Hammock et al. Neural Development 2010, 5:32 http://www.neuraldevelopment.com/content/5/1/32 RESEARCH ARTICLE Open Access Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain Elizabeth AD Hammock1,2*, Kathie L Eagleson3, Susan Barlow4,6, Laurie R Earls4,7, David M Miller III2,4,5, Pat Levitt3* Abstract Background: In an effort to identify genes that specify the mammalian forebrain, we used a comparative approach to identify mouse homologs of transcription factors expressed in developing Caenorhabditis elegans GABAergic neurons. A cell-specific microarray profiling study revealed a set of transcription factors that are highly expressed in embryonic C. elegans GABAergic neurons. Results: Bioinformatic analyses identified mouse protein homologs of these selected transcripts and their expression pattern was mapped in the mouse embryonic forebrain by in situ hybridization. A review of human homologs indicates several of these genes are potential candidates in neurodevelopmental disorders. Conclusions: Our comparative approach has revealed several novel candidates that may serve as future targets for studies of mammalian forebrain development. Background As with other cell types, the diversity of GABAergic Proper forebrain patterning and cell-fate specification neurons has its basis in different developmental origins, lay the foundation for complex behaviors. These neuro- with timing and location of birth playing key roles in developmental events in large part depend on a series of cell fate [1,6-8]. gene expression refinements (reviewed in [1]) that com- Despite the phenotypic variety of GABAergic neurons, mit cells to express certain phenotypic features that all use GABA as a neurotransmitter. In mammals, define circuit formation. Relatively subtle disturbances GABA is produced by one of two GABA-synthesizing in development may underlie the etiology of neurodeve- enzymes, glutamic acid decarboxylase (GAD)65 or lopmental disorders, especially when alternative cogni- GAD67. These closely related enzymes are orthologs of tive phenotypes do not have an apparent malformation the Caenorhabditis elegans protein UNC-25, which is at the gross anatomical level. In the forebrain, cells pro- found only in cells that produce GABA. Because UNC- ducing g-aminobutyric acid (GABAergic interneurons) 25/GAD and other components of the GABA synthetic have been implicated in neurodevelopmental disorders, pathway are highly conserved, it is likely that mamma- including autism and schizophrenia [2-4]. These neu- lian orthologs of some of the genes that specify rons are composed of a diverse class of cells providing a GABAergic cell fate in C. elegans embryogenesis may wide range of control of neural activity, and vary in neu- also control GABAergic fate specification during mam- roanatomical location, electrophysiological properties, malian embryogenesis. transcriptome/proteome and innervation patterns as We have explored this hypothesis in an effort to either local circuit or long-range projection neurons [5]. define new candidates for regulating forebrain GABAer- gic cell fate that may be highly conserved across evolu- tionarily distant taxa. This discovery-based approach * Correspondence: [email protected]; [email protected] 1 (Figure 1) complements existing analyses of the tran- Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA scriptomes of subpopulations of mammalian GABAergic 3Zilkha Neurogenetic Institute, Keck School of Medicine, University of cells [9-13]. Thus, by using data from the transcription Southern California, Los Angeles, CA 90089, USA profiling of GABAergic cells in embryonic C. elegans,in Full list of author information is available at the end of the article © 2010 Hammock et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hammock et al. Neural Development 2010, 5:32 Page 2 of 14 http://www.neuraldevelopment.com/content/5/1/32 Figure 1 Summary diagram of experimental approach. combination with bioinformatics analyses, we report isolates of the unc-25::GFP cells and from four replicates here transcripts with sequence homologs that may also of the reference samples. A comparison of the unc-25:: be involved in GABAergic fate specification in mam- GFP and reference data sets identified 673 transcripts mals. We focused our attention on transcripts with gene showing elevated expression (1.7×) in GABAergic neu- regulation ontologies. To probe the potential role of rons at a false discovery rate (FDR) ≤ 1% [14]). these conserved players in mammalian development, we mapped these gene products in the developing mouse Bioinformatics