Author response: Genetic and neuronal regulation of sleep by neuropeptide VF (2017)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Sleep is an essential and phylogenetically conserved behavioral state, but it remains unclear to what extent genes identified in invertebrates also regulate vertebrate sleep. RFamide-related neuropeptides have been shown to promote invertebrate sleep, and here we report that the vertebrate hypothalamic RFamide neuropeptide VF (NPVF) regulates sleep in the zebrafish, a diurnal vertebrate. We found that NPVF signaling and npvf-expressing neurons are both necessary and sufficient to promote sleep, that mature peptides derived from the NPVF preproprotein promote sleep in a synergistic manner, and that stimulation of npvf-expressing neurons induces neuronal activity levels consistent with normal sleep. These results identify NPVF signaling and npvf-expressing neurons as a novel vertebrate sleep-promoting system and suggest that RFamide neuropeptides participate in an ancient and central aspect of sleep control. https://doi.org/10.7554/eLife.25727.001 Introduction Sleep is an essential, phylogenetically conserved behavioral state whose regulation requires several brain regions that engage multiple neurochemical systems (Allada and Siegel, 2008; Cirelli, 2009). Historically, stereotactic lesion experiments have demonstrated a central role in sleep regulation for the hypothalamus (Saper et al., 2010; Weber and Dan, 2016), a structure that is anatomically and molecularly conserved between zebrafish and mammals (Biran et al., 2015; Chiu and Prober, 2013; Liu et al., 2015; Shimogori et al., 2010). More recent genetic approaches have corroborated a critical role for hypothalamic regulation of vertebrate sleep, due in part to production of specific wake- and sleep-promoting neuropeptides (Richter et al., 2014). However, only a small number of such neuropeptides have been identified, suggesting that additional neuropeptides remain to be discovered. Indeed, the hypothalamus produces many neuropeptides (van den Pol, 2012), but potential roles in sleep for most of these neuropeptides, and the neurons that produce them, are largely unknown. Sleep has primarily been studied using mammals, where electrophysiology can be used to define sleep and wake states. However, progress has been challenging, due in part to the complexity of mammalian brains, the significant cost and labor required to generate and test mutant and transgenic rodents, and the poor amenability of mammalian model systems for large-scale screens. To overcome these limitations, several groups have established invertebrate sleep models by using behavioral criteria to define sleep-like states (Allada and Siegel, 2008; Cirelli, 2009). First, sleep primarily occurs during specific periods of the circadian cycle during which animals usually adopt an inactive and specific posture. In mice, an inactive period of ≥40 seconds significantly correlates with electroencephalographic indications of sleep (Pack et al., 2007). Second, animals exhibit an increased arousal threshold during sleep, thus distinguishing sleep from quiet wakefulness. However, sleeping animals can still be aroused by strong stimuli, which distinguishes sleep from paralysis or coma. Third, sleep is controlled by a homeostatic system, which can be demonstrated by an increased need for sleep following sleep deprivation. Based on these criteria, rest in nematodes, flies and zebrafish (Hendricks et al., 2000; Prober et al., 2006; Raizen et al., 2008; Shaw et al., 2000; Van Buskirk and Sternberg, 2007; Yokogawa et al., 2007; Zhdanova et al., 2001) has been shown to be a sleep-like state (for simplicity, hereafter referred to as sleep). These model organisms have been used to perform genetic and genomic screens that have identified many genes that affect sleep (Chiu et al., 2016; Cirelli et al., 2005; Koh et al., 2008; Nath et al., 2016; Shang et al., 2013; Shi et al., 2014; Trojanowski et al., 2015), including several neuropeptides. However, hypothalamic neuropeptides implicated in regulating vertebrate sleep, including hypocretin (Hcrt) (Adamantidis et al., 2007; de Lecea et al., 1998; Lin et al., 1999) and melanin concentrating hormone (MCH) (Konadhode et al., 2013; Tsunematsu et al., 2014; Verret et al., 2003; Willie et al., 2008) lack clear invertebrate orthologs, suggesting that neuropeptidergic regulation of sleep may have evolved independently in vertebrates and