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      The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type

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          Abstract

          The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

          Author Summary

          The correct generation and maintenance of the nervous system is critical for the animal’s life. Dysregulation of these processes leads to multiple neurodevelopmental disorders. It has been a daunting challenge not only to identify the developmental mechanisms that determine neuronal cell fate, but also to understand the extent to which the mechanisms are evolutionarily conserved. Here, we describe a developmental mechanism that determines the fate of a specific cholinergic and peptidergic neuronal type in C. elegans. We show that the lim-4 LIM homeodomain transcription factor is necessary and sufficient to promote and maintain the specific cholinergic and peptidergic properties and functions via binding to unique DNA sequences. We also demonstrate that C. elegans lim-4 and human LHX6 show striking functional similarity; specifically, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in human neuronal cell lines. Given the high conservation of these transcription factors, these developmental mechanisms are likely to be generally applicable in the nervous system of other organisms as well.

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          Most cited references52

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          Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology.

          The use of primary mammalian neurons derived from embryonic central nervous system tissue is limited by the fact that once terminally differentiated into mature neurons, the cells can no longer be propagated. Transformed neuronal-like cell lines can be used in vitro to overcome this limitation. However, several caveats exist when utilizing cells derived from malignant tumors. In this context, the popular SH-SY5Y neuroblastoma cell line and its use in in vitro systems is described. Originally derived from a metastatic bone tumor biopsy, SH-SY5Y (ATCC(®) CRL-2266™) cells are a subline of the parental line SK-N-SH (ATCC(®) HTB-11™). SK-N-SH were subcloned three times; first to SH-SY, then to SH-SY5, and finally to SH-SY5Y. SH-SY5Y were deposited to the ATCC(®) in 1970 by June L. Biedler.Three important characteristics of SH-SY5Y cells should be considered when using these cells in in vitro studies. First, cultures include both adherent and floating cells, both types of which are viable. Few studies address the biological significance of the adherent versus floating phenotypes, but most reported studies utilize adherent populations and discard the floating cells during media changes. Second, early studies by Biedler's group indicated that the parental differentiated SK-N-SH cells contained two morphologically distinct phenotypes: neuroblast-like cells and epithelial-like cells (Ross et al., J Nat Cancer Inst 71:741-747, 1983). These two phenotypes may correspond to the "N" and "S" types described in later studies in SH-SY5Y by Encinas et al. (J Neurochem 75:991-1003, 2000). Cells with neuroblast-like morphology are positive for tyrosine hydroxylase (TH) and dopamine-β-hydroxylase characteristic of catecholaminergic neurons, whereas the epithelial-like counterpart cells lacked these enzymatic activities (Ross et al., J Nat Cancer Inst 71:741-747, 1983). Third, SH-SY5Y cells can be differentiated to a more mature neuron-like phenotype that is characterized by neuronal markers. There are several methods to differentiate SH-SY5Y cells and are mentioned below. Retinoic acid is the most commonly used means for differentiation and will be addressed in detail.
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            Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites.

            We describe the comprehensive characterization of homeodomain DNA-binding specificities from a metazoan genome. The analysis of all 84 independent homeodomains from D. melanogaster reveals the breadth of DNA sequences that can be specified by this recognition motif. The majority of these factors can be organized into 11 different specificity groups, where the preferred recognition sequence between these groups can differ at up to four of the six core recognition positions. Analysis of the recognition motifs within these groups led to a catalog of common specificity determinants that may cooperate or compete to define the binding site preference. With these recognition principles, a homeodomain can be reengineered to create factors where its specificity is altered at the majority of recognition positions. This resource also allows prediction of homeodomain specificities from other organisms, which is demonstrated by the prediction and analysis of human homeodomain specificities.
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              Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.

              The functions of the 11 classes of exposed chemosensory neurons of C. elegans were tested by killing cells with a laser microbeam. One pair of neurons, the ASE neurons, is uniquely important for chemotaxis: killing the ASE neurons greatly reduced chemotaxis to cAMP, biotin, Cl-, and Na+. Additional chemosensory function is distributed among several other cell types. Thus, 3 pairs of chemosensory neurons (ADF, ASG, and ASI) contribute to a residual response to cAMP, biotin, Cl-, and Na+ after ASE is killed. Chemotaxis to lysine similarly depends on the partly redundant functions of 4 pairs of chemosensory neurons (ASE, ASG, ASI, and ASK). The combined activity of several neuron types that act in parallel might increase the fidelity of chemotaxis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                25 August 2015
                August 2015
                : 11
                : 8
                : e1005480
                Affiliations
                [1 ]Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, Korea
                [2 ]Laboratory of Biochemistry and Cellular Engineering, Division of NanoBio Technology, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, Korea
                [3 ]Nano-Bio Electron Microscopy Research Group, Korea Basic Science Institute, Daejeon, Korea
                [4 ]School of Life Sciences, KNU Creative BioResearch Group (BK21 plus program), School of Animal BT Science, College of Natural Sciences, Kyungpook National University, Daegu, Korea
                [5 ]Department of Anatomy and Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan School of Medicine, Gyeonggi-Do, Korea
                [6 ]Department of Biology, City College of the City University of New York, New York, New York, United States of America
                University of California San Diego, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JK JY CL KKi. Performed the experiments: JK JY SKC YHH ZF. Analyzed the data: JK JY HSK WBJ CL KKi. Contributed reagents/materials/analysis tools: SJP MOK ZYR KKa. Wrote the paper: JK JY CL KKi.

                Article
                PGENETICS-D-15-01534
                10.1371/journal.pgen.1005480
                4549117
                26305787
                27366a34-83b1-4156-8445-b369f712eeb3
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 22 June 2015
                : 31 July 2015
                Page count
                Figures: 7, Tables: 1, Pages: 27
                Funding
                This work was supported by the NIH (5 R01 NS042459) and NSF (IOS 9808861 and IOS 9973581) (CL), from the DGIST R&D Program of the Ministry of Science, ICT and Future Planning (15-BD-06), the National Research Foundation of Korea (NRF-2012R1A1A2009385), and the T.J. Park Science Fellowship of POSCO T.J. Park Foundation (KKi), KBSI (E35700) and R&D Convergence Program of NST (National Research Council of Science & Technology) of Republic of Korea (CAP-14-3-KRISS) (HSK) and from the DGIST R&D Program of the Ministry of Science, ICT and Future Planning (14-NB-01), the National Research Foundation of Korea (NRF-2014R1A2A2A01005619) (WBJ), an intramural research fund from Sungkyunkwan University (S-2012-1188-000) and a research grant from Samsung Biomedical Research Institute (GE1-B3-011-1) (KKa). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
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                All relevant data are within the paper and its Supporting Information files.

                Genetics
                Genetics

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