screen forebrain, with a selective focus on the telencephalon. Genes in the list of GABAergic enriched transcripts with As a proof of principle, this strategy identified several Gene Ontology (GO) terms related to DNA and tran- gene products already known to play a role in the speci- scription regulation were analyzed for potential homol- fication of forebrain GABAergic interneurons in mam- ogy to mouse transcripts. Because functional homology mals. Additionally, our approach identified several is conserved at the protein level, we generated a list of previously unexplored gene products that serve as pro- C. elegans proteins from the list of corresponding tran- mising candidates for future investigation of forebrain scripts and then used BLASTP [16] analysis available at patterning. WormBase [17] from June 2005 to November 2008 (wormbase releases WS144 to WS196) to identify the Materials and methods closest matching mouse protein sequence homologs. We C. elegans transcription profiling then used this list of mouse protein homologs to gener- A microarray profiling of C. elegans cells (MAPCeL) ate the corresponding catalogue of mouse transcripts for strategy was used to obtain a transcriptome profile of C. in situ hybridization analysis. We did not distinguish elegans GABAergic neurons [14,15]. A complete among potential splice variants and/or protein isoforms description of the methods used for this study and the for a given single gene locus. To further rank potential GABAergic neuron expression profile will be reported candidates, we performed BLASTP in the reverse direc- elsewhere (S Barlow, L Earls, J Watson, C Spencer, K tion; after generating the list of mouse protein sequence Watkins, D Miller, manuscript in preparation). Briefly, homologs, those proteins were used to identify the best the unc-25::GFP marker was used to label C. elegans sequence homologs in the C. elegans proteome. GABAergic neurons. unc-25::GFP-expressing embryos were dissociated with chitinase and cultured for 24 Mouse care and use hours and viable unc-25::GFP labeled cells were isolated Timed pregnant C57Bl6j mice were bred in-house from by fluorescence activated cell sorting (FACS). Total founders originating from Jackson Labs under protocols RNA was purified from both the sorted unc-25::GFP approved by the Institutional Animal Care and Use positive cells and from the reference sample of all Committee of Vanderbilt University. Mice were main- embryonic cells. The RNAs were amplified and hybri- tained on a 12:12 light-dark cycle and were permitted dized to the Affymetrix C. elegans array. Average signal food and water ad libitum. Noon on the day following a intensities were calculated from three independent time-delimited overnight pairing was considered Hammock et al. Neural Development 2010, 5:32 Page 3 of 14 http://www.neuraldevelopment.com/content/5/1/32 embryonic day 0.5 (E0.5). Pregnant females were readily with VectaMount (Vector Laboratories, Burlingame, CA, identifiable at E14.5 and were deeply anesthetized with USA). isofluorane vapors followed by rapid decapitation in order to harvest embryos. Expression patterns of genes Light microscopy at this fetal age were analyzed because it is a mid-point Microscopy was performed using an Axioplan II micro- in the age-range for cortical GABAergic neuron produc- scope (Zeiss, Jena, Germany), and micrographs were tion and migration in the mouse forebrain [8]. Thus, we acquired with a Zeiss AxioCam HRc camera (Zeiss) in hypothesized that expression patterns related to Axiovision 4.1 software (Zeiss). Low-magnification GABAergic neuron specification and differentiation images were collected and linearly adjusted for bright- likely would be apparent at this age. ness and contrast using Adobe Photoshop (version 7.0, Adobe, San Jose, CA, USA). No other image alterations Riboprobe labeling other than resizing were performed. All figures were I.M.A.G.E. clones were obtained from ATCC (Manassas, prepared digitally in PowerPoint 2007 (Microsoft, Red- VA, USA) and Open Biosystems (Huntsville, AL, USA) mond, WA, USA). for the mouse transcripts (Additional file 1). The iden- tity of each I.M.A.G.E. clone was confirmed by sequen- Results cing at the Vanderbilt DNA Sequencing Facility. When Genes expressed in C. elegans GABAergic neurons necessary, due to cDNA size or the plasmid vector, we C. elegans embryonic GABAergic neurons were profiled subcloned the I.M.A.G.E. clone into a separate vector by the MAPCeL approach in which unc-25::GFP labeled (Additional file 2). These subclones were also sequenced cells were isolated by