Gene 557 (2015) 11–18

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The zinc finger RNA binding protein, ZFR, contributes to axon guidance in Caenorhabditis elegans Tine Kjærgaard a, Rasmus Desdorf b, Anders Heuck a, Anders Olsen b, Karin Lykke-Hartmann a,⁎ a b

Aarhus University, Department of Biomedicine, Wilhelm Meyers Allé 4, DK-8000 Aarhus, Denmark Aarhus University, Department of Molecular Biology and Genetics, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark

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Article history: Received 21 August 2014 Received in revised form 12 November 2014 Accepted 30 November 2014 Available online 2 December 2014 Keywords: ceZFR Axon guidance C. elegans ceZfr RNAi-induced axon midline crossing Axon defasciculation and cord commissures

a b s t r a c t ZFR is an ancient and highly conserved chromosome-associated protein from nematodes to mammals, embryologically expressed in most species, with the exception of the nematode Caenorhabditis elegans. The ZFR encodes zinc and RNA binding protein, and in rat, the nuclear-cytoplasmic shuttling ZFR has been found with transport and translation-associated RNA granule-like structures in the somatodendritic compartments of hippocampal neurons. The majority of axons cross the midline before projecting to their contralateral synaptic target and this crossing decision is under tight control. Molecular factors contributing to these processes have been identified, although the mechanisms are not fully understood. In this study, we tested the role of ceZFR in axon guidance using ceZfr RNAi-treated animals to analyse axon midline crossing, axon fasciculation and cord commissures. In adult stages, RNAi-induced depletion of the ceZfr transcript leads to several phenotypes related to axon guidance. A midline crossing defect was observed in the ventral nerve cord (VNC) in axon type D, DD/VD motoneuron axons and axon type 1, interneuron axons. We further detected a dorsal nerve cord (DNC) axon fasciculation. Some ceZfr RNAi-treated animals revealed that cord commissures fail to reach their synaptic target. We provide evidence that ceZFR has a role in axon guidance. When Zfr was depleted by RNAi, the phenotypes are characterized by defects in axon midline crossing, axon defasciculation and cord commissures. Our results thus support the hypothesis that ZFR has essential roles during neurogenesis, and could support early steps of RNA transport and localization through RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In the developing central nervous system (CNS) in several bilaterally-symmetric organisms, many interneurons project axons along the paths that are perpendicular or parallel to the midline (reviewed in Kaprielian et al., 2000, 2001). The ventral midline of the developing CNS represents a binary choice for pathfinding axons. At the midline, the axons have to decide to crossover to the contralateral side of the CNS or not. Thus, the midline is an important choice point for several classes of pathfinding axons (Kaprielian et al., 2001). Caenorhabditis elegans has emerged as a major model system for the genetic control of

Abbreviations: Aa, amino acid; BLAST, Basic Local Alignment Search Tool; bp, base pair; ceZFR, C. elegans ZFR; CNS, central nervous system; DC, dorsal cord; DNA, deoxyribonucleic acid; dsRBD, double-stranded RNA binding domain; FL, full length; hZFR, human ZFR; kb, kilo base; min, minutes; mRNA, messenger ribonucleic acid; mRNP, messenger ribonucleoprotein; mZFR, murine ZFR; NES, nuclear export signal/sequence; NGM, nematode growth media; NLS, nuclear localization signal/sequence; RNA, ribonucleic acid; RNAi, RNA interference; RNP,ribonucleoprotein;sec, second;snRNP,small nuclear ribonucleoproteins; ssDNA, single-stranded deoxyribonucleic acid; ssRNA, single-stranded ribonucleic acid; VNC, ventral nerve cord; zf, zinc finger; ZFR, zinc finger RNA binding protein. ⁎ Corresponding author. E-mail address: [email protected] (K. Lykke-Hartmann).

http://dx.doi.org/10.1016/j.gene.2014.11.063 0378-1119/© 2014 Elsevier B.V. All rights reserved.

