Invivo and Systematic Analysis of Random Multigenic Deletions
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bioRxiv preprint doi: https://doi.org/10.1101/2021.01.20.427453; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Invivo and systematic analysis of random multigenic deletions associated with 2 human diseases during epithelial morphogenesis using Drosophila 3 4 Usha Nagarajan*1, Shanmugasundaram Pakkiriswami*2 ,Sandiya Srinivasan3^, Niveditha 5 Ramkumar3^, Rajeswari Rajaraman3^, Swaminathan Sethuraman1, Kumarasamy Thangaraj *3 6 7 1Central University of Haryana, Department of Biochemistry, Mahendragarh, Haryana, 123029 8 India 9 2Department of Biochemistry and Molecular Biology, Dalhousie University, Dalhousie Medicine 10 New Brunswick, Saint John, NB, E2L4L5,Canada, 11 3 School of Chemical and Biotechnology (SCBT), SASTRA University, Thanjavur, 613401, India. 12 4Centre for Cellular and Molecular Biology (CCMB), Hyderabad, 500 007, India. 13 14 Correspondence to: [email protected] [email protected], [email protected] 15 16 Abstract 17 Random loss of multigenic loci on chromosomes, a crucial drive for evolution, occurs 18 frequently in all living organisms. Analysis of such chromosomal disruption and 19 understanding the consequences of their impact on the growth and development of 20 multicellular organisms is challenging. In this report, we have addressed this issue 21 using invivo mosaic analysis of deficiency lines in Drosophila. Genes on fly 22 deficiency lines were compared with human orthologs for their implications in 23 disease development during cytoskeletal processes and epithelial morphogenesis. 24 The cytoskeletal phenotypes from the fly has been utilized to predict the function of 25 human orthologs. In addition, as these Drosophila deficiency lines are equivalent to 26 human microdeletions, based on the clonal behaviour and phenotypes generated, a 27 systematic analysis has been carried out to establish the critical loci that correspond 28 to Microdeletion Syndromes and Mendelian Disorders in humans. Further we have 29 drawn the synteny that exists between these chromosomes and have identified 30 critical region corresponding to defects. A few potential candidates that might have 31 an implication in epithelial morphogenesis are also identified. 32 33 34 35 36 Introduction 37 Contiguous-gene deletion syndromes or multigenic deletion syndromes display 38 extremely variable clinical symptoms depending on the genes lost in the loci (Rauch 39 et al., 2001; Slavotinek, 2012). A great deal of evidence shows that random loss of 40 genes or spontaneous changes on chromosomes (like virus-mediated mutagenesis 41 in humans causing chromatid breakages, translocations, duplications, 42 deficiencies/deletions leads to developmental anomalies and growth defects 43 (Fortunato and Spector, 2003). “Less-is-more” hypothesis insists that gene loss is an 44 engine for evolutionary change and disease development (Olson, 1999; Olson and 45 Varki, 2003). Despite the fact that the adaptive role of gene loss has formed the 46 basis for evolution, impact of their adaptive biological functions on individual integrity 47 and stability remains elusive. Just as how loss and gain of Homeotic genes and their 48 clusters prove fundamental for the evolution of body shape and size (Ruddle et al., bioRxiv preprint doi: https://doi.org/10.1101/2021.01.20.427453; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 1994), the loss and gain of genes implicated in epithelial morphogenesis also 50 influence the integrity of body size and shape (Schock and Perrimon, 2002). In a 51 multicellular organism, epithelial morphogenesis is an inevitable process in which 52 epithelia, tissues that line the organ and body surfaces, determine organ-to-body 53 shape and size (Gumbiner BM.1992; Schock and Perrimon, 2002). Of all the 54 factors implicated in this process, cytoskeletal components that balance rigidity and 55 flexibility of the cell, play a critical role in maintaining organ size and growth of the 56 organism (Knust, 2005; Gibson and Perrimon, 2005; Shen and Dahmann, 2005; 57 Donohoe et al., 2018). Defects during this process lead to loss of tissue integrity; 58 development of genetic disorders and diseases (Olson and Varki, 2003; Knust, 2005 59 in Birchmeier and Birchmeier, 2005). 