s2fieldsofstudy: Biology Psychology

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The tyrosine kinase receptor Tyro3 enhances lifespan and neuropeptide Y (Npy) neuron survival in the mouse anorexia (anx) mutation

ABSTRACT Severe appetite and weight loss define the eating disorder anorexia nervosa, and can also accompany the progression of some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Although acute loss of hypothalamic neurons that produce appetite-stimulating neuropeptide Y (Npy) and agouti-related peptide (Agrp) in adult mice or in mice homozygous for the anorexia (anx) mutation causes aphagia, our understanding of the factors that help maintain appetite regulatory circuitry is limited. Here we identify a mutation (C19T) that converts an arginine to a tryptophan (R7W) in the TYRO3 protein tyrosine kinase 3 (Tyro3) gene, which resides within the anx critical interval, as contributing to the severity of anx phenotypes. Our observation that, like Tyro3−/− mice, anx/anx mice exhibit abnormal secondary platelet aggregation suggested that the C19T Tyro3 variant might have functional consequences. Tyro3 is expressed in the hypothalamus and other brain regions affected by the anx mutation, and its mRNA localization appeared abnormal in anx/anx brains by postnatal day 19 (P19). The presence of wild-type Tyro3 transgenes, but not an R7W-Tyro3 transgene, doubled the weight and lifespans of anx/anx mice and near-normal numbers of hypothalamic Npy-expressing neurons were present in Tyro3-transgenic anx/anx mice at P19. Although no differences in R7W-Tyro3 signal sequence function or protein localization were discernible in vitro, distribution of R7W-Tyro3 protein differed from that of Tyro3 protein in the cerebellum of transgenic wild-type mice. Thus, R7W-Tyro3 protein localization deficits are only detectable in vivo. Further analyses revealed that the C19T Tyro3 mutation is present in a few other mouse strains, and hence is not the causative anx mutation, but rather an anx modifier. Our work shows that Tyro3 has prosurvival roles in the appetite regulatory circuitry and could also provide useful insights towards the development of interventions targeting detrimental weight loss. Summary: The receptor tyrosine kinase Tyro3 improves weight and survival in the anorexia mouse model, and is one of the few factors known to sustain appetite regulatory circuitry.

INTRODUCTION
Detrimental appetite and weight loss are the defining features of the restrictive subtype of the eating disorder anorexia nervosa (ANR) (Kaye et al., 1998(Kaye et al., , 2000Walsh and Devlin, 1998), and also often accompany a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) (Ahmed et al., 2016), Alzheimer's disease (AD) (Ringman et al., 2015;Burke et al., 2016;Sergi et al., 2013) and Huntington's disease (HD) (Petersén et al., 2005;Gabery et al., 2010). Eating disorders have the highest mortality rate of any mental illness (Sullivan, 1995). Moreover, appetite and weight loss are linked to poor mood and quality of life in ALS patients and can be major issues for many during late disease progression. In spite of their impact, the neurobiological systems (Kaye et al., 2013) and genetic factors (Shih and Woodside, 2016;Clarke et al., 2012;Pinheiro et al., 2009) that contribute to reduced food intake in ANR remain poorly understood and are likely complex, and have been largely unexplored in neurodegenerative disorders.
Neuronal circuitry that links the hypothalamus with extrahypothalamic regions, including the parabrachial nucleus and the nucleus accumbens, monitors peripheral energy levels and regulates food intake (reviewed in Sternson, 2013;Morton et al., 2006). Within the hypothalamus, orexigenic neurons co-express and release neuropeptide Y (Npy) (Allen et al., 1983;Chronwall et al., 1985;de Quidt and Emson, 1986;Hahn et al., 1998) and agouti-related protein (AgRP) (Hahn et al., 1998;Shutter et al., 1997) to promote feeding by inhibiting anorexic neurons expressing pro-opiomelanocortin (Pomc) (Bloch et al., 1978;Watson et al., 1978) and cocaine-and amphetamine-regulated transcript (Cart, also known as Cartpt) (Elias et al., 1998;Kristensen et al., 1998). Serotonin can act directly on 5HT2c receptors present in hypothalamic orexigenic neurons and 5HT1b receptors in anorexigenic neurons (Garfield and Heisler, 2009;Heisler et al., 2002Heisler et al., , 2006Lam et al., 2008;Xu et al., 2008). In the adult mouse, acute ablation of arcuate nucleus AgRP neurons leads to starvation (Luquet et al., 2005;Xu et al., 2005;Wu et al., 2008). AgRP neurons also project long-range axons to other brain regions, including the parabrachial nucleus (PBN), which can augment or suppress AgRP neuron actions. Further analyses in the mouse have shown that the severe aphagia caused by acute adult AgRP neuron loss can be reversed by eliminating glutamatergic excitatory drive or increasing GABAergic inhibition from the PBN or by inhibiting serotonergic activation of the nucleus of the solitary tract, which innervates and activates the PBN (Wu et al., 2012). Thus, in brief, loss of hypothalamic NPY/AGRP neurons, increased glutamatergic signaling from the PBN or serotonergic hyperinnervation of the nucleus tractus solitarius (NTS) could individually or together promote pathological appetite and weight loss.
Understanding how appetite regulatory circuits are involved or have been compromised in individual ANR cases, let alone in ANR populations, is largely unknown. Because prior to their diagnosis ANR patients have already been undernourished for an extended period of time, it is difficult to distinguish between the neurobiological causes and effects of ANR. Furthermore, imaging deeper brain regions such as the hypothalamus can be more challenging (Val-Laillet et al., 2015). Nonetheless, data concerning appetite regulating peptide levels in ANR patients have been obtained by assessing cerebrospinal fluid and plasma levels, and persistent changes in appetite regulating neuropeptides, such as Npy (Connan et al., 2007;Lob et al., 2003;Bronsky et al., 2011;Støving et al., 2009;Gendall et al., 1999;Kaye et al., 1990) and in the serotonin (Bailer et al., 2005;Galusca et al., 2008) neurotransmitter system have been found a long time after weight restoration in restrictive anorexia nervosa. With respect to neurodegenerative disorders, reduced expression of the orexigenic Npy and its receptor has been associated with Alzheimer's disease and other neurodegenerative disorders (Spencer et al., 2016;Davies et al., 1990;Minthon et al., 1990Minthon et al., , 1996Martel et al., 1990;Koide et al., 1995;Nilsson et al., 2001;Alom et al., 1990;Martignoni et al., 1992). Thus, in both ANR and neurodegenerative disorders the appetite regulatory system is detectably impacted, albeit in an as-yet poorly defined manner.
Just as in severe ANR cases, mice homozygous for the recessive mouse anorexia (anx/anx) mutation restrict their eating and become severely emaciated (Maltais et al., 1984). Thus, genetic analyses of the anx mutation might identify factors important for the maintenance of appetite regulatory circuitry and could provide insights towards the development of interventions targeting detrimental weight loss. In anx/anx mice, hypothalamic Npyexpressing (Npy+) neurons begin to degenerate at postnatal day (P)10-12 . By P19-22 most Npy+ processes and soma have disappeared, and anx/anx mice have become severely emaciated and perish (Maltais et al., 1984). In addition, 5-HT immunoreactive fibers innervate their target areas abnormally in anx/anx mice (Huynh et al., 2011;Son et al., 1994), and their body tremors, head weaving and hyperactivity can be improved by administration of 5-HT antagonists (Maltais et al., 1984). Currently, the primary molecular cause for neurodegeneration in anx/anx mice is unknown. Microarray-based analyses of P21 hypothalamic mRNA identified a 50% decrease in two genes: RNA polymerase II associated protein 1 (Rpap1) and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 1 (Ndufaf1), which reside in the anx critical interval . Ndufaf1 is a complex I assembly factor protein required for the first step of mitochondrial respiration. Lindfors et al. claimed that the 50% decrease in Ndufaf1 impacted mitochondrial respiration sufficiently to be the primary anx causative event. However, further experimental support for this proposed mechanism, such as detection of an anxspecific molecular lesion affecting Ndufaf1 or by genetic noncomplementation or transgenic rescue experiments, is lacking. Hence, the role of Ndufaf1 downregulation in anx phenotypes is debatable and the identity of the anx causative mutation or any other genetic factor that might contribute to its phenotypes is unknown.
Given the Npy+ neuron degeneration in anx/anx mice, we considered whether the TYRO3 protein tyrosine kinase 3 (Tyro3) gene, which has prosurvival roles in neuronal culture (Funakoshi et al., 2002;Zheng et al., 2009;Zhong et al., 2010) and resides within the anx critical interval, might contribute to anx phenotypes. Tyro3, along with its related Axl and Mer (also known as Mertk) receptors, is a member of the TAM family of receptor tyrosine kinases (reviewed in Hafizi and Dahlback, 2006;Linger et al., 2008;Lemke, 2013), and was previously also known as Rse (Mark et al., 1994), Sky (Ohashi et al., 1994), Brt (Fujimoto and Yamamoto, 1994), Tif (Dai et al., 1994), Dtk (Crosier et al., 1996), and Etk-2 (Biesecker et al., 1995). It is composed of a signal sequence, two immunoglobulin-like domains and two fibronectin type III repeats in its extracellular domains, a transmembrane domain and an intracellular tyrosine kinase domain. Upon activation by its ligands growth-arrest specific 6 (Gas6) or protein S, Tyro3 initiates homophilic dimerization, cross-phosphorylation, and downstream signaling through the PI-3K/AKT or Ras/ERK signaling pathways (reviewed in Linger et al., 2008;Lemke, 2013;Pierce and Keating, 2014). Tyro3 is highly expressed in neurons (Funakoshi et al., 2002;Lai et al., 1994;Ohashi et al., 1994;Prieto et al., 2000). By contrast, Axl is expressed in the rostral migratory stream, whereas Mer mRNA expression is concentrated in glia (Ji et al., 2013). In mice (Duncan et al., 2003;Prasad et al., 2006;Maddox et al., 2011) and humans (Ostergaard et al., 2011;Shahzadi et al., 2010;Mackay et al., 2010;Brea-Fernandez et al., 2008), mutations in Mer cause retinal degeneration, but no other neurobiological phenotypes have been otherwise reported for mice lacking either Axl or Mer alone. However, in mice lacking both Axl and Tyro3 the migration and survival of gonadotrophin release hormone (GnRH)-expressing neurons into the hypothalamus is compromised (Pierce et al., 2008). Moreover, loss of Tyro3 accelerates retinal degeneration in Mer −/− mice (Vollrath et al., 2015). Triple Tyro3/Axl/Mer mutants exhibit cellular degeneration in the neocortex, hippocampus, cerebellum, and of rods and cones in the retina (reviewed in Ji et al., 2015). These studies illustrate the redundant reinforcing neuroprotective roles of TAM receptors, and open the door to the possibility that Tyro3 on its own might have significant neuroprotective potential.
Here, we identify a point mutation (C19T) in the signal sequence of Tyro3 (R7W-Tyro3), and detect a deficit in secondary platelet aggregation in anx/anx mice that resembles one previously reported in mice lacking Tyro3. We show that Tyro3 is expressed in brain regions known to be affected by the anx mutation: the hypothalamic arcuate nucleus, the dentate gyrus and serotonergic neurons, and detect anomalies in RNA localization in anx/anx brains at P19. In the presence of wild-type Tyro3 transgenes, but not of a C19T mutated Tyro3 transgene (referred to here as R7W-Tyro3), the weight and lifespans of anx/anx mice was doubled relative to that of their non-transgenic anx/anx littermates and hypothalamic Npy+ neuron degeneration was delayed. Finally, we show that in the cerebella of transgenic mice, R7W-Tyro3 protein appeared mislocalized. Additional sequencing analyses revealed that the R7W-Tyro3 variant is present in AKR/J, FVB/NJ and NZW/lacZ mice, and thus R7W-Tyro3 is not the primary anx causative event, but rather is a strain-specific modifier of anx phenotypes. Taken together, these studies identify Tyro3 as having important neuroprotective roles in the appetite regulatory circuitry.