neuronal development. The nectrin family was the first identified signalling pathway components regulating axon guidance and loss-of-function mutations in the Uncoordinated (unc)-6 that resulted in the failure of axonal growth to follow their normal dorsal and ventral pathways during development (Brenner, 1974). Similarly, defects in growth cone migration were reported for the unc-6/Netrin and its receptors unc-40 (Hedgecock et al., 1990; Adler et al., 2006) and unc-5 (Hedgecock et al., 1990) and axon outgrowth has also been connected to other proteins in C. elegans, such as the enhancer of unc, enu-3 (Yee et al., 2011) and the zinc finger homeodomain transcription factor zag-1 (Wacker et al., 2003). The zinc finger RNA binding protein, ZFR, was identified in a screen for RNA-binding proteins expressed during murine spermatogenesis and was shown to be an ancient and highly conserved murine chromosome-associated zinc finger protein (ZFP) (Meagher et al., 1999). The N-terminal C2H2 zinc finger motifs resemble the U1 zinc finger found in several U1 small nuclear ribonucleoprotein C (U1-C) proteins that binds to the pre-mRNA 5′ splice site (Nelissen et al., 1991). The C-terminal dsRBD is among the most common RNA-binding motifs (St Johnston et al., 1992; Tian et al., 2004) and is involved in transcription, RNA processing/editing, RNA maturation and localization, RNA translation and repression hereof (Green and Mathews, 1992; St Johnston et al., 1992; Ramos et al., 2000; Tian et al., 2004) and may act as a nuclear

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localization signal (NLS) as well as being involved in meditating nuclear export (Nakielny and Dreyfuss, 1999; Eckmann et al., 2001). Zfr mRNA is highly expressed in mouse brain, testis and ovary and, to a smaller extent, in the heart, kidney, liver and spleen (Meagher et al., 1999), and during mouse pre-implantation development associated with chromosomes (Meagher et al., 1999; Doganli et al., 2010). In line with this, homozygous knock-out mice die during gastrulation as a result of increased programmed cell death and decreased mitotic index (Meagher and Braun, 2001). The human ZFR appears to be involved in the regulation of alternative pre-mRNA splicing (Kleines et al., 2001) and was identified in a screen for factors that interact with the pre-mRNA splicing activator RNPS1 (Sakashita et al., 2004). Analogous to this, Drosophila ZFR (Zn72D) was shown to promote productive splicing of the maleless (Mle) transcript (Worringer and Panning, 2007), thus regulating the X chromosome dosage of Mle, in conjugation with the DEAD box helicase Belle (Worringer et al., 2009). However, in rat hippocampus neurons, ZFR was shown to be a native component of Staufen2-containing granules, likely to play a role during the early steps of RNA transport and localization (Elvira et al., 2006). Interestingly, in contrast to the nuclear localization of ZFR in mouse testis, ovary and embryos (Meagher et al., 1999; Doganli et al., 2010), ZFR in rat neurons was not only observed in the nucleus, but also in somatodendritic compartment of primary hippocampal neurons and in the cytoplasm, where it forms granule-like structures connected to RNA transport and translation (Elvira et al., 2006). The ceZFR ortholog in C. elegans (Y95B8A.7; ceZFR), was identified in a large-scale RNA interference (RNAi) screen for gene regulating axon guidance (Schmitz et al., 2007), which appears to compliment with the neuronal role of ZFR in rat hippocampus (Elvira et al., 2006). The screen was performed using a RNAi-hypersensitive C. elegans strain VH715: nre-1(hd20); lin-15b(hd126); hdIs17 I; hdIs10 V, where interneurons of the motor circuit, as well as several classes of motoneurons in the ventral cord (DA, DB, DD, VD) were fluorescently labelled (Schmitz et al., 2007). In C. elegans, coordinated locomotion is mediated through six major motor neurons (DA, DB, DD, VA, VB, and VD). Four of these motor neuron classes are excitatory (DA, DB, VA, and VB), whereas, DD and VD are inhibitory motor neurons (White et al., 1976). The ceZFR was identified in a large scale RNAi screen for genes affecting axon navigation in the motor circuit of C. elegans (Schmitz et al., 2007). This study analysed axons in the ventral and dorsal cords, as well as outgrowth and navigation of motoneuron commissures that connect ventral and dorsal cords. The RNAi screen also identified the Fat-like cadherin CDH-4 that was recently shown to control axon fasciculation, cell migration and hypodermis and pharynx development (Schmitz et al., 2008). In this study we show that the conserved ceZFR is associated with axon defasciculation and guidance using RNAi to knock-down the gene function. The knock-down of ceZfr mRNA by RNAi leads to several phenotypes specifically associated to axon guidance. Interestingly, in a midline crossing defect in the ventral nerve cord (VNC) in axon type D, DD/VD motoneuron axons, and axon type I, interneuron axon was observed in our ceZfr RNAi. We further detected a dorsal nerve cord (DNC) axon defasciculation. Some ceZfr RNAi revealed that cord commissures fail to reach target and we found that more than two commissures of the type D, DD/VD motoneuron commissures did not reach the dorsal. Ventral cord patterning abnormalities were also observed. 2. Material and methods

