ORIGINAL RESEARCH published: 17 February 2017 doi: 10.3389/fnana.2017.00006 Expression of sept3, sept5a and sept5b in the Developing and Adult Nervous System of the Zebrafish (Danio rerio) Frederik Helmprobst 1, Christina Lillesaar 2 and Christian Stigloher 1* 1Biocenter, Division of Electron Microscopy, University of Würzburg, Würzburg, Germany, 2Biocenter, Department of Physiological Chemistry, University of Würzburg, Würzburg, Germany Septins are a highly conserved family of small GTPases that form cytoskeletal filaments. Their cellular functions, especially in the nervous system, still remain largely enigmatic, but there are accumulating lines of evidence that septins play important roles in neuronal physiology and pathology. In order to further dissect septin function in the nervous system a detailed temporal resolved analysis in the genetically well tractable model vertebrate zebrafish (Danio rerio) is crucially necessary. To close this knowledge gap we here provide a reference dataset describing the expression of selected septins (sept3, sept5a and sept5b) in the zebrafish central nervous system. Strikingly, proliferation zones are devoid of expression of all three septins investigated, suggesting that they have a role in post-mitotic neural cells. Our finding that three septins are mainly expressed in non-proliferative regions was further confirmed by double-stainings with a proliferative marker. Our RNA in situ hybridization (ISH) Edited by: study, detecting sept3, sept5a and sept5b mRNAs, shows that all three septins Gonzalo Alvarez-Bolado, are expressed in largely overlapping regions of the developing brain. However, the Heidelberg University, Germany expression of sept5a is much more confined compared to sept3 and sept5b. In Reviewed by: Steffen Scholpp, contrast, the expression of all the three analyzed septins is largely similar in the adult Karlsruhe Institute of Technology, brain. Germany Mario F. Wullimann, Keywords: septin, RNA in situ hybridization, neuronal development, retinal development, sept3, sept5a, sept5b Ludwig Maximilian University of Munich, Germany INTRODUCTION *Correspondence: Christian Stigloher [email protected] Septins, a highly conserved family of small GTPases that form filaments, have various functions in normal physiology as well as pathology (Mostowy and Cossart, 2012; Dolat et al., 2014). Received: 06 October 2016 For instance, septins are reported to play a role in cytokinesis, axon growth and exocytosis. Accepted: 25 January 2017 Furthermore, they are involved in developmental processes, such as left-right-asymmetry Published: 17 February 2017 regulation, as well as several pathologies, such as cancer, neurodegenerative diseases and Citation: psychiatric disorders (Dash et al., 2014; Dolat et al., 2014; Zhai et al., 2014; Marttinen et al., 2015). Helmprobst F, Lillesaar C and However, the cellular roles and functions of septins, in particular how they are involved in the Stigloher C (2017) Expression physiology of the nervous system, still need to be fully elucidated. of sept3, sept5a and sept5b in the Septins were first discovered in cell division screens in budding yeast (Hartwell et al., 1970) Developing and Adult Nervous System of the Zebrafish (Danio rerio). and named after their localization at the septum (Byers and Goetsch, 1976; Sanders and Field, Front. Neuroanat. 11:6. 1994). Interestingly, septin homologs were identified later on in other eukaryotes like fungi, doi: 10.3389/fnana.2017.00006 worms, flies, zebrafish and mammals (Cao et al., 2007; Pan et al., 2007; Nishihama et al., 2011). Frontiers in Neuroanatomy| www.frontiersin.org 1 February 2017 | Volume 11 | Article 6 Helmprobst et al. Septin Expression in Zebrafish Brain Development In humans, there are 13 septin genes, which can be divided 2015; Willis et al., 2016). These are key advantages of this model, according to sequence similarity into four homology groups: as septins seem to play important roles in synaptic development SEPT2 (SEPT1, SEPT2, SEPT4 and SEPT5), SEPT3/9 (SEPT3, (Yang et al., 2010). SEPT9 and SEPT12), SEPT6 (SEPT6, SEPT8, SEPT10, SEPT11 Here we present a precise description of the expression and SEPT14), and SEPT7 (SEPT7; Kinoshita, 2003; Dolat et al., patterns of septin3, septin5a and septin5b at different 2014). In the zebrafish (Danio rerio) genome, there are currently developmental stages of zebrafish larvae as well as in adult 17 septin genes annotated (Willis et al., 2016). The increased brains. This study builds the basis for a thorough analysis of the diversity of septins is likely due to an teleost specific genome role of these septins in the nervous system using the vertebrate duplication event (Postlethwait et al., 2004), so that there are model organism zebrafish (Danio rerio). for instance two homologs of SEPT5 (sept5a and sept5b) present in the zebrafish genome (Willis et al., 2016). In addition, MATERIALS AND METHODS genes like zebrafish sept9a and sept9b also have additionally multiple isoforms due to alternative splicing (Landsverk et al., Zebrafish Culture and Nomenclature 2010). The overall structure of septins is highly conserved. They Zebrafish (Danio rerio) larvae and adults (4–6 months) of share a phosphoinositide-binding polybasic domain, followed by the wild-type strain (AB) were kept on a day/night cycle of the GTP-binding sites and the Septin Unique Element (SUE). 