RESULTS
A point mutation in the signal sequence of Tyro3 is associated with the anx phenotype The Tyro3 gene is located within the anx critical interval, which we had refined to a 3.5 Mb region between D2Mit484 and D2Mit3 by using simple sequence length polymorphisms (SSLPs) to analyze 335 progeny (670 meioses) from heterozygous intercrosses on C57BL6/J (n=111) and Mus molossinus/Ei (MOLF/EiJ) (n=224) strain backgrounds (Fig. S1). Upon sequencing the exons and flanking intronic regions of Tyro3, we identified a cytosine-to-thymine (C19T) mutation in the signal sequence of Tyro3 (Fig. 1A). This mutation converts an arginine to a tryptophan at the seventh amino acid position, and also eliminates an NlaIV restriction site (Fig. 1B), which we used in a PCR-based assay to genotype 995 additional animals. All 176 affected progeny were homozygous for the R7W-Tyro3 mutation, whereas 819 unaffected animals were R7W-Tyro3/+ or +/+ (Fig. 1C). This sequence variant was absent in seven other inbred strains (C57BL6/J, C3H/HeJ, Balbc/J, MolfEi/J, CastEi/J, A/J, 129Sv/J) analyzed at the time. We identified two additional single nucleotide polymorphisms that led to nonsynonymous changes (SNP rs47863852: Val811Leu and SNP rs13459232: Gly824Ser, ENSEMBL build GRCM38), but these were present in Tyro3 from some of the other strains sequenced here. Thus, we decided to test whether the R7W-Tyro3 variant might affect anx phenotypes.
anx/anx mice exhibit platelet deficits similar to those reported in Tyro3 nulls As a first step to test whether the R7W-Tyro3 variant might have functional biological consequences, we tested whether anx/anx mice exhibit deficits in platelet aggregation, as had been observed in Tyro3 −/− mice (Angelillo-Scherrer et al., 2005;Gould et al., 2005). When we examined platelet aggregation in anx/anx, anx/+ and +/+ mice, we found that, upon activation with thrombin, P-selectin levels on the platelet surface were significantly reduced in anx/anx platelets ( Fig. 2A-D). In platelets lacking Tyro3, Axl or Mer, reductions in P-selectin levels were observed upon ADP stimulation. The effect of thrombin on P-selectin levels has not been reported for Tyro3 −/− platelets, nor did we examine the effect of ADP-stimulation on anx/anx platelets to further delineate the parallels of these aggregation deficits. Electron microscopy revealed that anx/anx platelet morphology appears normal, and dense and alpha granules are present in equivalent numbers per platelet surface area (Fig. 2E,F). However, anx/anx platelets appear smaller than +/+ and anx/+ platelets (P<0.001) and 17.5±5.9% (mean±s.e.m.) (57-63 platelets per replicate; n=4) of anx/anx platelets contained large vacuoles, which were never observed in +/+ or anx/+ platelets (Fig. 2F). Given the presence of these platelet deficits in anx/anx mice, we pursued the possibility that R7W-Tyro3 might further contribute to anx phenotypes.
Tyro3 expression in areas affected by the anx mutation and in anx/anx mutant brains Next, we examined Tyro3 expression in wild-type and anx/anx brains. Overall levels of wild-type and mutant Tyro3 RNA were not affected, as determined by qRT-PCR and northern blot analyses performed on +/+ and anx/anx brains at P21 (Fig. S2A). Previously, Tyro3 mRNA had been detected in the subventricular zone and layers 2/3, 5 and 6 of the cerebral cortex, the CA1 region of the hippocampus, the median eminence of the hypothalamus, and granule cells of the cerebellum in the adult mouse brain (Lai et al., 1994;Schulz et al., 1995;Gely-Pernot et al., 2012). Deficits in these regions have not been reported in anx/anx mice. Using RNA in situ hybridization on P10 and P21 wild-type brains, we confirmed Tyro3 expression in these regions (Fig. 3A,E,Q; Fig. S3), but also detected Tyro3 mRNA in areas affected by the anx mutation. Tyro3 was strongly expressed in the arcuate nucleus, ventromedial nucleus and, at lower levels, in the paraventricular nucleus of the (A-C) The effect of (A) +/+, (B) anx/+ and (C) anx/anx platelets on CD62 (P-selectin) expression, a marker for α-granule secretion, was detected using flow cytometry. After activation with thrombin, CD62 levels on the platelet surface were significantly reduced in (C) anx/anx platelets. Black lines represent resting platelets and red shading denotes thrombin-stimulated platelets. Representative examples of at least four independent experiments are shown. (D) The percentage of secondary platelet aggregation in +/+ and anx/+ animals was 56.4±3.2% (n=17) and anx/anx animals 23.9±9.8% (n=6) (Mann-Whitney Test, P=0.026). Data represented as mean±s.e.m. (E,F) Electron microscopy analyses revealed that (F) anx/anx platelets appeared normal, but often contain large vacuoles (indicated by asterisks) absent in (E) +/+ platelets. The abnormal vacuoles were observed in 17.5± 5.9% of anx/anx platelets (counted 57-63 platelets per replicate (n=4). In mice carrying the anx mutation, a C-to-T mutation in Tyro3 converts an arginine-to-tryptophan at amino acid 7 in Tyro3 protein (R7W-Tyro3) and (B) eliminates an NlaIV site. (C) Mice with the anx phenotype are homozygous for R7W-Tyro3 (n=176), whereas R7W-Tyro3/+ (n=584) and +/+ (n=235) mice appear normal.
hypothalamus and in ependymal cells lining the third ventricle (Fig. 3I). In the hippocampus, Tyro3 was expressed in CA1, but also CA3 and, in the dentate gyrus, intensely in the molecular layer and in distinct cells within the granule cell and polymorphic layers (Fig. 3E). Tyro3 mRNA was present in neuronslikely 5-HT oneslocated within all raphe nuclei (Fig. 3M). In the cerebellum, Tyro3 expression was seen in granule and Purkinje cells, faintly outlining basket and stellate cells, and in processes in the molecular layer (Fig. 3Q,R).
At P10, the expression pattern of Tyro3 appeared equivalent throughout the brains of anx/anx and +/+ animals (data not shown). However, at P21, anx/anx brains showed marked differences in Tyro3 expression. At low magnification, mutated (C19T) Tyro3 expression appeared overall more diffuse in specific cell populations and throughout the brain, and mislocalized in some regions (Fig. 3B,F,J, N,S). Most noticeably, in the dentate gyrus, mutant Tyro3 was highly expressed in the molecular layers of +/+ and anx/anx mice, but In anx/anx mice, C19T-Tyro3 RNA localization appears disorganized in the soma of cortical neurons. In the hippocampus of (E) +/+ and (F) anx/ anx mice, Tyro3 RNA is present in CA1, CA3, and the dentate gyrus (DG). (G) At higher magnification, Tyro3 is expressed in cells within the granule cell and polymorphic layers of the +/+ DG. (H) In anx/anx, only a few cells in the granule cell and polymorphic layers of the DG express C19T-Tyro3. (I) In the +/+ hypothalamus, Tyro3 is expressed in the median eminence, arcuate nucleus (ac), and ventromedial hypothalamus (VMH), and at lower levels in the paraventricular nucleus of hypothalamus (PVN). (J) In the anx/anx hypothalamus, C19T-Tyro3 expression appeared so diffuse at low magnification that it is difficult to distinguish it in specific subregions. At higher magnification, (K) in the +/+ arcuate nucleus, Tyro3 RNA is localized in processes emerging from neuronal soma, but (L) in the anx/anx arcuate nucleus, C19T-Tyro3 RNA localization appears disorganized. Tyro3 is expressed in neurons within the dorsal raphe nuclei of (M) +/+ and (N) anx/anx mice. At high magnification, Tyro3 RNA is seen at the edges of (O) +/+ neuronal soma and in their emerging processes, but appears disorganized in (P) anx/anx neurons within the raphe nuclei. (Q) In the +/+ cerebellum, Tyro3 is expressed in granule cells and Purkinje cells, shown at higher magnification in (R) (marked with arrows). In the cerebellar molecular layer, Tyro3-expressing processes can be seen at high magnification. Tyro3 RNA faintly outlines the soma of cellslikely cerebellar stellate and basket cells (marked with arrowheads in R). (S) In anx/anx cerebella, C19T variant Tyro3 RNA is present in granule and Purkinje cells. (T) At higher magnification, mutant Tyro3-positive cell bodies (marked with arrowheads), which are likely stellate or basket cells, can be seen in the molecular layer. Mutant Tyro3-expressing processes are not apparent. Asterisks in A,B,E,F,I,J,M, N,Q and S indicate regions shown at higher magnification directly below each respective panel in C,D,G,H,K,L,O,P,R and T. relative to +/+ littermates (Fig. 3E,G), the granule and polymorphic layers from anx/anx mice contain very few cells expressing C19Tmutated Tyro3 (Fig. 3F,H). At higher magnification, subcellular localization of the mutated mRNA appeared aberrant in P21 anx/anx brains. Generally, Tyro3 mRNA appeared excluded from the nucleus, concentrated instead to regions within the soma from which processes were extending and, at times, could be detected in processes. In anx/anx neurons, C19T variant Tyro3 RNA appeared concentrated in soma and was often seen in aggregates (Fig. 3D,H,L, P). For example, in wild-type cerebella, Tyro3 mRNA was seen in processes in the molecular layer and only faintly outlining presumptive stellate and basket cell neurons (Fig. 3Q,R). However, in anx/anx cerebella the mutant Tyro3 mRNA was detected only minimally in processes and was concentrated in the soma of presumptive basket and stellate cells (Fig. 3S,T). In summary, Tyro3 was expressed in all neuronal populationsspecifically the arcuate nucleus of the hypothalamus, the dentate gyrus of the hippocampus and presumptive serotonergic neurons in the raphe nucleifor which anomalies have been reported in anx homozygotes, and the localization of C19T variant Tyro3 RNA seemed to be affected throughout the brains of P21 anx/anx mice.