25 μl PCR reaction containing 12.5 μl 2× reaction mix (0.4 mM of each dNTP, 2.4 mM MgSO4), using 0.5 μM gene specific oligonucleotides for CeZfr (acc nr: NM_058385) (5′ GCAAGATCTATGGCAAAACAGGGCAT ACC and reverse 5′ GAAAGATCTAATATCGGCGGGCTCCTCG) containing a BglII restriction site, 1 μg total worm RNA, 0.5 μl SuperScript™ III RT/ Platinum® Taq High Fidelity Enzyme Mix and 10 μl RNase free H2O (Ambion). The cDNA synthesis was performed using the following PCR programme: 1 cycle of 30 min at 55 °C and 2 min at 94 °C, followed by 40 cycles of 15 s at 94 °C, 30 s at 55 °C and 5 min at 68 °C with a final extension of 20 min 68 °C. 2.2. ZFR cloning The amplified Zfr cDNA was gel extracted by using NucleoSpin® Extract II (Macherey-Nagel) following the manufacturer's protocol and cloned into the pTZ57R vector (Fermentas). N- and C-terminal sequences were digested from the full length of Zfr using XhoI and XhoI and KpnI, respectively. PCR and digestion products were cloned into the feeding vector L4440 (pPD129.36) (Timmons and Fire, 1998) and transformed into the HT115 (DE3) (tetracycline-resistant, RNaseIII (−) strain (Timmons et al., 2001)). The three respective clones are referred to as ZFRRNAi–FL81–1904, ZFRRNAi–N417–1066 and ZFRRNAi– C1073–1904 for full length, N- and C-terminal sequences of ZFR, respectively. All clones were verified by DNA sequencing (data not shown). 2.3. RNAi feeding and screening The C. elegans strain Bristol N2 was used as a standard wild type strain. Worms were grown and maintained according to standard protocols (Brenner, 1974; Stiernagle, 2006). Eggs were isolated from gravid hermaphrodites by alkalinehypochlorite treatment (Motohashi et al., 2006). L1 larvae were isolated by allowing eggs to hatch in S-basal (food-free medium) by incubating them overnight at room temperature. The following day the L1 larvae were collected. For isolation of adult hermaphrodites, eggs were placed on NGM plates spotted with concentrated Escherichia coli OP-50 bacteria. After about 72 h the adult hermaphrodites were collected (Motohashi et al., 2006). RNAi feeding was performed as previously described (Kamath et al., 2001) with the following modifications. The strain used for RNAi experiments was VH715 (Schmitz et al., 2007). The RNAi experiments were conducted on standard NGM plates (Hope, 1999) (0.3 w/v% NaCl, 0.25 w/v% peptone, 1.7 w/v% agar), (1 mM CaCl2, 1 mM MgSO4, 5 μg/ml cholesterol in EtOH, 25 mM KPO4 pH 6.0), and isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM) and ampicillin (100 μg/ml) were added before pouring the plates (Kamath et al., 2001) (referred to as RNAi plates). The feeding bacteria were generated by picking a single colony of HT115 bacteria containing cloned L4440 plasmids and was grown in culture in LB media with 100 μg/ml Amp overnight (12–16 h) (Kamath et al., 2001). The RNAi analysis was performed as described (Schmitz et al., 2007) using the RNAi sensitive strain VH715: nre-1(hd20); lin-15b(hd126); hdIs17[glr-1::YFP+unc-47::YFP+unc-129::YFP+rol-6(su1006)]; hdIs10 [unc-129::CFP+glr-1::YFP+unc-47::DsRed+hsp-16::rol-6], using ampicillin as selection on the RNAi plates. L3 stages hermaphrodites from the maintenance plates to the RNAi plates and left to incubate for 5 days at 20 °C, and the adult hermaphrodites are removed from the plates after 3 days. The phenotypic analysis was performed by placing 30–50 worms in a 20 μl drop of S-basal containing 3 mM levamisole on a glass slide, which were covered with a coverslip and immediately analysed under a fluorescent light microscope using LAS software (Leica).