invertebrates. Thus, it remains to be determined whether neuropeptides that regulate invertebrate sleep are functionally conserved in vertebrates. Members of a neuropeptide family that contain a C-terminal Arg-Phe-NH2 motif, known as RFamides, play key roles in promoting invertebrate sleep (He et al., 2013; Iannacone et al., 2017; Lenz et al., 2015; Nagy et al., 2014; Nath et al., 2016; Nelson et al., 2014; Shang et al., 2013; Turek et al., 2016). Vertebrates possess several RFamide family members (Elphick and Mirabeau, 2014), including the hypothalamic neuropeptide VF (NPVF). Mammalian NPVF (also known as RFRP or GnIH) was first identified in a bioinformatic screen for human RFamide neuropeptides (Hinuma et al., 2000). The human NPVF preproprotein produces three mature peptides, known as RFamide-related peptide (RFRP) 1, RFRP2, and RFRP3 (Hinuma et al., 2000). In rodents, intracerebroventricular infusion of synthetic RFRP1 or RFRP3 has been reported to affect hormone levels, food intake, sexual behavior and nociception (Kim, 2016; Kriegsfeld et al., 2015; Liu et al., 2001; Tsutsui et al., 2015; Ubuka et al., 2012). RFRP1 and RFRP3 can bind to NPFFR1 and NPFFR2 in mammalian cell culture, but RFRP2 does not and its cognate receptor(s) remains unknown (Hinuma et al., 2000; Liu et al., 2001). In the brain, NPVF is enriched within and around the dorsomedial hypothalamus in rodents (Liu et al., 2001; Poling et al., 2012) and humans (Ubuka et al., 2009), a region associated with sleep regulation (Saper et al., 2010). However, genetic gain- or loss-of-function sleep studies of mammalian NPVF-derived neuropeptides or npvf-expressing neurons (hereafter referred to as NPVF neurons) have not been reported. Here we address the role of NPVF in regulating vertebrate sleep using the zebrafish, a vertebrate that allows for rapid and cost-effective testing of genes for effects on behavior (Chiu et al., 2016). The zebrafish has been shown to exhibit a diurnal pattern of sleep/wake states using the behavioral criteria described above. Based on locomotor activity and arousal threshold assays, one or more minutes of inactivity correspond to a sleep state in zebrafish larvae (Elbaz et al., 2012; Prober et al., 2006). The zebrafish has genetic (Cirelli, 2009; Richter et al., 2014; Sehgal and Mignot, 2011), pharmacological (MacRae and Peterson, 2015) and neuroanatomical (Kaslin and Panula, 2001; McLean and Fetcho, 2004; Prober et al., 2006) similarities to mammalian sleep, suggesting that findings in zebrafish will translate to mammals. Using genetics, pharmacology, chemogenetics and optogenetics, we show that NPVF signaling and NPVF neurons are both necessary and sufficient to promote sleep, and that stimulation of NPVF neurons induces levels of neuronal activity consistent with normal sleep. These results establish RFamide neuropeptides as evolutionarily conserved sleep-promoting systems. Results Overexpression of NPVF promotes sleep in zebrafish larvae Recent studies using Drosophila melanogaster (Drosophila) and Caenorhabditis elegans (C. elegans) demonstrated that several RFamide peptides, as well as the neurons that produce them, are necessary and sufficient to promote invertebrate sleep (He et al., 2013; Iannacone et al., 2017; Lenz et al., 2015; Nath et al., 2016; Nelson et al., 2014; Shang et al., 2013; Trojanowski et al., 2015; Turek et al., 2016). A library of peptides was previously used to screen for zebrafish sleep phenotypes, but the library did not include RFamide peptides (Chiu et al., 2016). We therefore set out to test the hypothesis that RFamide peptides can also promote sleep in vertebrates. One C. elegans sleep-promoting RFamide peptide, FLP-13, is sufficient to induce sleep (Nath et al., 2016; Nelson et al., 2014) and is necessary for sleep in response to cellular stress (Nelson et al., 2014). The vertebrate RFamide whose mature peptide sequences most closely resemble those of FLP-13 is NPVF. The three mature peptides derived from NPVF (RFRP1-3) contain a conserved C-terminal LPXRF motif (where X = L or Q) (Figure 1A) that is critical for peptide function (Findeisen et al., 2011). The nine peptides derived from FLP-13 contain a similar C-terminal PXIRF motif (where X = L or F) (Figure 1—figure supplement 1A). Figure 1 with 3 supplements see all Download asset Open asset NPVF overexpression decreases locomotor activity and increases sleep. (A) Multiple sequence alignment of human (Hs), mouse (Mm), and zebrafish (Dr) NPVF preproproteins. Colored boxes demarcate identified or predicted mature peptide sequences. Note that the mouse NPVF protein lacks RFRP2. (B–I) NPVF overexpression decreased locomotor activity (B,D) and increased sleep (C,E) in transgenic animals compared to WT siblings and to pre-HS. Yellow bar indicates heat shock. Pre- and Post-HS data is calculated for the day of HS. White and black bars under behavioral traces indicate day (14 h) and night (10 h), respectively. NPVF overexpression decreased wake activity (F), decreased sleep latency (time to first sleep bout following lights on in the morning Pre-HS, or following HS in post-HS measurement) (H) and increased sleep bout number (I), and also caused a trend of increased sleep bout length (G). Mean ± SEM from one representative experiment (B,C), or three pooled experiments (D–I) are shown. n = number of animals. ***p<0.0001 by Two-way ANOVA with Holm-Sidak test. See also Figure 1—figure supplements 1–3. https://doi.org/10.7554/eLife.25727.002 To determine whether NPVF overexpression affects sleep, we generated a stable transgenic zebrafish line in which a heat shock-inducible promoter regulates expression of the zebrafish npvf open reading frame. After heat shock-induced overexpression of NPVF, we found that these animals were 71% less active, had 62% less wake activity (amount of activity while awake), and slept 350% more during the day compared to their non-transgenic wild-type (WT) siblings (Figure 1B–1F) (activity, wake activity and sleep: p<0.0001, Two-way ANOVA, Holm-Sidak test). The increase in sleep was primarily due to an increase in the number of sleep bouts (+331%, p<0.0001, Two-way ANOVA, Holm-Sidak test), along with a modest increase in the length of sleep bouts (+21%) that did not reach statistical significance (Figure 1G and I). NPVF overexpression also drove the transition from wake-to-sleep by 2850% compared to WT siblings, with the average transgenic animal falling asleep in 3.7 ± 0.4 min immediately following heat shock compared to 108.9 ± 16.0 min for WT siblings (p<0.0001, Two-way ANOVA, Holm-Sidak test, Figure 1H). Interestingly, induction of NPVF overexpression in the middle of the day did not affect sleep the following night. A previous study suggested that the circadian system prevents premature sleep onset in the evening, when homeostatic sleep drive is high (Cavanaugh et al., 2016). However, when we induced NPVF overexpression at the end of the day, fifteen minutes before lights off, we observed significantly decreased locomotor activity (−36%) and increased sleep (+20%) during the night, as well as the following day (Locomotor activity: −14%; Sleep: 124%) compared to WT siblings (all: p<0.0001, Two-way ANOVA, Holm-Sidak test, Figure 1—figure supplement 2), although the effect during the following day was smaller than that observed when NPVF overexpression was induced in the middle of the day. These observations suggest that the absence of a phenotype at night following heat shock during the middle of the day is due to declining levels of overexpressed NPVF protein, rather than circadian regulation of NPVF function. This interpretation is supported by our observation that the level of npvf mRNA, assayed using RT-qPCR and in situ hybridization (ISH), does not vary in a circadian manner (p>0.05, One-way ANOVA, Figure 1—figure supplement 3A and B). To further test whether NPVF-induced sleep is regulated by circadian cues, we raised and tested animals in either constant light (LL) or dark (DD) from birth, which prevents the establishment of circadian rhythms (Gandhi et al., 2015; Hurd and Cahill, 2002; Kaneko and Cahill, 2005). Following heat-shock in LL, NPVF-overexpressing animals were 60% less active and slept 75% more than their WT siblings (Figure 1—figure supplement 3C–G) (activity and sleep: p<0.0001, Two-way ANOVA, Holm-Sidak test). To test whether light affects this phenotype, we raised and tested animals in DD, and observed similar effects on overall activity and sleep as in LL (Figure 1—figure supplement 3J–N). Interestingly, we observed differences in sleep architecture between LL and DD. In LL, the increase in NPVF-induced sleep was due to a 200% increase in the number of sleep bouts compared to WT siblings (p<0.0001; Two-way ANOVA, Holm-Sidak test) (Figure 1—figure supplement 3H and I). In DD, increased sleep in NPVF-overexpressing animals was due to a 75% increase in sleep bout length compared to WT siblings (p<0.0001, Two-way ANOVA, Holm-Sidak test), with no effect on the number of sleep bouts (Figure 1—figure supplement 3O and P). Together, these results suggest that NPVF-induced sleep does not require circadian rhythms or depend on lighting conditions, although effects on sleep architecture are influenced by lighting conditions. These results indicate that the ability of RFamide neuropeptides to promote sleep is conserved in invertebrates (He et al., 2013; Iannacone et al., 2017; Lenz et al., 2015; Nath et al., 2016; Nelson et al., 2014; Shang et al., 2013; Trojanowski et al., 2015; Turek et al., 2016) and vertebrates. We next asked whether the C. elegans sleep-promoting RFamide FLP-13 is sufficient to affect behavior in zebrafish. To answer this question, we generated transgenic zebrafish in which expression of the C. elegans flp-13 open reading frame is regulated by a heat shock-inducible promoter. We found that overexpression of FLP-13 decreased locomotor activity by 26% compared to WT siblings (Figure 1—figure supplement 1B) (p<0.0001, Two-way ANOVA, Holm-Sidak test). However, FLP-13 overexpression did not significantly affect sleep (Figure 1—figure supplement 1C). These results are consistent with the notion that RFamide peptides have an evolutionarily conserved role in suppressing motor behaviors, even though the behavioral effect of overexpressing FLP-13 in zebrafish is weaker than that of its potential zebrafish homolog NPVF. Mature peptides derived from NPVF synergistically promote sleep In humans and zebrafish, npvf encodes for a preproprotein that contains three mature peptides (RFRP1-3) (Ubuka and Tsutsui, 2014). To determine whether individual mature peptides are sufficient to promote sleep, we generated transgenic zebrafish that contained heat-shock inducible transgenes in which the amino acid sequence of one or more mature peptides was scrambled to abolish its function (Figure 2). Heterozygous transgenic animals were mated to WT animals, and their heterozygous transgenic and WT larval progeny were assayed for locomotor activity and sleep phenotypes. Overexpression of a transgene in which all three peptides were scrambled had no behavioral effect (Figure 2D–F and N), indicating that one or more peptides are required for NPVF overexpression-induced sleep. Compared to their WT siblings, overexpression of transgenes containing only RFRP1 or RFRP3 decreased activity by 18% or 14%, respectively (both: p<0.0001, One-way ANOVA, Holm-Sidak test), but had no significant effect on sleep (Figure 2D, E, G, I, O and Q). Overexpression of RFRP2 (present in the human but not rodent npvf ortholog) had a stronger effect, decreasing locomotor activity by 56% and increasing sleep by 120% (activity and sleep: p<0.0001, One-way ANOVA, Holm-Sidak test) (Figure 2D, E, H and P). Compared to their WT siblings, overexpression of transgenes containing any combination of two RFRP peptides induced significantly larger activity and sleep phenotypes, similar to overexpression of WT NPVF, with >70% decreased locomotor activity and >200% increased sleep (activity and sleep: p<0.0001, One-way ANOVA, Holm-Sidak test) (Figure 2D, E, J–M and R–U). Figure 2 with 2 supplements see all Download asset Open asset Individual RFRP peptides act synergistically to promote sleep. (A) Schematic diagram of the NPVF preproprotein and its three RFRP peptides. (B) Eight heat shock-inducible transgenes containing all possible combinations of one, two or three scrambled RFRP peptides tested. Colors correspond to those used in panels (C–U). (C) Amino acid sequences of zebrafish Tg(hs:NPVF) transgenes containing single or multiple scrambled RFRP peptides. RFRP peptide sequences are predicted based on conservation with peptides isolated in other species and on the location of glycine amidation signals and lysine or arginine endoproteolytic cleavage sites. The C-terminus of each predicted peptide contains a LPXRF motif, where X = L or Q. Gray shading indicates scrambled sequences. Daytime activity (D) and sleep (E) of transgenic animals normalized to their WT siblings following afternoon heat-shock-induced transgene overexpression are shown as mean ± SEM for two pooled experiments, with at least 64 animals per genotype. Examples of activity and sleep data for each transgene are shown in (F–U). Yellow bars indicate heat shock. White and black bars under behavioral traces indicate day (14 hr) and night, respectively. n = number of animals. n.s. = not