Rescue of anx phenotypes by Tyro3 transgenes
Next, we performed transgenic rescue experiments. Because phylogenetic sequence comparisons between Tyro3 non-coding regions showed highest conservation within the first 9.5 kb upstream of exon 2c, we postulated that these regions might suffice to direct transgene expression in endogenous Tyro3expressing domains, and used these regions from C57BL/6J genomic DNA to generate the T3Xpressn vector (Fig. 4A). We generated three varieties of transgenic mice: five lines expressing mouse Tyro3 protein C-terminally tagged with GFP (Tyro3-GFP), a line expressing mouse R7W-Tyro3 C-terminally tagged with GFP (R7W-Tyro3-GFP) and three lines expressing untagged human TYRO3 (HuTyro3) (Fig. 4B). The R7W-Tyro3 expression constructs were identical to the normal Tyro3 cDNA constructs except for the C19T polymorphism. GFP-tagged lines can facilitate tracking protein localization, but the GFP tag might compromise protein function. Untagged human TYRO3 should have full protein function, and its RNA can be distinguished from endogenous murine Tyro3. Tyro3-GFP and R7W-Tyro3-GFP protein localization in P21 +/+ brain regions recapitulated endogenous Tyro3 RNA expression, as shown for the hypothalamus (Fig. S4) and serotonergic neurons (Fig. S5). In the arcuate nucleus and median eminence, a subset of Tyro3-GFP-and R7W-Tyro3-GFPexpressing neurons co-express Npy (Fig. S4). In the hindbrain raphe nuclei, Tyro3-GFP-and R7W-Tyro3-GFP-expressing neurons express the 5-HT marker tryptophan hydroxylase 2 (Tph2) (Zhang et al., 2004), thus identifying them as 5-HT neurons (Fig. S5). +/+ and anx/+ mice transgenic for HuTyro3 or Tyro3-GFP were indistinguishable from non-transgenic +/+ and anx/+ littermates. Tyro3 transgenic wild-type mice showed no adverse effects with regards to viability, fertility and overall health.
Notably, all anx/anx mice transgenic for Tyro3-GFP or HuTyro3 showed mild or no neurobiological anx-related behaviors such as ataxia, shaking and headweaving at P21, and survived at least until P35, as shown for all Tyro3-GFP and HuTyro3 transgenic lines ( Fig. 4C; P<0.01). By contrast, the R7W-Tyro3-GFP transgene, even though expressed at levels similar to some of the rescuing Tyro3 transgenes (Fig. S2B), could not delay onset of the behavioral phenotypes by P12-15 or extend the lifespans of anx/anx mice past P21 (Fig. 4C).
Because Npy+ neuron degeneration has been linked to appetite deregulation in anx/anx mice , we examined the effects of the Tyro3-GFP transgene on Npy+ neurons in anx/anx mice. Normally, Npy+ neurons are found in the arcuate nucleus and innervate the parvocellular and magnocellular paraventricular nucleus of hypothalamus (PVN) and dorsomedial nucleus of the hypothalamus, as detected by silver-enhanced immunoperoxidase staining (Fig. 5A,D). At P19, the percentage of Npy+ soma present in the arcuate nucleus of anx/anx mice had been reduced to 20.2± 1.8% relative to wild-type littermates, whereas in Tyro3-GFP#1; anx/anx mice 83.58±7.9% were still present. Moreover, at P19, Npy-immunoreactive cell bodies are consistently clustered aberrantly within and adjacent to the median eminence in anx/anx mice. In all anx/anx mice examined no punctate Npy staining suggestive of Npy localization in processes could be seen extending out of the arcuate nucleus towards the PVN or dorsomedial nucleus of the hypothalamus (Fig. 5B,E). In Tyro3-GFP#1; anx/anx mice, Npy+ soma were distributed throughout the arcuate nucleus and punctate Npy staining suggestive of Npy+ processes extended towards and into the PVN as in +/+ mice (Fig. 5C,F). However, qualitatively, Npy immunoreactivity still appeared stronger in Tyro3-GFP; anx/anx neuronal soma than in those of +/+ mice. Thus, in the presence of the Tyro3-GFP transgene, more Npy+ neurons and Npy+ processes can be detected within the arcuate nucleus in P19 anx/anx mice an observation consistent with a delay in neurodegeneration. This increase in Npy-containing processes and hence Npy signaling is likely responsible for the increased weight gain seen in anx/anx; Tyro3-GFP mice. In summary, rescue of these anx phenotypes by HuTyro3 and Tyro3-GFP transgenes, but not the R7W-Tyro3 transgene, suggest that the C19T Tyro3 mutation might contribute to the anx phenotypes.