2.1. RNA isolation and RT-PCR 2.4. Reverse Transcription and Quantitative PCR Total RNA was isolated from C. elegans using Trizol® according to the manufacturer's instructions (Invitrogen) (Chomczynski and Mackey, 1995). Reverse transcriptase (RT)-PCR was performed on 1 μg of total RNA using SuperScript™ III One Step RT-PCR system (Invitrogen) in a

SuperScript™ III Reverse Transcriptase (Invitrogen) was used to generate the 1st strand oligo d(T) cDNA from total RNA from different worm stages by following the manufacturer's protocol. 4 μg of total

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RNA was used to generate cDNA. Primers were designed for C. elegans Zfr (acc nr: NM_058385) and actin-1 (act-1) (NM_073418.4) using an exon-spanning approach from Pearl Primer software [125]. Several primer pairs were tested for Zfr and after optimization the primer pair qPCR_ZFR_1171_F_C and qPCR_ZFR_1417_R_C was chosen, which generated a qPCR product of 246 base pair (bp). The primer pair qPCR_978_act1F_C and qPCR_1109_act1R_C for amplification of Act-1 generated a qPCR product of 131 bp. The quantification of Zfr and Act-1 transcripts was performed by using real-time quantitative PCR. Each cDNA sample was amplified in triplicate using Brilliant® SYBR Green Master Mix (Stratagene) in a total reaction mixture of 25 μl reaction containing 12.5 μl SYBR Green, 1 μl of 10 μM Zfr (forward and reverse) or act-1 (control) (forward and reverse) primers, 1 μl of template cDNA (egg-, L1- and adult stages) and 10.5 μl of nuclease-free H2O. The PCR was run on a LightCycler®96 (Roche), using the following programme: 1 cycle of 10 min at 95 °C; stage 2, 45 cycles of 30 s at 95 °C, 1 min at 60 °C and 1 min at 72 °C; and stage 3 (dissociation), 1 cycle of 15 s at 95 °C, 1 min at 60 °C, 15 s at 95 °C and 15 s at 60 °C. A non-template control was included in each qPCR run. Primer efficiencies were tested by dilution series of a cDNA template (mix worm culture) for Zfr and Act-1. The analysis of the qPCR data was performed on Prism6 (GraphPad). 3. Results 3.1. ceZfr gene structure and design of ceZfr RNAi ZFR is a protein encoded by 1074 amino acids and is composed of three widely spaced C2H2 zinc finger motifs with the CxxCx(12)Hx(6) H consensus sequence and a double-stranded RNA binding domain

