Jun 24;23(6):774-80. Epub 2014 Sep 24.

Cellular and Genetic Medicine Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia.

Williams-Beuren Syndrome (WBS) is a rare genetic condition caused by a hemizygous deletion involving up to 28 genes within chromosome 7q11.23. Among the spectrum of physical and neurological defects in WBS, it is common to find a distinctive response to sound stimuli that includes extreme adverse reactions to loud, or sudden sounds and a fascination with certain sounds that may manifest as strengths in musical ability. However, hearing tests indicate that sensorineural hearing loss (SNHL) is frequently found in WBS patients. The functional and genetic basis of this unusual auditory phenotype is currently unknown. Here, we investigated the potential involvement of GTF2IRD1, a transcription factor encoded by a gene located within the WBS deletion that has been implicated as a contributor to the WBS assorted neurocognitive profile and craniofacial abnormalities. Using Gtf2ird1 knockout mice, we have analysed the expression of the gene in the inner ear and examined hearing capacity by evaluating the auditory brainstem response (ABR) and the distortion product of otoacoustic emissions (DPOAE). Our results show that Gtf2ird1 is expressed in a number of cell types within the cochlea, and Gtf2ird1 null mice showed higher auditory thresholds (hypoacusis) in both ABR and DPOAE hearing assessments. These data indicate that the principal hearing deficit in the mice can be traced to impairments in the amplification process mediated by the outer hair cells and suggests that similar mechanisms may underpin the SNHL experienced by WBS patients.
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http://dx.doi.org/10.1038/ejhg.2014.188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795059PMC
June 2015

RAI1 transcription factor activity is impaired in mutants associated with Smith-Magenis Syndrome.

PLoS One 2012 18;7(9):e45155. Epub 2012 Sep 18.

John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, Florida, USA.

Smith-Magenis Syndrome (SMS) is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1), located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1) and no ability to activate the transcription through an endogenous target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045155PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445574PMC
February 2013

Definition of a critical genetic interval related to kidney abnormalities in the Potocki-Lupski syndrome.

Am J Med Genet A 2012 Jul 25;158A(7):1579-88. Epub 2012 May 25.

Division of Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada.

Potocki-Lupski syndrome is a genomic disorder caused by duplication of 17p11.2. It is characterized by failure to thrive, intellectual disability, hypotonia, and behavioral difficulties. Structural renal anomalies have been observed in <10% of affected individuals. We present detailed clinical and molecular data on six patients with Potocki-Lupski syndrome, two of whom had renal abnormalities, and investigate the prevalence of kidney abnormalities in the mouse model for the syndrome. In contrast to affected humans, the mouse model does not demonstrate a renal phenotype. Comparison of the duplicated segment in patients with Potocki-Lupski syndrome and the renal phenotype and the syntenic duplicated region in the mouse model allowed us to suggest a 0.285 Mb critical region, including the FLCN gene that may be important for development of renal abnormalities in patients with this duplication.
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http://dx.doi.org/10.1002/ajmg.a.35399DOI Listing
July 2012

Copy number variation and susceptibility to complex traits.

EMBO Mol Med 2011 Jan 23;3(1):1-4. Epub 2010 Dec 23.

John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL, USA.

Copy number variations (CNV) within the genome are extremely abundant. In this closeup, Canales and Walz discuss how CNV are associated with normal variation, genomic disorders, genome evolution, adaptive traits and how the use of a novel screen described by Ermakova et al in this issue that is designed to identify human diseaserelevant phenotypes associated with CNV in the mouse can help elucidating susceptibility or predisposition to diseases loci.
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http://dx.doi.org/10.1002/emmm.201000111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401997PMC
January 2011

Podocyte-specific overexpression of wild type or mutant trpc6 in mice is sufficient to cause glomerular disease.

PLoS One 2010 Sep 20;5(9):e12859. Epub 2010 Sep 20.

Centro de Estudios Científicos (CECS), Valdivia, Chile.

Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2-3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012859PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2942896PMC
September 2010

Functional and cellular characterization of human Retinoic Acid Induced 1 (RAI1) mutations associated with Smith-Magenis Syndrome.

BMC Mol Biol 2010 Aug 25;11:63. Epub 2010 Aug 25.

John P Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, Florida, USA.

Background: Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1.

Results: We generated the full-length cDNA of the wild type protein and five mutated forms: RAI1-HA 2687delC, RAI1-HA 3103delC, RAI1 R960X, RAI1-HA Q1562R, and RAI1-HA S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1. Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity.

Conclusion: Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains.
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http://dx.doi.org/10.1186/1471-2199-11-63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939504PMC
August 2010

Abnormal social behaviors and altered gene expression rates in a mouse model for Potocki-Lupski syndrome.

Hum Mol Genet 2008 Aug 10;17(16):2486-95. Epub 2008 May 10.

Centro de Estudios Científicos, CECS, Valdivia, Chile.

The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here, we investigated the social behavior and gene expression pattern of this mouse model in a pure C57BL/6-Tyr(c-Brd) genetic background. Dp(11)17/+ male mice displayed normal home-cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target versus an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes, we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model, we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mice.
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http://dx.doi.org/10.1093/hmg/ddn148DOI Listing
August 2008