Effects of the R7W-Tyro3 variant
To understand the possible consequences of the C19T mutation on Tyro3 localization and function, we examined whether the mutation impinged on signal sequence function in vitro. Point mutations within signal sequences can impact protein secretion and function in a variety of ways. The Signal P program (www.cbs.dtu.dk/services/ SignalP) (Bendtsen et al., 2004), which examines amino acid sequences for characteristics of protein signal sequences (Bendtsen et al., 2004), did not predict that R7W-Tyro3 would substantially alter the signal sequence cleavage site (Fig. S6). Signal sequence mutations can also affect translocation from the cytoplasm to the endoplasmic reticulum and post-translational modifications, including N-glycosylation, that occur during protein secretion. In accordance with predictive analysis, when we assessed proteolytic processing of in vitro transcribed and translated Tyro3 and R7W-Tyro3 proteins mixed with signal peptidase from canine pancreatic rough microsomes, the extent of signal sequence cleavage and glycosylation of the R7W-Tyro3 protein seemed equivalent to that of Tyro3 (Fig. S7). Thus, the C19T mutation in Tyro3 does not impact signal sequence function in vitro.
In the presence of GAS6, an established Tyro3 ligand present in serum, Tyro3 activation leads to Akt phosphorylation (Lan et al., 2000;Zhong et al., 2010). Therefore, we examined whether serumdependent Akt phosphorylation was affected in vitro and in vivo. We detected no differences in Tyro3-or R7W-Tyro3-dependent Akt phosphorylation in the absence or presence of serum in transiently transfected COS7 cells, in stably transfected Neuro2A cells or in protein extracts from +/+ and anx/anx cerebella or hippocampi ( Fig. 6 and data not shown). In the absence or presence of serum, immunoprecipitation of Tyro3-RFP and R7W-Tyro3-RFP using an anti-RFP antibody and subsequent immunoblot analysis with an anti-phosphotyrosine antibody also revealed no detectable differences in tyrosine phosphorylation (Fig. 6). Thus, signaling function does not appear to be overtly compromised by the C19T Tyro3 mutation.
When we examined whether Tyro3-GFP and R7W-Tyro3-GFP protein localization differed in transiently transfected Neuro2A, COS7 and HEK293T cells or in stably transfected Neuro2A cells, we found that in all instances their subcellular localization appeared indistinguishable (Fig. S8 and data not shown). Using Fig. 4. anx/anx lethality is delayed by Tyro3 transgenes, but not by an R7W-Tyro3 transgene. (A) Tyro3 transgenes were constructed using a 9.5 kb BamHI-NotI fragment upstream of the translational start site located in exon 2c (adapted from Biesecker et al., 1995). The signal sequence of TYRO3 is indicated in black. (B) The T3Xpressn vector was used to generate transgenic mice expressing mouse Tyro3-GFP, R7W-Tyro3-GFP and human TYRO3 (HuTyro3). The number of independent founder lines generated and analyzed is shown for each transgenic construct. (C) Analyses of survival of transgenic and non-transgenic anx/anx progeny. All anx/anx homozygous mice die by P21. In all mouse lines transgenic for Tyro3-GFP or huTyro3, transgenic anx/anx progeny survived past P35. anx/anx mice transgenic for R7W-Tyro3-GFP did not survive past P21. (D-F) Effect of Tyro3 transgenes on bodyweight. For (D) Tyro3-GFP line #1, (E) HuTyro3 line #1, and (F) the sum total of all HuTyro3 and Tyro3-GFP transgenic lines, transgenic anx/anx mice weigh more than non-transgenic littermates at P21. Groups with significant differences relative to each other are indicated with brackets and a single asterisk for anx/anx versus anx/anx; Tyro3 transgenic; # for +/+ and anx/+ vs anx/anx, andf or +/+ and anx/+ vs anx/anx; Tyro3 transgenic. P<0.01 for all groups by unpaired t-one-way ANOVA data represented as mean±s.e.m.
immunofluorescence with an anti-Tyro3 antibody on cultured cortical and hippocampal neurons from anx/anx and +/+ littermates, endogenous R7W-Tyro3 and Tyro3 localization also appeared indistinguishable (Fig. S9). However, when we used silverenhanced immunohistochemistry with an anti-GFP antibody to survey Tyro3-GFP and R7W-Tyro3-GFP protein distribution in the brains of P10 transgenic +/+ mice, we detected distinctly different localization patterns in cerebella (Fig. 7). At P10, Tyro3-GFP; +/+ and R7W-Tyro3-GFP; +/+ Purkinje cells appear normal, as determined by calbindin immunofluorescence. Tyro3-GFP protein was distributed in tiny puncta throughout the molecular layer of transgenic +/+ cerebella (Fig. 7B,B′). However, R7W-Tyro3-GFP protein was predominantly found in Purkinje cell soma and in the major shafts extending from the soma in large, sparse puncta (Fig. 7D,D′). The stereotypic organization and large dendritic arbors of Purkinje cells facilitated detection of this mislocalization, which was otherwise not obvious in other brain regions. Thus, the R7W-Tyro3 variant causes protein mislocalization in the postnatal cerebellum.

R7W-Tyro3 is a strain-specific modifier of the anx phenotype
The anorexia mutation arose spontaneously on a mixed Swiss Webster/Mus poschiavinus background (Maltais et al., 1984) and has subsequently been maintained on a C57Fe/a background. Recently whole genome sequence has become available for 17 inbred mouse strains in addition to C57BL/6J (Yalcin et al., 2012;Keane et al., 2011;Wong et al., 2012). We found that the R7W-Tyro3 variant is also present in FVB/NJ, AKR/J and NZW/lacJ strains, which are related to the Swiss Webster strain. As none of these strains exhibit anx-like phenotypes, the R7W-Tyro3 variant cannot be the primary genetic lesion responsible for anx phenotypes. All three of these strains also share other sequence variations, such as the deletion of a U6 pseudogene in the Tyro3 promoter region (Fig. 4). This pseudogene is present in all Tyro3 and R7W-Tyro3 transgenic constructs, and hence does not contribute to the rescue of anx phenotypes observed in our Tyro3 transgenic lines. All of these observations indicate that the anx mutation arose on an ancestral Swiss Webster-related background and that Tyro3 acts as a strain-specific linked modifier of anx phenotypes.

DISCUSSION
Our study reveals Tyro3 as the first validated, linked gene that contributes to anx phenotypes and can delay hypothalamic neurodegeneration in anx/anx mice. As aforementioned, previous microarray analysis of the degenerating arcuate nucleus from P21 anx/anx mice  revealed twofold differences in the levels of Ndufaf1 and Rpap1. We found that cDNAs of both Ndufaf1 and Rpap1 are 100% identical to those of AKR/J mice. We did find a shared intronic SINE insertion within the Ndufaf1 gene and a 222 bp deletion located 1296 bp upstream of the Rpap1 transcriptional start site in both anx/anx and AKR/J mice and these differences could conceivably interfere with Ndufaf1 and Rpap1 transcription, respectively. Furthermore, whereas mitochondrial dysfunction can lead to neurodegeneration, abnormal mitochondrial respiration and localization can also be a consequence of neurodegeneration (Martin, 2012). Thus, although it remains a possibility that decreases in Rpap1 and/or Ndufaf1 levels might influence anx phenotypes, in contrast to Tyro3, at present any direct experimental support for the involvement of either is lacking.
Our findings reveal roles for Tyro3 in maintaining Npy+ neurons within the appetite regulatory circuitry. Unlike anx/anx mice, no abnormalities in size or appetite have been reported for Tyro3 −/− or TAM −/− mice. A series of studies in which AgRP neurons were ablated at different times and over varying timeframes might help explain why anx/anx mice show acute hypothalamic deficits and why, if such deficits are present in Tyro3 −/− or TAM −/− mice or other mouse mutants, they might have been missed. AgRP/Npy+ neuron ablation that occurs acutely at birth or gradually over a period of a few months leads to only mild decreases in weight (Bewick et al., 2005;Xu et al., 2005). By contrast, acute AgRP/Npy+ neuron ablation in adult mice (Gropp et al., 2005;Luquet et al., 2005) or in anx/anx mice starting at P10-P12, a time by which arcuate nucleus neuron projections have formed functional connections with other hypothalamic regions (Bouret et al., 2004), inhibits feeding behavior and causes starvation. Thus, appetite regulatory circuitry can adjust and compensate for gradual adult or acute neonatal loss, and gradual Npy+ neuron loss in Tyro3 −/− or TAM −/− mice would have gone unnoticed. Considering that other genes within the anx critical interval do not play known roles in Tyro3 signaling, it seems likely that Tyro3 boosts neuronal survival in general rather than by interacting specifically with the as-yet-unknown primary anx causative gene.

Tyro3 and restrictive anorexia nervosa
To date no unequivocal, well-replicated genes or loci associated with ANR have been identified in humans (reviewed in Yilmaz et al., 2015). With respect to neurotrophic factors and tyrosine kinase receptor signaling, candidate gene approaches have obtained mixed results for linkage of the rs6265 (V66M) polymorphism in brain-derived neurotrophic factor (BDNF) with ANR (Dardennes et al., 2007;Ribasés et al., 2005Ribasés et al., , 2004Ando et al., 2012;Brandys et al., 2013;Rybakowski et al., 2007). In the case of appetite-regulating neuropeptides, so far missense mutations in POMC have been reported in one individual with ANR, but no associations with common variants have been detected in ANR populations (Yilmaz et al., 2014). Furthermore, several genome-wide linkage studies found association of the serotonin receptor 1D and opiod receptor delta 1 with ANR (Bergen et al., 2003;Brown et al., 2007). Considering that body mass index remains low in recovered individuals with ANR, and ANR predominantly affects females, a genome-wide association meta-analysis cross-referencing these traits uncovered possible associations with, amongst other genes, CTBP2 and NBEAL1 (Hinney et al., 2016). Both CTBP2 and NBEAL1 show altered hypothalamic expression in response to fasting and diet-induced obesity and are postulated to act upon or at synapses. Finally, epoxide hydrolase 2 (EPHX2) emerged as a candidate for ANR associated with increased depression and anxiety in a high-throughput sequencing study (Scott-Van Zeeland et al., 2014). The paucity of known genetic factors likely reflects the heterogeneity among ANR cases and the genetic complexity of ANR specifically and psychiatric disorders in general. Because onset of ANR is accompanied by a range of psychiatric manifestations, including anxiety, body dysmorphic disorder, and often heightened sensitivity to negative stimuli, which are not of hypothalamic origin, and because these often persist to some degree even upon recovery, it seems unlikely that (E) Serum-activated tyrosine phosphorylation of Tyro3-GFP and R7W-Tyro3-GFP seems equivalent in transiently transfected COS7 cells. In the absence and presence of serum, immunoprecipitation of Tyro3-GFP and R7W-Tyro3-GFP was carried out with an anti-GFP antibody. Subsequent analysis with an anti-phosphotyrosine antibody revealed no differences in tyrosine phosphorylation as indicated by equivocal banding patterns. *P<0.05, **P<0.01 by unparied t-test; data represented as mean±s.e.m.
hypothalamic degeneration serves as an initiating event in ANR. Nonetheless, should any hypothalamic neurodegeneration occur in ANRpossibly as a secondary effectnormal Tyro3 signaling might offset this and perhaps improve chances of recovery.