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Table 1 Summary of phenotype observation for each RNAi construct used. Axon crossing midline

Axon defasciculation

Cord commissures fail to reach target

ZFRRNAi–FL81–1904 ZFRRNAi–C1073–1904

ZFRRNAi–FL81–1904 ZFRRNAi–N417–1066 ZFRRNAi–C1073–1904

ZFRRNAi–FL81–1904

(dsRBD). The ceZFR protein is similar to orthologous proteins from Homo sapiens, Mus musculus, Danio rerio, Xenopus laevis, and Drosophila melanogaster, and, although the ceZFR appears shorter, this is most likely due to incomplete sequence information available for the ceZFR gene (Doganli et al., 2010). The ceZFR sequence was assembled from two database entries (NM_058385 and BJ799994) that consist of 1928 nt and a 637 amino acid (aa)-long open reading frame (ORF) containing two C2H2-type zinc binding motifs, a dsRDB domain and a NLS (Fig. 1A), with specific domain distributions (schematically shown in Fig. 1B). Several C. elegans RNAi libraries exist and are widely used to screen for gene-specific phenotypes (Kamath et al., 2003; Sonnichsen et al., 2005). It is interesting to note that a phenotype of ceZFR RNAi has not previously been detected in RNAi screens performed on the wild type strain N2. This of course might be explained by the fact that no phenotypes could be observed upon knock-down of ceZFR or perhaps that ceZFR does not exist in any of the libraries and therefore no phenotypes were observed. However, this could also be due to the nature of neuronal phenotypes that often are rarely detectable using the N2 strain. This hypothesis was confirmed by the use of a RNAi hypersensitive strain, which identified several genes regulating axon guidance, including ceZFR (Schmitz et al., 2007). We generated three RNAi knock-down

A …………Y C E V C K I S C A G G I T Y K E H L E G Q R H K K K E A M A K Q G I P S T S L A K N K LSYRCDLCDVTCTGQDTYSAHVRGGKHLKTAQLHKKLGKPVPEDVPTII APGADGPTETKAKPKWHQQALPGGKIVIGINTVNFVGGTKLNSTGQLEE KKREVAAAVSSVGRKTGGAAATTTIEVEDEKLRAMIAAEEVQPVGEEHVT EERDATGKLVQFHCKLCDCKFSDPNAKEIHIKGRRHRVSYRQKIDPTLVV DVKPSNKRSQEKRKNQLPAVHEPPSFMKTPWFAPPAPEGREFNIVDDRT INEKYAGLNPGVEFISNVDRLISDINESLKYVSDKIERDVRKIPEDVVELPT TTTTTEQPPRTVLGCSRVGIIAKGTFIKGDRCAEVVLTCTPVPTSGLVEQIR RLFGESTTSLTIEPDPESPSSLIVTANYFPNMKCRILITSAVVRKDDDSIVTG CAADKDLCIYALASIRNTKWYDSHCQYLNSCQSVIRLLRDLRNKYPEVAC LDDYKMELIVSNIIDSSPMSLGLSDAFKRIVEALASGYLYSAILSDPCETSR PNVLDALTDEQKHSLTALAQNFVRQIAFNQIHEILGIDRLQDTIDLPEDAP MLKRPLESNENAENAENLDDSPVSKKEKLDEEPADI

B ceZFR (637 aa)

B

DR

S

NL

C2H2-type zinc binding motif (spanning 23 aa each) DRB (spanning 47 and 241 aa, respectively) NLS (spanning 24 aa) Fig. 1. Schematic representation of the ceZFR protein sequence. (A) Protein sequences highlighting the conserved protein domains found within ceZFR, with red, blue and pink letters indicating the zinc binding domains 1–3, the dsRBD and the NLS1–2, respectively (B) The distributions of the protein domains in ceZFR, indicating the locations of the zinc binding domains, the dsRBD and the NLSs.