60 61 Compared to loss of individual essential genes and frameshift mutations, gains and 62 losses of multiple (more than one) genes bring about greater dramatic genetic 63 repercussions. Random loss of contiguous (loss of genes in the same order) genes 64 would disrupt the normal development, affecting growth and patterning of the tissue, 65 which otherwise occur concurrently. Loss of such contiguous genes that may occur 66 due to any kind of genetic aberrations might regulate both cell-autonomous and non- 67 autonomous functions of the tissue giving rise to heterogeneity in cell population 68 (mosaicism). It is intriguing to understand the influence of such sudden, random loss 69 of genes causing mosaicism and to compare their effect with other organisms. 70 71 Genomic studies suggest that genetic mosaicism is a very common phenomenon 72 during epithelial morphogenesis. Although such lesions to the genome are random 73 and generally asymptomatic in initial stages, affected individuals subsequently 74 develop varying levels of metabolic disorders and disabilities (Chial, 2008). Also, 75 since such incidences occur sporadically, chances of undertaking systematic 76 analysis is very low or may not be feasible. In the past, rigorous systematic analysis 77 of genes (Reiter et al., 2001) and genetic screening (St Johnson, 2002; Yamamoto 78 et al., 2014) have been undertaken to understand genes implicated in disease 79 development and their functions. However, how these individuals subjected to such 80 sudden and random loss of genes manage without their gene function and what 81 impact does it have on the individual is not clear. In multigenic deletions, the 82 synergistic and antagonistic effect of genes in maintaining homeostasis or disease 83 development and the alternate pathways that operate to compensate the loss is not 84 known yet. Although it is intriguing to understand the consequence of random loss of 85 multiple genes that leads to defects and genetic disorders, it is extremely 86 challenging. 87 88 We hypothesized that those limitations can be overcome and unanswered questions 89 can be addressed by using the available Drosophila deficiency line collections, one 90 of the unique genetic tools well-utilized extensively for genetic screening, mapping 91 and characterization processes. Two types of molecularly defined deficiency lines, 92 whose details including ends of the genes lost as well as cytological location on the bioRxiv preprint doi: https://doi.org/10.1101/2021.01.20.427453; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 chromosome are known, are available (Ryder et al., 2004; Parks et al., 2004; Cook 94 et al., 2012; Roote and Russell, 2012). In addition, as chromosomes of these 95 deletion-bearing fly stocks resemble those chromosomes with microdeletion or 96 chromosomal deletions of contiguous genes (loss of genes in the same order) in 97 other organisms, the collection has been utilized for systematic and comparative 98 analysis to establish the critical loci that correspond to Mendelian Disorders and 99 Microdeletion Syndromes in humans. 100 101 Based on this logic, in this work, we set out a pilot project by subjecting these 102 deficiency lines for mitotic clonal analyses in Drosophila wing imaginal discs to 103 identify and understand their role in cytoskeletal organization during epithelial 104 morphogenesis. Clonal analysis technique allows to analyse behaviour of two 105 different population of cells in a tissue (Lee and Luo, 2001; Wu and Luo, 2006; 106 Smith-Bolton et al., 2009; Rodriguez et al., 2012; Powell and Piddini, 2016). Here we 107 have utilized this property to understand the behaviour of randomly eliminated 108 genes, thereby identify the critical gene in that locus implicated in epithelial 109 morphogenesis. During this process of clonal analysis, we have also followed the 110 cytoskeletal defects produced by these deficiency lines. We also undertook a 111 systematic functional analysis of the genes in the fly deficiency lines, compared them 112 with human orthologs and analysed their implications in cytoskeletal processes and 113 epithelial morphogenesis. Further we have drawn the synteny that exist between 114 these chromosomes and identified critical region of epithelial defects. Here, in this 115 report, few potential candidates that might have an implication in epithelial 116 morphogenesis are discussed. 117 118 Results and Discussion 119 (i) Recombination of Deletion-bearing chromosomes with FRT chromosomes 120 and generation of mitotic clones 121 To address the behavioural property of the tissue subjected to multigenic deletions 122 we generated mitotic clones using the available deficiency lines in Drosophila wing 123 imaginal discs. We recombined X and II chromosome deficiency lines with their 124 respective FRT chromosomes. We have subjected ~67 FRT-recombined X 125 chromosome