Tyro3 and appetite dysregulation in neurodegenerative disorders
Whether hypothalamic degeneration accompanies the ingestive dysfunctions observed in some patients with neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), which, in familial cases, is often caused by mutations in Cu/Zn superoxide dismutase (SOD1) or hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), is unknown. The observation that ALS shows great variability in disease onset and progression even within families (Penco et al., 2011;Fogh et al., 2007;Kim et al., 2007) has fueled the pursuit of genetic disease modifiers in humans, mice, flies and yeast (Couthouis et al., 2011;Zhang et al., 2015;reviewed in Casci and Pandey, 2015;Paul and Gitler, 2014). As TAM receptors are unique to chordates, Tyro3 would not have been identified in invertebrate or yeast-based screens. A few receptor tyrosine kinase (RTK) signaling pathways, which promote cell survival and act protectively on motor neurons both in in vivo and in vitro ALS models, have been uncovered and include glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and insulin-like growth factor 1 (IGF-1) (Tovar et al., 2014;Kasai et al., 2011;Riboldi et al., 2011). Moreover, for variants in the VEGFA and EphA4 RTK signaling pathways, findings from animal models align well with their linkage to disease severity in individuals with ALS (Lambrechts et al., 2003;Van Hoecke et al., 2012). Given our findings, it might be warranted to examine whether Tyro3 variants are linked with the occurrence and severity of ingestive dysfunction in neurodegenerative disorders or, for that matter, with the general onset and severity of ALS or other degenerative disorders in general.

Tyro3 as a strain-specific modifier
In some instances, some of the other mouse strains that carry the R7W-Tyro3 variant have shown enhanced vulnerability to neurodegenerative disorders. For example, mice, which lack the SMN1 RNA binding protein and act as a model for human spinal muscular atrophy (SMA), survived twice as long on a pure C57BL/ 6N background than on the R7W-Tyro3-carrying FVB/N background (Ackermann et al., 2013). Moreover, mice lacking the amyotrophic lateral sclerosis 2 (Als2) gene exhibited a shorter life span than wild-type littermates when they were on a FVB/N than on a C57BL6/J background (Hadano et al., 2010). Thus, the R7W-Tyro3 variant might be one of the factors that contribute to increased disease susceptibility in these instances.

Possible consequences of the R7W-Tyro3 variant
Although bioinformatic analysis did not predict an appreciable impact on Tyro3 processing and we did not observe dramatic changes in the glycosylation state of the extracellular domain or localization of the mutant protein in cell culture, the R7W-Tyro3 variant appears to have consequences in vivo. Often, the biochemical effects of signal sequence mutations that are associated with human disorders correlate well with the disease state, as shown for prepro-parathyroid hormone (PTH) (Arnold et al., 1990). However, sometimes, even though a human disease is apparent, signal sequence function seems normal in in vitro assays. For example, a subtle mutation in the signal sequence of the cell surface calcium-sensing receptor (CASR) resulted in less protein at the plasma membrane, but the in vitro co-translational processing of the CASR mutant protein was unaffected (Pidasheva et al., 2005). Nonetheless the individual carrying this mutation, along with others carrying more dramatic mutations in the signal sequence of CASR, suffered from hypocalciuric hypercalcemia. Another example is a mutation in the signal sequence of thyroxin-binding globulin (TBG), which is associated with a partial deficiency of TBG-Allentown. Even though the Signal P program predicted no change in cleavage site, TBG was lowered in the blood of individuals with this deficiency (Fingerhut et al., 2004). Thus, C19T mutation in Tyro3 might compromise signal sequence function subtly in general or significantly in specialized, long-lived cell types, such as neurons.
Another possibility for the apparent lack of impact of the R7W-Tyro3 variant on in vitro signal sequence function is that it impacts RNA-sequence-based functions. Although, conventionally, the main roles of signal sequences have been based on their peptide sequences, the RNA sequence of the signal sequence coding region of some secretory proteins can activate nuclear mRNA export (Palazzo et al., 2007). Consistent with the possibility of an RNAbased mechanism, comparison of all sequenced Tyro3 orthologues revealed that the cytosine mutated in the C19T Tyro3 mutation is invariant and located within a larger conserved 21 nucleotide sequence. Current programs designed to identify RNA binding protein sites (RBPDB: http://rbpdb.ccbr.utoronto.ca/index.php and RegRNA: http://regrna.mbc.nctu.edu.tw/html/prediction.html) did not score disruption or creation of a binding site for a known RNA binding protein (RBP) by the C19T Tyro3 mutation. Moreover, the RNA binding profiles for some neuronally important RNA binding proteins (RBPs), such as FMRP1 (Miyashiro et al., 2003;Darnell et al., 2001Darnell et al., , 2005, Nova (Ule et al., 2003), TDP-43 (Sephton et al., 2011), RbFox1 (Lee et al., 2016) and SRRM4/nSR100 (Irimia et al., 2014), have begun to be determined, but none have recovered wildtype Tyro3 mRNA. Endogenous Tyro3 protein is localized in neuronal processes (Fig. S9) (Prieto et al., 2007). Many regulatory elements known to govern localization of neuronal mRNAs, such as BDNF  and Arc (Rao et al., 2006), reside in 3′ UTRs. However, the transgenic Tyro3 expression constructs contain only Tyro3 coding sequences and 154 nt of the 5′UTR, and the C19T Tyro3 mutation resides near the translational start site. Taken together, these observations might be more in line with a disruption of translational regulation. Given that we could only observe protein and possible RNA mislocalization in vivo and not in culture, neuron-specific RNA-binding and/or translational regulatory factorspossibly ones dependent on neuronal activitymight be involved, and these might either be absent in cell culture or not have been activated under normal culture conditions. However, although an intriguing speculation, it is currently unknown whether an RNAbased mechanismand perhaps a novel onemight play a role in R7W-Tyro3 mislocalization.
Regardless of mechanism, the mislocalization of R7W-Tyro3-GFP protein in the cerebella of wild-type mice does suggest that the R7W variant causes protein mislocalization in vivo and that, given that these mice appear normal, this effect is not due to overtly compromised neuronal health. The molecular mechanism by which the R7W-Tyro3 variant disrupts Tyro3 function(s) remains unknown, and given that its effect is only apparent in vivo in the mouse and most striking in anx/anx mice, identification and validation of such a mechanism is more challenging.

CONCLUSION
In conclusion, here we have identified a mutation in the signal sequence of the Tyro3 tyrosine kinase receptor (R7W-Tyro3) as enhancing anx phenotypes, and shown that normal Tyro3 acts protectively to sustain the appetite regulatory circuitry in anx/anx mice. Whereas predictive programs and in vitro analyses could not predict or detect appreciable differences, the biological consequences of both the R7W-Tyro3 variant and normal Tyro3 are apparent in vivo. Furthermore, our findings suggest the existence of subtle, quite possibly often missed, mechanisms that might regulate protein and/or RNA subcellular localization. To date platelet function has not been systematically examined in ANR. Our observations suggest that, in the future, systematic analyses of platelet function in eating disorders and possibly other psychiatric and neurodegenerative disorders might prove useful and help categorize these into specific biological subtypes. Overall, our observations highlight the importance of analyses in the mouse or other model organisms as a testbed during the search for human disease modifiers and for genetic variants with measurable biological effects. Finally, enhancing Tyro3 expression or signaling could provide a strategy for counteracting deficits in the appetite regulatory circuitry that might contributein a primary or secondary mannerto the ingestive dysfunctions seen in ANR and some neurodegenerative disorders.

MATERIALS AND METHODS
Mice anx/+ mice maintained on a B6C3Fe a/a hybrid background were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed onto C57Bl6/J and Molf/EiJ backgrounds. To identify mice carrying the C19T-Tyro3 mutation, polymerase chain reaction (PCR)-based assay using primers 5′-GATGGCGCTGAGGCGGAGCATG and 5′-CGCGGC-CGGAGGTCTGGCAG, and subsequent digestion with NlaIV was used.
All animal experiments were performed in strict adherence to guidelines for experimentation with laboratory animals set by the Canadian Animal Care Center (CACC).

Platelet aggregation assays
Platelet aggregation assays were performed as in Angelillo-Scherrer et al. (2005). In brief, 1×10 6 platelets per mouse were incubated with 1 U thrombin (Sigma) for 10 min at room temperature (RT), labeled with FITCconjugated anti-P-selectin antibody (1:1000; BD Biosciences-Pharmingen Cat. no. 561849 and analyzed by flow cytometry using a FACs instrument and Cell Quest software. Platelet aggregation was analyzed for six or more mice of each genotype (anx/anx, anx/+, +/+) and data analyzed using the Mann-Whitney Test.

Platelet electronmicroscopy
Isolated platelets were fixed with 4% paraformaldehyde and 2% gluteraldehyde in 0.1 M sodium cacodylate buffer pH 7.3. Sectioned samples were analyzed by Tecnai 20 transmission electron microscopy followed by data analysis using Excel Docu (Soft Imaging Systems). For morphologic analyses, 56-63 platelets from each of four anx/anx and three anx/+ and +/+ animals were analyzed. Data was analyzed using the Mann-Whitney Test.