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Fig. 2. Axonal defects in the dorsal cord nerve the ceZfr RNAi. (A) Axons do not cross the midline in the RNAi control group, but frequently observed to crossover into the contralateral axon bundle in ceZfr RNAi-treated animals. (B) Axon crossing the midline in the ventral nerve cord in ZFRRNAi–FL81–1904 RNAi and (C–D) axon crossing the midline in the dorsal nerve cord in ZFRRNAi–C1073–1904. Higher magnifications of the axon midline crossing are shown in (E–G), indicated by arrows.

constructs to facilitate specific knock-down of ceZFR in C. elegans. The first design included the full length (FL) of the open reading frame for ceZFR (ZFRRNAi–FL81–1904) and then two additional designs containing inserts of N- (ZFRRNAi–N417–1066) and C-terminal (ZFRRNAi–C1073–1904) regions of ceZFR, respectively. The N-terminal insert is 656 bp long and covers the region from the second zinc finger motif to where the dsRBD begins and the C-terminal insert is 850 bp long and includes the dsRBD. The empty L4440 vector was used as a negative control.

3.2. ceZfr RNAi-induced knock-down reveal axon guidance defects A C. elegans strain that showed an enhanced RNAi in the nervous system was used as an RNAi hypersensitive strain to identify genes regulating axon guidance (Schmitz et al., 2007). In this study, the C. elegans Y95B8A.7 clone was described to be a ZFR1 homolog and to have a role in axon guidance. We developed Zfr-deficient C. elegans to further investigate the role of ceZFR in axon guidance using different double-

Fig. 3. Axonal defects in the ventral nerve cord. Defasciculation in ceZfr RNAi. (A) The ventral cord is tightly fasciculated in the ventral nerve cord at the vulva in the control RNAi control group, but frequently defasciculated in the ceZfr RNAi. (B) Axon crossing the midline in the ventral nerve cord in ZFRRNAi–FL81–1904 RNAi and (C–D) axon crossing the midline in the ventral nerve cord in ZFRRNAi–N417–1066. Higher magnifications of the axon midline crossing are shown in E–G, indicated by arrows.

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stranded (ds)RNA targets to trigger an RNAi response and obtained related phenotypes, as described below. 3.3. ceZfr RNAi revealed defects related to axon midline crossing, axon defasciculation and cord commissures Several specific phenotypes related to axon guidance were observed and included axon midline crossing, axon defasciculation and commissure phenotypes, as will be described below. Table 1 summarizes the phenotypes observed for each RNAi construct. 3.4. ceZfr RNAi shows axon midline crossing defects The midline crossing defect in the ventral nerve cord phenotype describes the crossing over of neuron processes or nerve bundles to the contralateral side. In the RNAi control group, none of the neuron processes were observed to crossover from the ventral to the dorsal side (Fig. 2A). In contrast, this phenotype was observed in both ZFRRNAi– FL81–1904 and ZFRRNAi–C1073–1904 knock-down C. elegans (Fig. 2B and C). From the ZFRRNAi–FL81–1904 knock-down, an axon was observed to crossover into the contra lateral axon bundles (Fig. 2B and E) further supported by ZFRRNAi–C1073–1904 knock-down, which also revealed other axon crossing over into the contralateral axon bundle in G1 and G3 (Fig. 2F and G). 3.5. ceZfr RNAi displays axon defasciculation The axon defasciculation in the dorsal nerve cord is a phenotype where neuron processes are separated from each other, and therefore debundles. The axon defasciculation phenotype was observed for ZFRRNAi –FL81–1904, ZFRRNAi–N417–1066 and ZFRRNAi–C1073–1904, whereas the controlRNAi group displayed no phenotype (Fig. 3A). Two types of axon defasciculations were observed. A clear axon defasciculation was observed, where the separation or debundling of the processing neurons is large (Fig. 3B, C, indicated by arrowheads in E and F). In addition to this single defasciculation, multiple-separated/ debundled processing neurons were observed, which appear to be a kind of net linking several fasciculations (Fig. 3D, indicated by arrowheads in G). 3.6. Cord commissures failed to reach target in ceZfr RNAi-treated animal In ceZfr mutants, the cord commissures failed to complete the connection between the two nerve cords. Several defects in cord commissures were observed for the ZFRRNAi–FL81–1904 mutants and the phenotypes observed can be described as cord commissures fail to complete axon tract connections between the two nerve cords (Fig. 4B and C; commissures indicated by arrowheads), which clearly shows an example of a commissure that failed to reach the dorsal nerve cord, in contrast to the RNAi control group (Fig. 4A). 3.7. qPCR analysis of ceZfr-RNAi-treated C. elegans The ceZfr transcript levels were analysed by a quantitative real-time PCR experiment. This revealed that the ceZfr transcript levels in RNAitreated C. elegans with ZFRRNAi–FL81–1904, ZFRRNAi–N417–1066 and ZFRRNAi –C1073–1904, respectively, were comparable (Fig. 5), and indifferent from non-treated C. elegans as well (data not shown). We also assessed the ceZFR protein levels by Western blotting, however, we did not observe any cross reactivity with commercial available antibodies (data not shown). 4. Discussion The molecular basis for the navigation of axons towards their target was previously explored using a high-throughput RNAi screen in the