significant, ***p<0.0001 by One-way ANOVA compared to scrambled NPVF peptide control (black) using Dunnett’s multiple comparison test. See also Figure 2—figure supplements 1 and 2. https://doi.org/10.7554/eLife.25727.006 To test whether the behavioral effects induced by overexpression of two or more RFRP peptides is due to a dosage effect, we compared sleep in animals containing 0 (WT), 1 (heterozygous transgenic) or 2 (homozygous transgenic) copies of the hs:RFRP1, hs:RFRP3 or hs:RFRP1-3 transgenes. Following heat-shock induced overexpression, we observed no difference in locomotor activity or sleep amount between WT, Tg(hs:RFRP1)/+ and Tg(hs:RFRP1)/Tg(hs:RFRP1) animals (Figure 2—figure supplement 1A–D). Similarly, we observed no difference in amount of locomotor activity or sleep between Tg(hs:RFRP3)/+ and Tg(hs:RFRP3)/Tg(hs:RFRP3) animals (Figure 2—figure supplement 1F and H). We observed a small decrease in locomotor activity in Tg(hs:RFRP3)/Tg(hs:RFRP3) compared to +/+ animals (Figure 2—figure supplement 1E and G), but the effect was much smaller than that observed for Tg(hs:RFRP1,3)/+ compared to +/+ animals (Figure 2K and S). Finally, we compared +/+, Tg(hs:RFRP1-3)/+, and Tg(hs:RFRP1-3/hs:RFRP1-3) animals and observed that RFRP1-3 overexpression increased sleep and decreased locomotor activity to a similar extent in heterozygous and homozygous transgenic animals compared to +/+ siblings (Figure 2—figure supplement 1I–L). Taken together, these results suggest that the larger phenotypes observed following overexpression of 2 or more different RFRP peptides compared to single peptides is due to the synergistic action of these peptides rather than a dosage effect. To compare the effect of overexpressing different RFRP peptides on neuronal activity in the brain, we performed ISH using a probe specific for c-fos, which can be used as a marker of neuronal activity (Guzowski et al., 2005). One hour after heat shock, we observed robust induction of c-fos expression along the brain ventricular lining in transgenic animals that overexpressed WT npvf, whereas their non-transgenic siblings lacked c-fos expression in this region (Figure 2—figure supplement 2A–C). In several vertebrates, including fish, hypothalamic RFamidergic neurons have been described as cerebrospinal fluid (CSF)-contacting neurons (Castro et al., 2001), suggesting that activation of cells along the brain ventricle by RFRP peptides could drive behavioral state changes by inducing signals in the CSF to promote sleep, as has been described for several sleep regulators (Krueger et al., 2007). Consistent with the behavioral phenotypes induced by overexpression of different RFRP peptides, we observed robust ventricular c-fos expression in animals that overexpressed any combination of two RFRP peptides (Figure 2—figure supplement 2J and data not shown) or RFRP2 alone (Figure 2—figure supplement 2H), reduced c-fos expression in animals that overexpressed RFRP1 or RFRP3 (Figure 2—figure supplement 2G and I), and no c-fos expression in animals that overexpressed a transgene in which all three peptides were scrambled (Figure 2—figure supplement 2F). Taken together with the behavioral data (Figure 2 and Figure 2—figure supplement 1), these changes in c-fos expression suggest that NPVF overexpression-induced sleep results from synergistic effects of RFRP peptides. Loss of NPVF signaling results in reduced sleep We next tested the hypothesis that NPVF signaling is required for normal sleep levels. Using CRISPR/Cas9, we generated a zebrafish npvf mutant that contains a 7 bp deletion after the first amino acid of RFRP2 (Figure 3—figure supplement 1A). This mutation results in a shift in the translational reading frame and produces a protein that contains RFRP1 but lacks RFRP2 and RFRP3. Consistent with the NPVF overexpression phenotype, we found that npvf -/- animals were 27% more active and slept 10% less than their npvf +/+ siblings at night (sleep: p<0.05; activity: p<0.005, One-way ANOVA, Holm-Sidak Test) (Figure 3A–D). These effects were due to a 10% reduction in sleep bout length and a 19% increase in wake activity in npvf -/- animals compared to their npvf +/+ siblings (p<0.01 and p<0.005, respectively, One-way ANOVA, Holm-Sidak test) (Figure 3—figure supplement 1B–E). npvf +/- animals exhibited an intermediate sleep phenotype that was not significantly different from that of their npvf -/- siblings (night: p=0.3, One-way ANOVA, Holm-Sidak test) (Figure 