Quantitative RT-PCR
RNA from P19 cortices from anx/anx, anx/+ and +/+ mice was isolated using Trizol reagent (Invitrogen) according to manufacturer's instructions. cDNA was synthesized from total RNA using oligo-dT primers and the Superscript III First Strand Synthesis System (Invitrogen) according to manufacturer's instructions (2 µg of RNA used in 20 µl reversetranscription reaction). Primers specific for Tyro3 (5′-AATGCCGAGAT-TTACAACTACCTCAT-3′ and 5′-GTTCCATTCGACAGACACGTGAA-GCTT-3′) and GAPDH (5′-TGTGAACGGATTTGGCCGTAT-3′and 5′-CATGTAGACCATGTAGTTGAG-3′) were used. Reactions were run with SYBR green PCR master mix (Invitrogen) in an AB 7500 fast real-time PCR system and relative gene expression levels were calculated using Sequence Detection System 2.2 Software (Applied Biosystems). Expression values were normalized relative to sample Gapdh mRNA expression. RNA from five anx/+ and +/+ and three anx/anx animals was analyzed. Data were analyzed using nonparametric t-tests and Mann-Whitney test.
RNA in situ hybridization on sections RNA in situ hybridization on sections was performed on brains perfused with 4% paraformaldehyde in diethyl-pyrocarbonate-treated 1×PBS and embedded in optimal cutting temperature medium (Sakura Finetek, Torrance, California, USA). Coronal sections of 14 µm were cut using the Leica VT1000 cryostat. RNA in situ hybridization was performed on tissue sections as described in Storm and Kingsley (1996). Sense and antisense probes were generated from a 588 bp HindIII fragment, containing the partial coding sequence of the Tyro3 intracellular kinase domain, cloned into pBluescript KSII. The sense probe was generated with XhoI digestion and T3 RNA polymerase transcription, the antisense probe upon EcoRI digestion and in vitro transcription with T7 RNA polymerase (Roche, Basel, Switzerland).

Generation of transgenic animals
Tyro3 and R7W-Tyro3 cDNA was generated from RNA from +/+ and anx/ anx brains using Superscript First-Strand Synthesis RT System (Invitrogen), amplified by real-time PCR, and inserted into pDONR201 with BP Clonase (Invitrogen). To generate an untagged human TYRO3 construct, human TYRO3 cDNA (generously provided by Dr Kensaku Mizuno, Tohoku University, Sendai, Japan) was inserted into filled-in SalI sites of pDONR201. Using LR Clonase, these versions of Tyro3 were cloned into the T3Xpressn vector. The T3Xpressn vector consists of 9.5 kb BamHI-NotI fragment 5′ of and containing the transcriptional start site for signal sequence-containing exon 2c (Biesecker et al., 1995) and 154 nt of the 5′UTR, attR recombination sites, an in-frame C-terminal GFP, and Sv40 IVS polyA. Resulting expression constructs were digested with NruI, purified with GeneClean (Qbiogene, Irvine, California, USA), and used to generate transgenic mice by standard pronuclear microinjection into FVBN/J oocytes. Positive transgenic mice were identified by Southern blot analysis and by a PCR-based assay using the following primers: GWTyro3-F1: GGGGACA-AGTTTGTACAAAAAAGCAGGCTGCCGCCGATGGCGCTGAGGCGG-AGCATGGGGTG and GWTyro3-R1: GGGGACCACTTTGTACAAGAA-AGCTGGGTTAACTGCTACTGTGAGGCAGTAGCCCTTG.

Immunostaining
In all cases, a minimum of three mice of a given genotype and stage were analyzed. Adult brain cryosections were blocked in 10% normal donkey serum (NDS) in PBS with 0.2% Triton X-100 (PBST) for 1 h at RT. Antibodies were diluted in 2% NDS PBST and incubated overnight at 4°C. Samples were washed and incubated with Cy3 or Alexa Fluor 488 secondary antibodies (1:500; Invitrogen) for 1 h at RT. Samples were mounted with Vectashield plus DAPI (Vector Labs).
For immunofluorescence, primary neuronal culture samples were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 PBS for 5 min at RT. All samples were blocked in histoblock [3% BSA, 1% BM blocking solution (0.01 M maleic acid, 15 mM NaCl, pH 7.5), 20 mM MgCl2 and 0.3% Tween 20 in PBS] for 1 h at RT, incubated overnight in primary antibodies. Secondary antibodies were applied and samples processed as described above.
The following antibodies were used: anti-Npy (1:1000; Peninsula Labs, T4068.0500), anti-Tyro3 (1:1000; Abcam, ab79778) and mouse anti-MAP2 (1:2000; Sigma, M4403), rabbit anti-calbindin (1:1000; Chemicon, AB1778), anti-5HT (1:8000; Immunostar, 20080) For silver nitrate enhanced immunoperoxidase staining, 20 µm sections were cut through hypothalami of P19 +/+, anx/anx and Tyro3-GFP; anx/anx mice for anti-Npy staining, and through cerebella of P19 and P10 Tyro3-GFP; +/+ and R7W-Tyro3-GFP;+/+ mice for anti-GFP staining. Immunoperoxidase staining was performed with the Vectastain kit (Vector Laboratories) and enhancement with silver nitrate was performed as described in Kitt et al. (1987). Slides were scanned using a Zeiss Axio Scan.Z1 microscope. For analyses of Npy-expressing neurons, hypothalami of P19 mice were sectioned completely and Npy+ soma from nine sections containing the arcuate nucleus, median eminence and highest number of Npy+ neurons for each of three animals per genotype were counted. In wild-type mice Npy+ soma were identified by the presence of circular or semi-circular Npy staining in the soma. By contrast, Npy staining can be clearly detected throughout the soma from anx/anx mice and as a more pronounced circular staining within the soma of anx/anx; Tyro3-GFP transgenic animals. Thus, it is possible that the numbers of Npy+ soma is underestimated in wild-type animals, and that the difference between wild-type and anx/anx Npy+ neurons is even greater than recorded here. Pairwise comparisons were performed using the Wilcoxon signed-rank test (alpha<0.05; P<0.05) (Social Science Statistics, 2015: http://www. socscistatistics.com/tests/signedranks/Default2.aspx; accessed on 4.19.2016).

Co-translational processing
In vitro transcription/translation (TnT) with a Tyro3 and R7W-Tyro3 template containing all eight predicted N-glycosylation sites coupled with the addition of canine pancreatic microsomal membranes (CMM) and subsequent addition of endoglycosidase H was used to examine the cotranslational processing of R7W-Tyro3. Cell-free expression of Tyro3 and R7W-Tyro3 protein was performed using a TnT Coupled Reticulocyte Lysate System (Promega, Madison, WI, USA). DNA templates of Tyro3 and R7W-Tyro3 protein containing primary sequence for the first 410 amino acids of the extracellular domain, and, thus, all eight predicted Nglycosylation sites, were generated by digestion with NheI. TnT was performed in the presence and absence of Canine Pancreatic Microsomal Membranes (Promega) according to the manufacturer's instructions. To remove any N-acetylglucosamine residues, some products were denatured in denaturing buffer (0.5% SDS, 5 mM 2-mercaptoethanol) at 37°C for 15 min and subsequently digested with 500 U of endoglycosidase H (New England Biolabs, Beverly, MA, USA) at 37°C for 1 h. Samples were analyzed by 15% SDS-PAGE and autoradiography. β-lactamase from Escherichia coli was used to assay for signal peptidase activity, and α-factor from Saccharomyces cerevisiae to assay for core glycosylation activity.
This article is part of a special subject collection 'Neurodegeneration: from Models to Mechanisms to Therapies', which was launched in a dedicated issue guest edited by Aaron Gitler and James Shorter. See related articles in this collection at http://dmm. biologists.org/collection/neurodegenerative-disorders.


extfieldsofstudy:
Computer Science
Medicine

text:
Synthetic circuits that process multiple light and chemical signal inputs

Background Multi-signal processing circuits are essential for rational design of sophisticated synthetic systems with good controllability and modularity, therefore, enable construction of high-level networks. Moreover, light-inducible systems provide fast and reversible means for spatiotemporal control of gene expression. Results Here, in HEK 293 cells, we present combinatory genetic circuits responding to light and chemical signals, simultaneously. We first constructed a dual input circuit converting different light intensities into varying of the sensitivity of the promoter to a chemical inducer (doxycycline). Next, we generated a ternary input circuit, which responded to light, doxycycline and cumate. This circuit allowed us to use different combinations of blue light and the two chemical inducers to generate gradual output values over two orders of magnitude. Conclusions Overall, in this study, we devise genetic circuits sensing and processing light and chemical inducers. Our work may provide insights into bio-computation and fine-tuning expression of the transgene. Electronic supplementary material The online version of this article (doi:10.1186/s12918-016-0384-y) contains supplementary material, which is available to authorized users.