RNAi hypersensitive C. elegans strain VH715 (nre-1(hd20) lin15b(hd126)) (Schmitz et al., 2007). This screen identified almost 100 new genes associated with cord fasciculation and cord commissure outgrowth defects. It was suggested that the majority of the genes identified have a specific role in the navigation of certain classes of axons rather than a general role in axon guidance affecting every neuron, and that different axons extending in the same axon bundle apparently used different combinations of signals to navigate and stay on course. The conserved fat-like cadherin CDH-4 was identified as a gene involved in axon guidance and displayed midline crossing defects in the VNC and DNC defasciculation (Schmitz et al., 2008), and moreover, the classic cadherin gene hmr-1, was shown to be required in the regulation of axon fasciculation (Broadbent and Pettitt, 2002). Despite the fact that ZFR is a highly conserved protein and the overall protein domain organization of ZFR suggests a role in DNA and RNA processing (Doganli et al., 2010), several roles have been suggested for ZFR in various species and cell type populations, which indicate that ZFR plays not only with the splicosome to regulate mRNA slicing (Kleines et al., 2001; Sakashita et al., 2004; Worringer and Panning, 2007), but also with RNA granules in neurons supporting mRNA transport (Elvira et al., 2006). Initially, the role of ceZfr was searched in genomewide RNAi screens (Fraser et al., 2000; Kamath et al., 2003; Sonnichsen et al., 2005) performed on the wild type strain N2, however, this did not allow us to identify ceZFR in those existing RNAi libraries. Using a C. elegans strain with a highly sensitized background allowed for an axon navigation phenotype in ceZFR-deficient C. elegans, although, not all genes associated with axon navigation are presently known. All three RNAi constructs (ZFRRNAi–FL81–1904, ZFRRNAi–N417–1066 and ZFRRNAi–C1073–1904) used to knock-down the ceZfr gene displayed comparable axon navigation phenotype, although, variations were observed in the degree of the phenotype severity. We tried to assess the degree of RNAi-induced knock-down by applying quantitative (q)PCR, however, due to high background of non-knock-down ceZfr in unaffected cells, we could not use this to monitor the degree of knock-down. This might also be reflected in the cord commissure phenotype, where this phenotype was observed only in the ZFRRNAi–FL81–1904-treated animal and not in the ZFRRNAi–N417–1066- or ZFRRNAi–C1073–1904-treated animals. Moreover, our data supports the finding from large-scale RNA interference (RNAi) screen for genes regulating axon guidance (Schmitz et al., 2007). The phenotypes observed were midline crossing defects in the ventral nerve cord, where an axon crossed over into the contralateral side, and axon defasciculation, a debundling of neuron processes. This is in line with a recent study where rat ZFR is suggested to have a neuronal function in rat hippocampus neurons, where it is essential for the nucleo-cytoplasmic shuttling of Stau2 in neurons, and localizes not only to the nucleus but also to the cytoplasm (Elvira et al., 2006). Compared to this, the human ZFR ortholog was suggested to be involved in mRNA splicing activities, since it has been shown to be in complex with the splicing activator complex containing the RNPS1 protein (Sakashita et al., 2004). Analogous to this, the D. melanogaster ZFR ortholog has been shown to promote productive splicing of the maleless transcript (Elvira et al., 2006). Together this might suggest that the role of C. elegans ZFR in axon guidance is to assist in both pre-mRNA splicing and shuttling of RNA, and this could be assigned to different neuron cells rather than a dual-role in general neuronal populations. Basing on the knowledge we have about the function of ZFR at this point, one option does not exclude the other, and ZFR might be a dual player at all times, assisting in both mRNA splicing and shuttling. In this regard it is very interesting to note that despite the evolutionary conserved nature of ZFR in different species, the expression profiles observed are distinct (Doganli et al., 2010). In particular, the expression of the C. elegans Zfr mRNA was notably different when comparing to M. musculus, H. sapiens, X. laevis, D. rerio and D. melanogaster. In line with this, it is not surprising that phenotypes observed in ZFR knockout mice are different from Zfr-deficient C. elegans.