14 h light and 10 h darkness. Larvae were kept at 28.5◦C A coiled-coil domain is found in septins belonging to the and staged as reported previously (Kimmel et al., 1995) and SEPT2, SEPT6 and SEPT7 groups. Furthermore, septins form were raised in 30% Danieau’s solution (Westerfield, 2000). The heterofilaments consisting of two or more types of septins pigmentation of zebrafish larvae was inhibited by 0.2 mM (Kinoshita, 2003; Mostowy and Cossart, 2012; Dolat et al., 2014). 1-phenyl-2-thiourea treatment (Karlsson et al., 2001). The In the mammalian central nervous system Septin3 and brain structure nomenclature is conforming to reference atlases Septin5 have been attributed specific roles. Septin3 (Xue et al., (Wullimann et al., 1996; Mueller and Wullimann, 2015). 2004) and Septin5 (Kinoshita et al., 2000) are enriched at All experiments were performed according to the animal presynaptic terminals. Septin5 can inhibit exocytosis and interact welfare regulations of the District Government of Lower with presynaptic proteins (Beites et al., 1999, 2005). Furthermore, Franconia. Septin5 function has an effect on social behavior of mice (Suzuki et al., 2009; Harper et al., 2012). Septin5 is located in the human Phylogenetic Analyses genome at position 22q11.2, a region that is the site of the most The human homologs of zebrafish Sept3 (NP_001019589.1), common micro-deletion syndrome in humans. This deletion Sept5a (NP_956282.1), and Sept5b (NP_001003782.1) sequences is associated with multiple phenotypes including cardiac and (Willis et al., 2016) were aligned to Septin3 and Septin5 protein palatal anomalies, intellectual disabilities and other disorders sequences from mouse (Sept3: NP_036019.2; Sept5: affecting the nervous system (Guna et al., 2015). Moreover, for NP_998779.2) and human (Sept3 isoform B: NP_061979.3; Septin5 a fine-regulated expression at later developmental stages Sept5 isoform 1: NP_002679.2), as well as to the two known has been reported (Maldonado-Saldivia et al., 2000). septins from C. elegans (UNC-59: NP_493388.1; UNC-61 Despite these indications of the important roles of Septin3 and isoform a: NP_872156.2). Alignment was performed by the Septin5 in brain function and development, comparably little ‘‘One Click’’ Method using the web tool with GBlocks1 is known about the temporal expression of these genes during (RRID:SCR_010266, Dereeper et al., 2008) combining several early vertebrate brain development. Previously, it was shown that algorithms (Castresana, 2000; Guindon and Gascuel, 2003; zebrafish Septin5b is expressed in 1 day post-fertilization (dpf) Edgar, 2004; Anisimova and Gascuel, 2006; Chevenet et al., 2006; embryos (Gomez et al., 2012) and Septin3 is generally expressed Dereeper et al., 2010). in neural tissues (Thisse and Thisse, 2004). However, a precise temporal description of septin expression in early vertebrate brain development is currently missing. Such information is RNA Extraction and Reverse Transcription necessary and opens up a high resolution view into the cellular Zebrafish RNA was isolated from 1 to 4 dpf zebrafish larvae using role of septins in neurons. the RNeasy Mini Kit (Qiagen Gmbh, Düsseldorf, Germany). The The zebrafish is an established model organism well suited for RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, studies of early vertebrate brain development (Wullimann et al., Braunschweig, Germany) was used to generate cDNA. 1996; Mueller and Wullimann, 2015) and activity (Akerboom et al., 2012). Due to the accessible development and the large Molecular Cloning number of transparent offspring, zebrafish is a very fitting Specific PCR primers for sept3 (RefSeq: NM_001024418), sept5a model for the systematic investigation of gene expression during (RefSeq: NM_199988), and the forward primer (fwd) for sept5b development using RNA in situ hybridization (ISH; Thisse and (RefSeq: NM_001003782) were generated. Thisse, 2008). In addition to that, zebrafish is very suited for The sept5b reverse primer (rev, CGGTCCTGCTGCTTC systematic high resolution and functional analysis to shed light GGCTC) was designed according to previously published