Background
Synthetic biology adopts the concepts of engineering and computational science into biological systems, aiming to generate artificial genetic circuits and systems with desirable functions. It offers a promising way to address global challenges, for example, clean energy, environment restoration, and increasing medical needs. During the past decade, a remarkable development of synthetic biology has been achieved. Various synthetic devices and systems have been established, including biological oscillators, switches, counters, as well as logic gates [1][2][3][4]. However, it is still challenging to generate complex synthetic systems with good controllability and programmability. For instance, like electronic or mechanical systems that can be fine-tuned by various inputs and produce predictable outputs. Therefore, one can program the systems to act in a desirable way by altering input information. Development and characterization of standard modules, which sense and convert multiple input signals into cellular responses will help address the challenges [5,6].
In natural biological systems, multi-signal processing is a fundamental aspect. For example, the bow-tie (also called hourglass) architecture, which refers to systems that receive a diversity of inputs and convert the input signals through an intermediate "core", and finally generate a variety of outputs. Since the intermediate "core" is composed of relatively few universal components, the overall structure of the system resembles a bow-tie or hourglass [7]. For instance, in metabolic networks, multiple input nutrients are converted into multiple biomass components by a small number of mediator factors [7]. Previous work suggests that the recurrence of bow-tie architecture in various biological systems indicates its significance on enhancing the robustness of the biological systems [8]. In the counterpart electronic systems, modules for multi-input integration are also widely used, for example, a module called "digital-toanalog" converter (DAC) is commonly used in audio or video devices for converting multiple digital-input signals into the analog output signals [9].
Previous work reported some chemically-inducible expression systems in mammalian cells [10,11]. Recently, Optogenetics has demonstrated that light is an ideal source of signal for spatiotemporal control of gene expression [12][13][14][15]. The combination of chemical inducer and light inducer, for example by generating chimeric promoters that consist chemical-responsive and lightresponsive elements, can achieve spatial and stringent control of transgenes [16]. Using light as inducer can avoid drawbacks of using chemical inducers. For instance, the chemical inducers are needed to be transported into cells by passive or active manner before they encounter the sensors, which causes a delay of target gene expression. The delay may lead to undesirable cellto-cell variation. However, using light does not result in this problem. In addition, recent work demonstrated that light can be used as a communication signal between the computer and modified E. coli cells [17,18]. Connecting synthetic biology systems with a computer, and then monitor and control the behaviors of the circuits by a computer program can tremendously increase programmability of synthetic systems.
In this study, we first developed a 2-input circuit that exhibited different sensitivity to doxycycline (Dox) upon different doses of blue light illumination. Specifically, a blue light-inducible system, called LightOn system [14], was used to control the expression level of a transcriptional repressor TetR. A reporter GFP was driven by TetR-repressible promoter. The repression of TetR can be relieved by adding Dox. Therefore, light and Dox acted as inducers of this circuit. Next, we generated a 3-input circuit for conversion of the binary input sequence, consisting of light and chemicals, into graded output promoter activities. Specifically, this circuit was composed of a cumate-inducible promoter driving a modified rtTA (hereafter, rtTAm) [19], a light-inducible promoter driving the TetR co-repression peptides (hereafter, TCP) [20]. TCP-rtTAm complex activates the output TRE3G promoter. Therefore, Light-inducible system and cumate-switch system form an AND-gate. On the other hand, Dox also can trigger the DNA-binding of rtTAm. Therefore, Dox-inducible system and cumateswitch system also compose an AND-gate. Previous work suggests that short peptide inducer may be less efficient than Dox [21]. Moreover, it has been reported that the peptide competes with Dox for the tc-binding pocket of TetR [20]. Thereby, the potency of TCP fusion protein might be much lower than Dox as rtTAm inducer, and the presence of TCP fusion protein could inhibit Dox inducing ability.

Results
The dual input circuit converting illumination dose into sensitivity variations of a promoter to Dox In this circuit, LightOn system was used to control the expression of TetR. And a reporter GFP was driven by the CMV(tetO2) promoter (Life Technologies, T-REx system, and Additional file 1: Supplementary note) containing two copies of tet operator 2 (Fig. 1a). The LightOn system comprises a synthetic photoactive transactivator GAVPO and its cognate synthetic promoter U5 [14]. GAVPO monomers form a homodimer upon blue light illumination. The GAVPO dimer then binds to the UAS G element in the U5 promoter to recruit general transcription factors and coactivators to bind to the U5 promoter.
Our data indicated the expression level of the TetR::m-Cherry::NLS fusion could be tuned by adjusting the exposure of blue light (Fig. 1b-d). We examined the spatial resolution of this circuit. Specifically, we illuminated a small square area of the dish, while the other area of the dish was kept in dark. The result showed that cells in the illuminated area were TetR::mCherry::NLS positive and GFP low, while cells in the adjacent dark area were TetR::mCherry::NLS negative and GFP high. The light-induced TetR::mCherry::NLS suppressed the expression of GFP, and addition of Dox relieved this repression (Fig. 1c). Furthermore, we illuminated the cells with different doses of blue light before Dox treatment. Cells exposed to various amounts of light showed different activation thresholds of Dox induction. The increase in the level of the repressor resulted in an increase in the Dox threshold. Whilst, the dynamic range of the promoter was not affected (Fig. 1d).
Design and construction of a multi-input circuit for conversion of light and chemical binary information into different promoter activities Next, we attempted to design and construct a circuit for integration and conversion binary combinations of light and chemical signals into graded output values (Fig. 2a).
The circuit consists of two AND-gates that response to blue light, cumate and Dox, respectively ( Fig. 2a, b). However, the output strengths of the two AND-gates were not equivalent. The addition of saturated cumate and Dox resulted in strong output while the addition of cumate together with light illumination led to moderate output (Fig. 5).
The components composing the circuit are as following: LightOn system which was introduced above; the cumate-inducible system consists of a transcription repressor CymR and the repressible promoter CMV5CuO promoter (pCMV5CuO). CymR binds to the operator sequence (CuO) downstream of a strong promoter CMV5 and inhibits transcription. The addition of a cumate relieve the repression by CymR [19]; and a TetR co-repression peptide (TCP) -inducible system. Specifically, the mCherry-TCP fusion (Additional file 1: Supplementary note) can aid in DNA-binding of rtTAm. The rtTAm is a chimeric protein composed of a reverse TetR variant and 3 copies of VP16 activation domain (Additional file 1: Supplementary note). The TCP binds to the tc-binding pocket of the reverse TetR variant and triggers allosteric conformational change in the reverse TetR variant, leading to binding of the latter to its cognate DNA [20]. The responsive TRE3G promoter contains seven repeats of tet operator site (tetO) upstream of a CMV minimal promoter (Fig. 2b).
The circuit can be divided into three layers. The first layer receives light and cumate signals. In this layer. constitutively expressed GAVOP (it does not appear in the scheme) activates expression of mCherry-TCP upon blue light illumination. Constitutively expressed CymR (it does not appear in the scheme) suppresses the expression of rtTAm. Cumate is required to switch on the expression of rtTAm. The second layer is the information integration node. Specifically, the rtTAm, representing the presence of cumate, and mCherry-TCP, representing the presence of blue light, interact and form a protein complex, which can activate the output promoter. On the other hand, Dox also can aid binding of rtTAm to output promoter. The third layer is the responding (output) node, in which there is a luciferase gene driven by the TRE3G promoter (Fig. 2b).

Characterization of the ternary input circuit
We first characterized each inducible expression node, separately. And identified the saturation dose for each inducer. Then we characterized the complete circuit.
To our knowledge, there is no demonstration of induction of rtTA (in our case, it is rtTAm) by TCP in mammalian cells, to date. We first attempted to test whether intracellularly expressed TCP fusion protein, i.e., mCherry-TCP fusion, could induce rtTAm-dependent expression from the TRE3G promoter. To this end, we used CMV promoter to control the expression of mCherry-TCP and rtTAm. And used TRE3G promoter to drive a GFP gene (Fig. 3a). We introduced the circuit DNA into HEK293 cells by transfection and observed the transfected cells by fluorescent microscope, 72 h after transfection. In the control group, the mCherry-TCP fusion was replaced by mCherry. We observed GFP signal in the mCherry-TCP cells but not in the mCherry cells ( Fig. 3b and Additional file 2: Figure S1).
To characterize the LightOn system regulated expression of mCherry-TCP, we generated a stable cell line integrated with a modified circuit without expression of CymR. However, the other components were the same as the complete circuit (Fig. 4a). We illuminated the cells with blue LED (1.25 W m −2 ) for 24 h and then put the cells back in a dark environment for 0 h, 24 h, or 48 h (the total incubation time was 24 h, 48 h or 72 h, respectively). Next, we examined the mCherry-TCP expression levels by flow cytometry (Fig. 4b). At the meantime, we examined luciferase expression induced by mCherry-TCP. The data suggested that after 24 h illumination, the expression of mCherry-TCP reached the highest level, and then it start to decrease. However, the maximum expression level of luciferase was observed 48 h after the illumination started (Fig. 4c). In another experiment, we examined the kinetics of the circuit at earlier phases after illumination with constitutively expressed rtTAm. The results show that 10 h after illumination a moderate increase of luciferase activity was detected (Additional file 3: Figure S2A). And the response of the circuit to blue light was slower than the response to Dox (Additional file 3: Figure S2B).
Also, we characterized the cumate-inducible node in the circuit (Fig. 4d). We varied cumate concentrations, Fig. 2 The design of the 3-input circuit. (a) Illustration of the conversion of input signals including Dox, cumate and light into graded output signals, which are promoter activities. The system consists of three inducible systems formed Boolean logic gates. (b) The scheme of the circuit. It can be divided into three layers. In the first layer, LightOn system controls the expression of mCherry-TCP fusion; cumate-switch system controls the expression of rtTAm (co-expressed with EYFP linked by 2A peptide). In the second layer, Dox or mCherry-TCP fusion protein can serve as an inducer to trigger the binding of rtTAm to the cognate TRE3G promoter. In the third layer, rtTAm serves as transcription activator to initiate the transcription of luciferase from TRE3G promoter and then examined the expression of EYFP. The data suggested that addition of 30 μg/ml of cumate in the medium was enough to produce the maximal expression level of rtTAm and EYFP from pCMV5CuO (Fig. 4e). The kinetics of the circuit responding to cumate induction with constitutive mCherry-TCP expression was also characterized. In the experiment, the cells were continuously illuminated, meanwhile were treated with 30 μg/ml of cumate for different time durations. 10 h after cumate addition, a moderate increase of luciferase activity was detected (Additional file 3: Figure S2C).
Finally, we characterized the complete circuit. We applied all the eight combinations of inputs to the cells and measured the output values, i.e., the luciferase activities. Expression levels of mCherry-TCP and rtTAm (indicated by EYFP) were also examined. The data showed that the circuit responded to different input combinations and generated different output values, which evenly distributed in a range of two orders of magnitude. The output signal induced by TCP was weaker than the signal induced by Dox. In agreement with previous work [20], TCP inhibited the rtTAm-binding of Dox, which might explain that the input combination of "Light +, Cumate +, Dox +" induced lower luciferase level than the combination of "Light -, Cumate +, Dox+" (Fig. 5). We also introduced a conditional positive feedback loop into the third layer. A trans-activator, i.e., tTA was placed downstream of TRE3G promoter. The binding of tTA to TRE3G promoter can be blocked by Dox, but not by TCP (data not shown). The result suggested that the conditional positive Constitutively expressed mCherry-TCP and rtTAm (both driven by CMV promoter) interact with each other, and then the complex bind to the tetO elements within TRE3G promoter (consist of 7 × tetO elements and a minimal CMV promoter). A GFP gene is placed downstream of TRE3G promoter. (b) mCherry-TCP induced the rtTAm-dependent expression of GFP. GFP-positive cells were observed in cells co-transfected with mCherry-TCP and rtTAm coding plasmids, but not in cells co-transfected with mCherry and rtTAm coding plasmids. The scale bar is 50 μm feedback circuit responded to the input combinations similarly as the circuit without feedback (Additional file 4: Figure S3).