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ZFRRNAi-FL81-1904 Fig. 4. Cord commissure defects in ceZfr RNAi. Commissures in the (A) RNAi control group complete their connection to the dorsal nerve cord, in contrast to the commissures in the (B) ZFRRNAi– FL81–1904. A higher magnification of the failure to reach the dorsal nerve cord is shown in (C), indicated by an arrow.

ZFR is essential for gastrulation, growth and survival in mouse. Loss of ZFR function leads to increased cell death and decreased mitotic index resulting in embryonic death by eight to nine days of gestation (Meagher and Braun, 2001), suggesting a maternal ZFR function during pre-implantation development. The expression profile of ceZFR does not suggest similar role of ZFR in C. elegans, in agreement with the observed axon guidance-related phenotypes, although this does not

exclude the fact that additional phenotypes could be observed. In addition, the knock-down of the ceZfr transcript by RNAi did not cause lethality of the worms and the movement of the worms did not seem to be affected be the knock-down. 5. Conclusion In an initial attempt to assess the role of ceZFR in axon guidance, we used RNAi to knock-down the function of ceZFR in C. elegans using three different constructs encoding either the full length, the N- or the C-terminal parts of ZFR. The ceZfr RNAi-treated animal had several defects related to axon guidance suggesting incomplete neuronal differentiation. The most prominent defects observed were failures in axon midline crossing and axon defasciculation. In addition, the cord commissures fail to reach their target, suggesting that interneurons are affected. Studies of midline guidance continue to provide important insights into the molecular mechanisms supporting these functions, and this study serves to further contribute towards the molecular requirements for correct axon guidance perhaps mediated through RNA transport and localization by contributing to RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling. Acknowledgement

Fig. 5. Evaluation of ceZfr transcript levels by qPCR. ceZfr mRNA levels were addressed in C. elegans treated with RNAi against the ZFRRNAi–N417–1066 (RNAi N), ZFRRNAi–C1073–1904, (RNAi N) and ZFRRNAi–FL81–1904 (RNAi N) regions, respectively. β-Actin was used as internal reference genes for qPCR normalization. Ev; empty vector, mean standard deviation is used (n = 6 for each assay, P N 0.05, Tukey's test).

We would like to thank the members of both A.O. and K.L.-H. laboratories for good discussion and helpful suggestions. Special thanks go to Lone Vedel Schøler for her help in the C. elegans work. This work was supported by a STENO stipend from the Danish Council for Independent Research in Natural Sciences Grant from the Danish Agency for Science,

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