3C and D). This result suggests that NPVF signaling is required for normal sleep levels. Figure 3 with 2 supplements see all Download asset Open asset Genetic and pharmacological inhibition of NPVF signaling increases locomotor activity and decreases sleep. (A–D) npvf -/- animals are more active during the day and night (A,B), and sleep less at night (C,D) compared to their npvf +/+ siblings. npvf +/- animals show an intermediate sleep phenotype that is not significantly different than npvf -/- siblings. inhibition of NPVF signaling by the and increased locomotor activity and decreased sleep during the day and night compared to siblings. Mean ± SEM from one representative experiment three pooled experiments or pooled experiments (B,D) are shown. White and black bars under behavioral traces indicate day (14 h) and night (10 h), respectively. n = number of animals. by One-way ANOVA with Holm-Sidak test (B,D) and See also Figure 3—figure supplements 1 and 2. the behavioral phenotype of npvf -/- animals, which lack NPVF is we used as an to NPVF We tested two pharmacological of NPFFR1 and the that bind RFRP1 and RFRP3 in (Hinuma et al., 2000; Liu et al., 2001). of WT animals with the et al., 2006) in more activity and less sleep than animals with control during both the day and night (p<0.0001, (Figure in sleep were associated with and sleep bouts at night, but only to sleep bouts during the day (Figure 3—figure supplement 1G and H). also significantly increased wake activity during the day and night and sleep latency after lights at night compared to (p<0.0001, (Figure 3—figure supplement 1F and I). Similarly, of WT animals with an additional of NPFFR1 and NPFFR2 et al., 2015), in significantly increased locomotor activity and decreased sleep during the day and night (p<0.0001, (Figure the effects of were smaller than those of had similar effects on sleep architecture as (Figure 3—figure supplement and These results suggest that NPVF signaling is required for both the and of normal levels of sleep during the day and night. To that and are by we tested whether these sleep induced by overexpression of the cognate of these In behavioral experiments, animals and their WT siblings were with either or control in overexpression induced sleep post-HS compared to in animals (Figure 3—figure supplement and In overexpression-induced sleep was post-HS compared to in animals with (Figure 3—figure supplement and and by (Figure 3—figure supplement 2G and H). These results are consistent with in data that RFRP1 and RFRP3 bind NPFFR1 and NPFFR2 (Hinuma et al., 2000; Liu et al., 2001). These data suggest that decreased sleep due to with these can be to inhibition of NPVF NPVF signaling affects arousal threshold Sleep is from quiet by an increased arousal which we assayed by of to zebrafish larvae while their behavior (Gandhi et al., We the of animals that to the at several and used this data to Based on these we calculated the at which the response We observed that the for NPVF-overexpressing larvae was than that of their WT siblings (Figure by test). the for or animals was or than that of their siblings, respectively (Figure and (both: by test). These results that NPVF overexpression increases arousal whereas inhibition of NPVF signaling decreases arousal Figure Download asset Open asset NPVF signaling affects arousal response generated using a in Tg(hs:NPVF) animals and their WT siblings following heat shock in and WT larvae and in and WT larvae data indicates mean ± the for each or (A) Tg(hs:NPVF) animals had an of for WT siblings by test). (B) animals had an of for siblings by test). (C) animals had an of for siblings by test). n = number of animals. of a genomic that expression in NPVF neurons In mammalian brains, npvf is enriched in the dorsomedial hypothalamus (Hinuma et al., 2000; Liu et al., 2001; Ubuka et al., 2009). Similarly, using ISH we found that npvf is in the zebrafish dorsomedial hypothalamus at (Figure similar to previous observations et al., 2017; et al., However, zebrafish larvae only have NPVF neurons per brain an of than the number of NPVF neurons in et al., 2012). Using in situ hybridization we observed that npvf-expressing neurons also the (Figure ± n = and ± n = and not the marker not n = suggesting that this neuronal is in We further identified a genomic region to the npvf open reading frame that is sufficient to drive expression of protein in npvf-expressing with ± = of cells npvf mRNA, and ± =