Discussion
We observed leaky expression in the 3-input circuit, specifically, the leakage of rtTAm and mCherry-TCP was observed (Fig. 5). The leaky expression usually causes undesired basal expression of regulated proteins in the absence of the respective inducers and results in compromise of the circuits [22,23]. However, the leaky expression here might lead to the differential responses of the circuit to individual input combinations, which is not completely undesirable. It is possible that due to the leaky expression of rtTAm and mCherry-TCP, each inducer, i.e., light, cumate or Dox, exhibited different effects to the system. For instance, when cumate was added in the medium, the elevated rtTAm level could enhance leaky expression of TRE3G promoter, in the absence of the other two inducers; when the cells were illuminated, increased mCherry-TCP interacted with leaky rtTAm, and resulted in higher rtTAm-dependent expression of luciferase. Previous study demonstrated the way to fine-tune basal and/or maximal expression of LightOn system [24], which provides insights for the future modification of the circuit, especially when stringent control of a specific promoter is needed. Similarly, lower leaky expression level would be achieved by modifying CMV5(CuO) promoter using another weaker enhancer element to replace the strong CMV enhancer or increase the level of CymR by using a stronger promoter.
Our circuits can be used as building blocks in a synthetic programmable system. For instance, it can be utilized in a synthetic bow-tie structure, which is designed to sense and convert multiple input signals into  [17,18]. In their work, the light sequence generated by computer were used to control dynamics of synthetic circuits. Our circuit also can be modified to respond to a computer-generated sequence of light of different wavelengths. For instance, a red-or far-red light-responsive system [25], can be used to replace the cumate-inducible part in the current circuit. Thereby it may allow us to convert digital codes generated by an electronic-function-generator into gradual varying of cellular activities. It has been suggested that some master regulators displayed quantitative effects on cell fate decision, for instance, Oct/4 quantitatively influences differentiation, dedifferentiation or self-renewal of embryonic stem cells [26]; P53 quantitatively control cell fate decision between apoptosis and growth arrest [27]. Therefore, a synthetic circuit, which responds to various input programs and produced gradual output values, has the potential to be used as computer-aided cell fate controller. In addition, our 3-input circuit that responds to light and chemicals can be used to express a therapeutic gene at a specific place and time, meanwhile, exhibits minimized undesirable expression at other places, which would improve the safety of the therapy.
In natural biology systems, analog behaviors are common, for instance, stimulation of stress-responsive gene could be operated in an analog regime [28] and neurons perform both digital and analog information processing [29]. Moreover, the analog computing system has been demonstrated in E. coli [30][31][32]. As mentioned above, DAC is widely used in electronic engineering. A biological DAC-like module that combines multiple signals, and process the digital combinations of stimuli into graded output values for reconstruction of analog signal is needed to achieve sophisticated bio-computation functions, for example, programmable logic controller and reliable environmental sensor. In this study, by constructing a circuit that converts discrete input signals into varying of transcriptional activity of the output promoter, we attempted to explore the possibility that confers DAC merit, to a certain degree, to mammalian cells.
However, the output steps of our circuit were nonmonotonic, therefore it may not act as a real DAC (Fig. 5 and Additional file 4: Figure S3). We hypothesize that modification of TCP sequence to increase its efficiency of rtTAm induction might be a potential way to increase the linearity of the output steps. Previous work has suggested the method to improve the function of TetRinducing short peptides [20,33]. Also, a further modification is required to expand the rationale to implement more inputs.

Conclusion
We presented mammalian circuits that processed multiinput of blue light and chemical molecules. The 2-input circuit displayed blue light illumination dose-dependent shifting of Dox response threshold. The results suggested that increased expression of the upstream repressor (TetR) resulted in an increased activation threshold with similar basal expression level and dynamic range to that of the downstream TetR-repressible promoter. The 3-input circuit converted the sequence of blue light and two chemical molecules into varying of promoter activities over two orders of magnitude.

Construction of DNA plasmid
The details of DNA cloning are described in Additional file 1: Supplementary note.

Cell culture, transient transfections, and generation of stable cell lines
Human Embryonic Kidney (HEK) 293 cells were obtained from the American Type Culture Collection (Manassas, VA), and were used in our previous study conducted by Dr. Zai Wang et al. [34]. The cells were grown in High Glucose Dulbecco's modified Eagle's medium (DMEM, Life Technologies, catalog number: 12800-017) supplemented with 10% FBS (Life Technologies, catalog number: 10270-106). The cells were sustained at 37°C, in 5% CO 2 environment. In this study, all transfections were conducted by using Lipofectamine 2000 Transfection Reagent (Life Technologies, catalog number: 11668-019). 24 h before DNA transfection, 0.5 × 10 6 cells were seeded in each well of 6 well cell culture multiwell plate. On the day of DNA transfection, 5 μg DNA diluted in 500 μl Opti-MEM (Thermo Scientific, catalog number: 31985-070) mix with 10 μl Lipofectamine 2000 Transfection Reagent diluted in 500 μl Opti-MEM. The DNA-lipid complex was incubated at room temperature for 20 min, and then was added to the cells. To establish the desirable stable cell lines, 400 μg/ml Zeocin (InvivoGen, catalog number: ant-zn-1), 200 μg/ml Hygromycin B (Sigma-Aldrich, catalog number: H3274-50MG), and/or 2 μg/ml Puromycin (InvivoGen, catalog number: ant-pr-1) were correspondingly used to selected the cells for 10 to 14 days. Doxycycline (Sigma-Aldrich, catalog number 24390-14-5) and/or cumate solution (System Biosciences, catalog number: QM100A-1) were correspondingly used to treat the cells. For light-inducible expression, cells were sustained in a dark environment for 7 days before illumination with blue LED. To induce the LightOn controlled gene expression, the cells were illuminated for 24 h and then were put back to the dark environment for further 48 h, 24 h or 0 h culture before the measurements.

Inducible gene expression
For light-inducible expression, cells were kept in darkness for 7 days before illumination with blue LED. Cells were exposed to blue light (1.25 W m −2 ) for 5 min daily to maintain a minimal level of TetR::mCherry::NLS for the suppression of downstream GFP, thereby ensuring cells remained in the same condition (GFP negative) before Dox induction. In the tuning activation threshold experiment, the cells were exposed to blue light for different time durations and then kept in darkness before treatment with various concentrations of Dox. For cumate-inducible expression, the inducer was added into the culture medium 72 h before measurement of gene expression.

Fluorescence microscopy
The cells for microscopy images collection were grown in 60 mm dish, sustained in DMEM medium supplemented with 10% FBS, treated with the particular inducers, i.e., doxycycline, cumate and blue light. Images were taken at 72 h after induction started. Microscopy images were acquired on a Nikon TE2000-E inverted fluorescence microscope. The filter for fluorescent images of GFP was FITC (Ex 465-495 nm, Em 515-555 nm), exposure time was 3000 ms; for mCherry was Cy3 (Ex 530-560 nm, Em 573-648 nm), exposure time was 6000 ms; and for EYFP was YFP (Ex490-500, Em 520-560), exposure time was 3000 ms. Data processing was performed with software Image J.

Flow cytometry
The flow cytometry analysis was carried out on BD LSR Fortessa Analyzer. Before the analysis, the cells were trypsinized using 0.25% trypsin-EDTA and then were centrifuged. 100,000 to 150,000 cells were suspended in 700 μl PBS supplemented with 1% FBS before loading onto the analyzer. GFP was measured with a 488 nm blue laser and an FITC (530/30 nm) emission filter, whereas mCherry was measured with a 561 nm yellowgreen laser and a PE-Texas Red (610/20 nm) emission filter. FlowJo software was used to perform the data collection and processing.

Luciferase assay
The Varioskan Flash Spectral Scanning Multimode Reader (Thermo Scientific) was used to measure the chemiluminescence catalyzed by luciferase. The Luciferase Assay System (Promega, catalog number E1500) was used for luciferase activity measurements. The measurements were carried out at 72 h after induction by following the kit instruments.

Additional files
Additional file 1: Supplementary note. This note describes the details of DNA cloning; information of plasmids used in this study; information of oligonucleotides used in this study; nucleic acid sequence of CMV(tetO2) promoter and CMV5(CuO) promoter; amino acid sequence of rtTAm, TCP, and mCherry-TCP. (DOCX 30 kb) Additional file 2: Figure S1. TCP fusion proteins induce rtTAmdependent expression of GFP. We transfected HEK cells with mCherry, mCherry-TCP, or mCherry-NLS-TCP constitutive expression plasmids, respectively, also transfected TRE3G promoter controlled GFP expression plasmid to all the three groups of cells. We divided mCherry transfected cells into two dishes and treated one dish of the cells with 1 μg/ml Dox at 24 h after transfection. The images were collected at 72 h after transfection. (TIF 532 kb) Additional file 3: Figure S2.